National Institute of Allergy and Infectious Diseases, NIH: Volume 1: Frontiers in Research (Infectious Disease) (Infectious Disease)

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National Institute of Allergy and Infectious Diseases, NIH Volume 1

Frontiers in Research

Infectious Disease Vassil St. Georgiev

For other titles published in the series, go to www.springer.com / humana click on the series discipline click on the heading “Series” click on the name of the series

National Institute of Allergy and Infectious Diseases, NIH Volume 1

Frontiers in Research

Edited by Vassil St. Georgiev, PhD Karl A. Western, MD John J. McGowan, PhD National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

Editors Vassil St. Georgiev, PhD Karl A. Western, MD John J. McGowan, PhD National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

Series Editor Vassil St. Georgiev National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

ISBN 978-1-934115-77-0 e-ISBN 978-1-59745-569-5 DOI: 10.1007/978-1-59745-569-5 Library of Congress Control Number: 2007941162 © 2008 Humana Press, a part of Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Adapted from Chapter 30, Fig. 30.3, showing the sCD4-17b bifunctional protein, which in turn is based on the atomic structure reported in Kwong et al., Nature, 393:648–659 (1998). Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Dedication

To the thousands of investigators who, for more than 50 years, have received the support of the National Institute of Allergy and Infectious Diseases (NIAID) and have dedicated their lives and careers to biomedical research.

RESEARCH IS NOT A SYSTEMATIC OCCUPATION BUT AN INTUITIVE ARTISTIC VOCATION Albert Szent-Györgyi

Preface

For more than 50 years, as part of the National Institutes of Health, the mission of the National Institute of Allergy and Infectious Diseases (NIAID) has been to conduct and support basic and applied research to better understand, treat, and prevent infectious, immunologic, and allergic diseases with the ultimate goal of improving the health of individuals in the United States and around the world. In recent years, NIAID has responded to new challenges including emerging and re-emerging infectious diseases, potential bioterrorism threats, and an increase in pediatric asthma prevalence. A cornerstone of NIAID-supported research also continues to be the discovery and improvement of vaccines focused on an array of infectious diseases with global public health importance. As part of its mission to foster biomedical discovery and to reduce the burden of human disease, NIH and NIAID in particular, are committed to encouraging the accelerated translation of biomedical discoveries into effective clinical care and public health practice throughout the world. In pursuit of this goal and its disease-specific scientific objectives, NIAID seeks to broaden research opportunities and collaborations involving scientists and institutions outside the United States. During 2006, special emphasis was given to fostering scientific collaboration between U.S. researchers and investigators in Central and Eastern Europe, the Baltic Region, Russia, Ukraine, and other newly independent states that were formerly part of the Soviet Union. Although the countries of Central and Eastern Europe have strong traditions in biomedical research, scientists from this region have been less successful than their Western European colleagues in competing for NIAID funding and in forming partnerships with U.S. scientists. To help address this situation, NIAID convened a research conference in Opatija, Croatia (June 24–30, 2006) so that U.S. and European scientists could explore shared research interests with a focus on microbiology and infectious diseases, HIV/AIDS, and basic and clinical immunology. In the field of microbiology and infectious diseases, major presentations at the conference focused on recent research developments in emerging and re-emerging infections (anthrax and other potential biological weapons, vector-borne infections, tuberculosis, and influenza). A number of presentations discussed ongoing research targeting the development of infectious disease prophylactics and therapeutics. One of the most serious problems worldwide that confronts efforts to control and treat infectious diseases is the increasing resistance of some pathogens to the current armamentarium of drugs. Microorganisms belonging to all four classes of infectious agents (bacteria, viruses, parasites, and fungi) have developed resistance to previously effective chemotherapeutics, thereby becoming serious threats to individual well-being and international public health. One striking example of drug resistance is the emergence of extensively drug-resistant tuberculosis. Several conference presentations were therefore focused on drug resistance. HIV/AIDS also remains a major infectious disease research priority and it was well addressed during the conference. Since the start of the HIV/AIDS pandemic in the early 1980s, nearly 20 million people worldwide have died of the disease. According to an estimate issued by the Joint United Nations Programme on HIV/AIDS (UNAIDS) by the end of 2003, about 38 million adults and children were living with HIV/AIDS and in many countries overall prevalence still is rising. Although much progress has been made in the treatment of AIDS and in understanding effective strategies to prevent HIV transmission, research is urgently needed on vaccines, microbicides, therapeutic agents, behavioral prevention strategies, and the management of HIVrelated co-morbidities. NIAID-funded research in basic and clinical immunology has led to significant discoveries that have guided the effective treatment of a host of immunological conditions. For example, “tolerance induction” research has enabled the selective blocking of inappropriate or destructive immune responses while leaving protective immune responses intact. Major presentations at vii

viii

Preface

the conference discussed various topics in immunomodulation, autoimmunity, infections and immunity, and vaccine development. Finally, two sessions at the research conference were designed to inform participants about NIAID’s research funding mechanisms and the NIH application process. With more than 100 participants, the 2006 NIAID Research Conference in Croatia clearly demonstrated NIAID’s commitment to a cutting-edge scientific exchange to help generate more research cooperation. Following the meeting, numerous research collaborations have been explored and numerous joint research applications have been prepared and submitted. NIAID is pleased to have supported this important and unusual meeting and it welcomes publication of the important scientific findings presented there. The future of science lies in cooperation across national borders. Therefore, it is particularly rewarding to see research partnerships grow between scientists from countries previously characterized by a lack of communication and mutual understanding. With a strong research base, talented investigators in the United States and abroad, and the availability of powerful new research tools, NIAID will continue to support scientists in the forefront of basic and applied infectious and immune-mediated disease research.

Vassil St. Georgiev Bethesda, MD

Acknowledgments

We would like to express our appreciation to Ms. Caroline Manganiello and the staff of technical writers for their help in the preparation of this volume.

ix

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vassil St. Georgiev

vii

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv

PART I

INTRODUCTION

National Institute of Allergy and Infectious Diseases (NIAID): An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karl A. Western PART II

MICROBIOLOGY AND INFECTIOUS DISEASES

Section 1

Emerging and Re-Emerging Infections

1

Biotools for Determining the Genetics of Susceptibility to Infectious Diseases and Expediting Research Translation into Effective Countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malak Kotb, Robert W. Williams, Nourtan Fathey, Mohamed Nooh, Sarah Rowe, Rita Kansal, and Ramy Aziz

3

13

2

Spore Surface Components and Protective Immunity to Bacillus anthracis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patricia Sylvestre, Ian Justin Glomski, Evelyne Couture-Tosi, Pierre Louis Goossens, and Michèle Mock

19

3

New Candidate Anthrax Pathogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serguei G. Popov

25

4

Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. L. Stevenson, N. Ismail, and D. H. Walker

37

5

Multiple Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) of Brucella spp. Identifies Species-Specific Markers and Insights into Phylogenetic Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn Y. Huynh, Matthew N. Van Ert, Ted Hadfield, William S. Probert, Bryan H. Bellaire, Michael Dobson, Robert J. Burgess, Robbin S. Weyant, Tanja Popovic, Shaylan Zanecki, David M. Wagner, and Paul Keim

6

Expression of the MtrC-MtrD-MtrE Efflux Pump in Neisseria gonorrhoeae and Bacterial Survival in the Presence of Antimicrobials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William M. Shafer, Jason P. Folster, Douglas E. M. Warner, Paul J. T. Johnson, Jacqueline T. Balthazar, Nazia Kamal, and Ann E. Jerse

47

55

xi

xii

Contents

Section 2

Tuberculosis

7

What can Mycobacteriophages Tell Us About Mycobacterium tuberculosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graham F. Hatfull

67

8

Clinical Mycobacterium tuberculosis Strains Differ in their Intracellular Growth in Human Macrophages . . . . . . . . Sue A. Theus, M. Donald Cave, and Kathleen D. Eisenach

77

9

Mechanisms of Latent Tuberculosis: Dormancy and Resuscitation of Mycobacterium tuberculosis . . . . . . . . . . . . . Galina Mukamolova, Elena Salina, and Arseny Kaprelyants

83

10

Separating Latent and Acute Disease in the Diagnosis of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Mark Doherty

91

11

Mutant Selection Window Hypothesis: A Framework for Anti-mutant Dosing of Antimicrobial Agents . . . . . . . . . . Karl Drlica and Xilin Zhao

101

Section 3 Avian Influenza 12

The NIAID Influenza Genome Sequencing Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lone Simonsen, Gayle Bernabe, Karen Lacourciere, Robert J. Taylor, and Maria Y. Giovanni

109

13

Lessons from the 1918 Spanish Flu Epidemic in Iceland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magnús Gottfredsson

115

14

Control of Notifiable Avian Influenza Infections in Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ilaria Capua and Stefano Marangon

123

15

Understanding the Complex Pathobiology of High Pathogenicity Avian Influenza Viruses in Birds . . . . . . . . . . . . . David E. Swayne

131

Section 4 Prophylactics and Therapeutics for Infectious Diseases 16 Development of Prophylactics and Therapeutics Against the Smallpox and Monkeypox Biothreat Agents . . . . . . . . Mark Buller, Lauren Handley, and Scott Parker 17 The Hierarchic Informational Technology for QSAR Investigations: Molecular Design of Antiviral Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. E. Kuz’min, A. G. Artemenko, E. N. Muratov, L. N. Ognichenko, A. I. Hromov, A. V. Liahovskij, and P. G. Polischuk

145

163

18

Antivirals for Influenza: Novel Agents and Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William A Fischer, II and Frederick Hayden

179

19

Anti-Infectious Actions of the Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA) . . . . . . . . . . . . . . . . . . . . . . . . . V. P. Lozitsky

193

20

A New Highly Potent Antienteroviral Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lubomira Nikolaeva-Glomb, Stefan Philipov, and Angel S. Galabov

199

Section 5

Russian Perspectives in Emerging and Re-Emerging and Infections Research

21

Reduction and Possible Mechanisms of Evolution of the Bacterial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . George B. Smirnov

205

22

Interaction of Yersinia pestis Virulence Factors with IL-1R/TLR Recognition System . . . . . . . . . . . . . . . . . . . . . . . . Vyacheslav M. Abramov, Valentin S. Khlebnikov, Anatoly M. Vasiliev, Igor V. Kosarev, Raisa N. Vasilenko, Nataly L. Kulikova, Vladimir L. Motin, George B. Smirnov, Valentin I. Evstigneev, Nicolay N. Karkischenko, Vladimir N. Uversky, and Robert R. Brubaker

215

Contents

xiii

23

IS481-Induced Variability of Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ludmila N. Sinyashina, Alisa Yu. Medkova, Evgeniy G. Semin, Alexander V. Chestkov, Yuriy D. Tsygankov, and Gennagiy I. Karataev

227

24

Microarray Immunophosphorescence Technology for the Detection of Infectious Pathogens . . . . . . . . . . . . . . . . . . Nikolay S. Osin and Vera G. Pomelova

233

25

Development of Immunodiagnostic Kits and Vaccines for Bacterial Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentina A. Feodorova and Onega V. Ulianova

241

Section 6

26

Perspectives in Emerging and Re-Emerging Infections—Research in Central Asia and Caucasus

Research in Emerging and Re-Emerging Diseases in Central Asia and the Caucasus: Contributions by the the National Institute of Allergy and Infectious Diseases and the National Institutes of Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katherine T. Herz

27

Disease Surveillance in Georgia: Benefits of International Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lela Bakanidze, Paata Imnadze, Shota Tsanava, and Nikoloz Tsertsvadze

28

Epidemiology (Including Molecular Epidemiology) of HIV, Hepatitis B and C in Georgia: Experience From U.S.–Georgian Collaboration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tengiz Tsertsvadze

29

The National Tuberculosis Program in the Country of Georgia: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Archil Salakaia, Veriko Mirtskhulava, Shalva Gamtsemlidze, Marina Janjgava, Rusudan Aspindzelashvili, and Ucha Nanava

PART III

251 253

257 263

HUMAN IMMUNODEFICIENCY VIRUS AND AIDS

30

Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS . . . . . . . . . . . . . . . . . . . Edward A. Berger

271

31

HIV Latency and Reactivation: The Early Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guido Poli

279

32

HIV-1 Sequence Diversity as a Window Into HIV-1 Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milloni Patel, Gretja Schnell, and Ronald Swanstrom

289

33

Human Monoclonal Antibodies Against HIV and Emerging Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dimiter S. Dimitrov

299

34

Biological Basis and Clinical Significance of HIV Resistance to Antiviral Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark A. Wainberg and Susan Schader

309

35

NIAID HIV/AIDS Prevention Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David N. Burns and Roberta Black

319

36

Epidemiological Surveillance of HIV and AIDS in Lithuania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saulius Caplinskas

327

PART IV

IMMUNOLOGY AND VACCINES

Section 1

Immunomodulation

37

TACI, Isotype Switching, CVID, and IgAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emanuela Castigli and Raif S. Geha

343

38

A Tapestry of Immunotherapeutic Fusion Proteins: From Signal Conversion to Auto-stimulation . . . . . . . . . . . . . . . Mark L. Tykocinski, Jui-Han Huang, Matthew C. Weber, and Michal Dranitzki-Elhalel

349

xiv

Contents

39

A Role for Complement System in Mobilization and Homing of Hematopoietic Stem/Progenitor Cells . . . . . . . . . . M. Z. Ratajczak, R. Reca, M. Wysoczynski, M. Kucia, and J. Ratajczak

40

Post-translational Processing of Human Interferon-γ Produced in Escherichia coli and Approaches for Its Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maya Boyanova, Roumyana Mironova, Toshimitsu Niwa, and Ivan G. Ivanov

Section 2

357

365

Autoimmunity

41

B-cell dysfunctions in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moncef Zouali

377

42

A Model System for Studying Mechanisms of B-cell Transformation in Systemic Autoimmunity . . . . . . . . . . . . . . Wendy F. Davidson, Partha Mukhopadhyay, Mark S. Williams, Zohreh Naghashfar, Jeff X. Zhou, and Herbert C. Morse, III

385

43

Breach and Restoration of B-Cell Tolerance in Human Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . . . . . Iñaki Sanz, R. John Looney, and J. H. Anolik

397

Section 3

Infection and Immunity

44

Dendritic Cells: Biological and Pathological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jacques Banchereau, John Connolly, Tiziana Di Pucchio, Carson Harrod, Eynav Klechevsky, A. Karolina Palucka, Virginia Pascual, and Hideki Ueno

409

45

Immunomic and Bioinformatics Analysis of Host Immunity in the Vaccinia Virus and Influenza A Systems . . . . . . Magdalini Moutaftsi, Bjoern Peters, Valerie Pasquetto, Carla Oseroff, John Sidney, Huynh Hoa-Bui, Howard Grey, and Alessandro Sette

429

46

Immunoreactions to Hantaviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alemka Markotic´ and Connie Schmaljohn

435

47

Innate Immunity to Mouse Cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Djurdjica Cekinovic´, Irena Slavuljica, Tihana Lenac, Astrid Krmpotic´, Bojan Polic´, and Stipan Jonjic´

445

Section 4

Vaccines

48

Research and Development of Chimeric Flavivirus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simon Delagrave and Farshad Guirakhoo

459

49

Correlates of Immunity Elicited by Live Yersinia pestis Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vivian L. Braciale, Michael Nash, Namita Sinha, Irina V. Zudina, and Vladimir L. Motin

473

PART V

BUILDING A SUSTAINABLE PERSONAL RESEARCH PORTFOLIO

50

Strategies for a Competitive Research Career . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hortencia Hornbeak and Peter R. Jackson

483

51

Selecting the Appropriate Funding Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priti Mehrotra, Hortencia Hornbeak, Peter R. Jackson, and Eugene Baizman

487

52

Preparing and Submitting a Competitive Grant Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peter R. Jackson and Hortencia Hornbeak

497

53

Identifying Research Resources and Funding Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eugene Baizman, Hortencia Hornbeak, Peter R. Jackson, and Priti Mehrotra

507

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

519

Contributors

Vyacheslav M. Abramov • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia J. H. Anolik • Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA A. G. Artemenko • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Rusudan Aspindzelashvili • National Center for Tuberculosis and Lung Diseases / National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Ramy Aziz • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Eugene Baizman • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Lela Bakanidze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Jacqueline T. Balthazar • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Jacques Banchereau • Baylor Institute for Immunology Research, Dallas, TX, USA Bryan H. Bellaire • Louisiana State University Health Science Center, Shreveport, LA, USA Edward A. Berger • Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Gayle Bernabe • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Roberta Black • Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Maya Boyanova • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Vivian L. Braciale • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Robert R. Brubaker • Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA Mark Buller • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Robert J. Burgess • Armed Forces Institute of Pathology, Washington, DC, USA

David N. Burns • Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Saulius Caplinskas • Lithuanian AIDS Center, Mykolas Romeris University, Vilnius, Lithuania Ilaria Capua • OIE/FAO Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy Emanuela Castigli • Division of Immunology, Children’s Hospital, Boston, MA, USA M. Donald Cave • Neurobiology and Developmental Science, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Djurdjica Cekinovic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Alexander V. Chestkov • State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia Evelyne Couture-tosi • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Wendy F. Davidson • Marlene and Stewart Greenebaum Cancer Center and Department of Microbiology and Immunology, and the Center for Vascular and Inflammatory Diseases, BioPark Building 1, University of Maryland, Baltimore, MD, USA Simon Delagrave • Acambis Inc., Cambridge, MA, USA Tiziana Di Pucchio • Baylor Institute for Immunology Research, Dallas, TX, USA Dimiter S. Dimitrov • Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA Michael Dobson • Armed Forces Institute of Pathology, Washington, DC, USA T. Mark Doherty • Statens Serum Institut, Department of Infectious Disease Immunology, Copenhagen, Denmark Michal Dranitzki-Elhalel • Hadassah Medical Center, Ein Kerem, Israel Karl Drlica • Public Health Research Institute, Newark, NJ, USA Kathleen D. Eisenach • Departments of Pathology, Microbiology and Immunology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA Valentin I. Evstigneev • Department of Biochemistry, Immunity, and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Nourtan Fathey • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

xv

xvi Valentina A. Feodorova • Scientific and Research Department, Saratov State University, Saratov Russia, Russia William A Fischer, II • Johns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland; and University of Virginia, Charlottesville, VA, USA Jason P. Folster • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Angel S. Galabov • Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Shalva Gamtsemlidze • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Raif S. Geha • Division of Immunology, Children’s Hospital, Boston, MA, USA Vassil St. Georgiev • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Maria Y. Giovanni • Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Magnús Gottfredsson • Department of Medicine, Landspitali University Hospital and University of Iceland School of Medicine, Reykjavik, Iceland Ian Justin Glomski • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Pierre Louis Goossens • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Howard Grey • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Farshad Guirakhoo • Senior Director, External Research & Development, Global Research and R&D, Sanofi Pasteur Acambis Inc., Cambridge, MA, USA Ted Hadfield • Armed Forces Institute of Pathology, Washington, DC, USA Lauren Handley • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Carson Harrod • Baylor Institute for Immunology Research, Dallas, TX, USA Graham F. Hatfull • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA Frederick Hayden • Johns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland and University of Virginia, Charlottesville, VA, USA Katherine T. Herz • Office of Global Research, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Huynh Hoa-bui • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Hortencia Hornbeak • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA A. I. Hromov • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Jui-han Huang • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Lynn Y. Huynh • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Contributors Paata Imnadze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Ivan G. Ivanov • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Peter R. Jackson • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Marina Janjgava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Ann E. Jerse • Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Paul J. T. Johnson • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Stipan Jonjic • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Nazia Kamal • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Rita Kansal • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN Arseny Kaprelyants • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Gennagiy I. Karataev • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Nicolay N. Karkischenko • Scientific Center of Biomedical Technologies RAMS, Russia Paul Keim • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Valentin S. Khlebnikov • Department of Biochemistry, Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Eynav Klechevsky • Baylor Institute for Immunology Research, Dallas, TX, USA, Technion–Israel Institute of Technology, Technion City, Haifa, Israel Igor V. Kosarev • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Malak Kotb • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Astrid Krmpotic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia M. Kucia • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA Nataly L. Kulikova • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia V. E. Kuz’min • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Karen Lacourciere • Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Tihana Lenac • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia A. V. Liahovskij • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Contributors R. John Looney • Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA V. P. Lozitsky • Ukrainian I.I. Mechnikov Research Anti-Plague Institute, Odessa, Ukraine Stefano Marangon • OIE/FAO Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy Alemka Markotic´ • University Hospital of Infectious Diseases, Zagreb, Croatia John J. McGowan • National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Alisa Yu. Medkova • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Priti Mehrotra • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Roumyana Mironova • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Veriko Mirtskhulava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia and Emory University, Atlanta, GA, USA Michèle Mock • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Herbert C. Morse, III • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA Vladimir L. Motin • Departments of Pathology/Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Magdalini Moutaftsi • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Galina Mukamolova • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Partha Mukhopadhyay • Marlene and Stewart Greenebaum Cancer Center and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA E. N. Muratov • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Ucha Nanava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Michael Nash • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Toshimitsu Niwa • Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Nagoya, Japan Lubomira Nikolaeva-glomb • Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Mohamed Nooh • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Zohreh Naghashfar • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA L. N. Ognichenko • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

xvii Carla Oseroff • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Nikolay S. Osin • Department of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia A. Karolina Palucka • Baylor Institute for Immunology Research, Dallas, TX, USA Scott Parker • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Virginia Pascual • Baylor Institute for Immunology Research, Dallas, TX, USA Valerie Pasquetto • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Milloni Patel • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Bjoern Peters • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Stefan Philipov • Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria Guido Poli • AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy Bojan Polic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia P. G. Polischuk • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Vera G. Pomelova • Laboratory of Molecular Diagnostics, Department of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia Serguei G. Popov • National Center for Biodefense and Infectious Disease, George Mason University, Manassas, VA, USA Tanja Popovic • United States Centers for Disease Control and Prevention, Atlanta, GA, USA William S. Probert • California State Department of Health Services, Microbial Diseases Laboratory, CA, USA J. Ratajczak • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA M. Z. Ratajczak • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA Sarah Rowe • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Archil Salakaia • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Elena Salina • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Iñaki Sanz • Department of Medicine, Division of Clinical Immunology and Rheumatology. University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Susan Schader • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada Connie Schmaljohn • U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA Gretja Schnell • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

xviii Evgeniy G. Semin • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Alessandro Sette • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA John Sidney • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Lone Simonsen • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Namita Sinha • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Ludmila N. Sinyashina • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Irena Slavuljica • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia George B. Smirnov • The Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia William M. Shafer • Department of Microbiology and Immunology and Laboratories of Microbial Pathogenesis, Emory University School of Medicine, Atlanta, GA, USA H. L. Stevenson • Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA Ronald Swanstrom • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA David E. Swayne • Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA, USA Patricia Sylvestre • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Robert J. Taylor • Office of the Director, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Sue A. Theus • Department of Pathology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Shota Tsanava • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Nikoloz Tsertsvadze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Tengiz Tsertsvadze • Infectious Diseases, AIDS, and Clinical Immunology Research Center, Tbilisi, Republic of Georgia Yuriy D. Tsygankov • State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia Mark Tykocinski • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

Contributors Hideki Ueno • Baylor Institute for Immunology Research, Dallas, TX, USA Onega V. Ulianova • Scientific and Research Department, Saratov State University, Saratov, Russia Vladimir N. Uversky • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Matthew N. Van Ert • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Anatoly M. Vasiliev • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Raisa N. Vasilenko • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia David M. Wagner • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Mark A. Wainberg • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada Matthew C. Weber • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA D. H. Walker • Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA Douglas E. Warner • Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, USA Karl A. Western • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Robbin S. Weyant • United States Centers for Disease Control and Prevention, Atlanta, GA, USA Mark S. Williams • Department of Microbiology and Immunology, University of Maryland School of Medicine, and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA Robert W. Williams • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis TN, USA M. Wysoczynski • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA M. Shaylan Zanecki • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Xilin Zhao • Public Health Research Institute, Newark, NJ, USA Jeff X. Zhou • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA Moncef Zouali • Inserm, Paris, University of Paris, France Irina V. Zudina • Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA

National Institute of Allergy and Infectious Diseases (NIAID): An Overview Karl A. Western

The National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH) is within the U.S. Department of Health and Human Services (DHHS; Figure 1). The NIH is the DHHS agency responsible for biomedical research and research training. In the U.S. federal system, health is considered primarily a local and state responsibility, with the federal government providing support and assistance as required. Biomedical research, however, is viewed as a federal responsibility. For that reason, the NIH size and budget have resulted in its becoming the largest of the DHHS agencies. The NIH consists of 27 institutes and centers, 24 of which carry out and fund biomedical research and three that support the NIH biomedical research endeavor (Figure 2). Each institute consists of two major components: the extramural and the intramural. Intramural programs consist of NIH scientists working in NIH government laboratories. Intramural research constitutes of about 10 to 20% of each institute’s research effort and budget. Intramural researchers select scientists to come to their laboratories for research training and conduct international research using the funding available to their laboratory. The extramural program of each institute is approximately 80 to 90% of its total funding and operates through both unsolicited and solicited research applications for grants, collaborative agreements, and contracts. Applications are submitted to the NIH Center for Scientific Review, which assigns each application to the appropriate initial review group for scientific peer review and to an institute according to the scientific content of the application and the research mission of the institute. NIH is unique among national biomedical research agencies in that nearly one-half of the intramural scientists are not U.S. citizens and that foreign scientists are eligible to apply directly or as a partner in extramural awards. From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

NIAID is similar in its organization to other NIH institutes in that it has three intramural divisions and five extramural divisions (Figure 3). The Division of Intramural Research heavily emphasizes basic biomedical research, while the Vaccine Research Center’s mission includes the discovery and early development of vaccine products. The Division of Clinical Research was established in 2006 to set up domestic and international sites to carry out human subject studies on new or improved diagnostic tests, drugs, vaccines and other prevention products. The Division of Microbiology and Infectious Diseases is responsible for all infectious and parasitic diseases except for the human acquired immunodeficiency syndrome (AIDS). The Division of AIDS is responsible for AIDS and related conditions. The Division of Allergy, Immunology, and Transplantation is concerned with the human immune system. The Division of Extramural Activities provides support to the other three extramural divisions through NIAID-organized initial review groups, grant and contract management, and award databases. The NIAID mission is to understand, treat, and ultimately prevent infectious, immunological, and allergic diseases that affect or threaten U.S. populations and hundreds of millions of people worldwide. The major areas of NIAID investigation currently are (in alphabetical order): AIDS; acute respiratory infections, including influenza; antimicrobial drug resistance, asthma and allergic diseases; civilian biodefense; emerging infectious diseases; enteric infections; genetics, transplantation, and immune tolerance; immune disorders; malaria and other tropical diseases; sexually transmitted diseases; tuberculosis, and vaccine development and evaluation. The evolution of the NIAID budget is summarized in Figure 4. Prior to the recognition of AIDS, NIAID was the seventh largest NIH Institute. As a result of its research responsibilities in infectious diseases and immunology, funding for AIDS and AIDS-related research rose to become one-half of the NIAID budget. Subsequent to the anthrax attacks in 2001, NIAID was given lead responsibility for the U.S. Civilian Biodefense Research Initiative. At the present time, NIAID is the second 3

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U.S. Department of Health and Human Services The Secretary Deputy Secretary

Administration for Children and Families (ACF)

Administration on Aging (AoA)

Food and Drug Administration (FDA)

Centers for Medicare and Medicaid Services (CMS)

Agency for Health Care Policy and Research (AHCPR)

Indian Health Services (IHS)

Centers for Disease Control and Prevention (CDC)

Agency for Toxic Substances and Disease Registry (ATSDR)

Substance Abuse and Mental Health Services Administration (SAMHSA)

Health Resources and Services Administration (HRSA)

National Institutes of Health (NIH)

Program Support Center (PSC)

Figure 1. (See Color Plates).

largest institute after the National Cancer Institute. NIAID research funding is approximately one-third AIDS, one-third civilian biodefense, and one-third non-AIDS/non-biodefense. Following a Congressional mandate to double the NIH budget in the 1990s, the NIH budget has been flat for the past several years, resulting in overall inflation-adjusted negative growth. During this period, NIAID funding for international research has maintained a slow and steady growth (Figure 5) so that international research now accounts for 10% of the total NIAID budget. This remarkable sustainability is due to the globalization of health problems, the relevance of health conditions globally to domestic U.S. health problems, humanitarian objectives, and the economic development, political stability, and increasing investment in international health on the part of key international partners such as Brazil, China, and India. This sustained interest and growth in international research is not seen across NIH. One major factor that fuels NIAID’s global research activities is that our mission in infectious diseases necessitates that we partner with countries that have heavier burdens of disease and/or different risk factors in the development of clinical sites and the evaluation of new or improved diagnostic tests, treatment modalities, or prevention products.

NIAID operates under five guiding principles in Global Health Research. First, every effort is made to target collaborative research efforts to the needs of the partner country or region. Second, it strives to develop collaborative relationships that begin with collaboration in basic research and discovery so that intellectual property can be shared and proceed through product development, the design of human subject studies, and the conduct of rigorous clinical trials that generate data resulting in approval of the product by regulatory agencies. Third, to achieve multidisciplinary research collaboration, research capacity must be built and sustained in the host country. Fourth, NIAID strives to stimulate scientific collaboration and global multi-sector partnerships. Finally, NIAID international collaboration must develop training, communication, and outreach programs. NIAID uses six approaches to support its international research. The first is through the NIAID intramural research divisions for pre- and postdoctoral research training. This research training frequently results in sustained collaboration once the visiting scientists have returned to their home countries. Intramural collaboration is limited by the resources available in each laboratory but has the advantages of being

NIAID: An Overview

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National Institutes of Health Office of the Director

National Institute on Aging

National Institute on Alcohol Abuse and Alcoholism

National Institute of Allergy and Infectious Diseases

National Institute of Arthritis and Musculoskeletal and Skin Diseases

National Cancer Institute

National Institute of Child Health and Human Development

National Institute on Deafness and Other Communication Disorders

National Institute of Dental and Craniofacial Research

National Institute of Diabetes and Digestive and Kidney Diseases

National Institute on Drug Abuse

National Institute of Environmental Health Sciences

National Eye Institute

National Institute of General Medical Sciences

National Heart, Lung, and Blood Institute

National Human Genome Research Institute

National Institute of Mental Health

National Institute of Neurological Disorders and Stroke

National Institute of Nursing Research

National Institute of Biomedical Imaging and Bioengineering

National Center for Complementary and Alternative Medicine

Fogarty International Center

National Center for Research Resources

National Library of Medicine

National Center on Minority Health and Health Disparities

Clinical Center

Center for Information Technology

Center for Scientific Review

Figure 2. (See Color Plates).

decentralized and scientifically driven, and it provides the opportunity to establish long-term collaboration with the NIAID laboratory and other researchers who have trained there. Because about 50% of NIH intramural scientists are from outside the United States and only 10% of intramural scientists become tenured, the intramural research training experience provides an opportunity to become part of a global network linking trainees and their home institutions with NIAID-tenured scientists, U.S. scientists who take academic or private sector appointments or join other U.S. agencies, and foreign scientists who return home to continue their research careers. Foreign investigators are encouraged to partner with U.S. extramural investigators in the submission of investigatorinitiated research applications or in response to solicited program announcements (PAs) and requests for applications (RFAs). This is how NIAID supports the bulk of its international research. If the collaboration is between U.S. scientists

and scientists in another industrialized country, there may be no NIAID funding involved. On the other hand, if the collaborating overseas scientist is from a middle- or lower-income country and/or does not have his or her own funding, NIAID will provide the U.S. investigator with research funds to support the overseas component. NIH is unique among national domestic research agencies in that foreign investigators are eligible to apply directly for investigator-initiated research awards. Foreign scientists and institutions are also eligible to apply for most solicited grant and collaborative agreement solicitations. There are no international set-aside funds, and foreign investigators must compete against experienced U.S. investigators. All unsolicited foreign applications with a competitive score must also be approved by the National Allergic and Infectious Diseases Council before funding. Because of the intense competition and grantsmanship required, NIAID does not encourage foreign investigators to apply directly unless their ideas are

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K. A. Western

NIAID Divisions and Offices (simplified) Board of Scientific Counselors Associate Director for Management & Operations

Office of Financial Management

Office of Management for New Initiatives

Office of Human Resources Management

Office of Administrative Services

Office of Technology Information Systems

Office of Policy Analysis

Office of the Director

National Advisory Allergy & Infectious Disease Counselors

Office of Biodefense Research

Office of Global Research

Office of Clinical Research

Office of Ethics Office of Technology Development

Division of Acquired Immunodeficiency Syndrome

Office of Communications & Public Relations

Division of Allergy, Immunology & Transplantation

Division of Microbiology & Infectious Diseases

Office of Equal Employment Opportunities

Division of Extramural Activities

Dale & Betty Bumpers Vaccine Research Center

Division of Intramural Research

Figure 3. (See Color Plates).

truly novel and the investigator has considerable experience preparing NIAID grant applications. NIH is obligated to follow U.S. contracting laws, so that foreign institutions can be funded in response to requests for proposals only if there is a prior determination that there is no viable U.S. source, or the foreign application is clearly superior to responses from U.S. institutions. NIAID also participates in a number of bilateral programs with foreign governments and institutions. These agreements may be developed at the Presidential, State Department, DHHS, or NIH levels in science and technology, health, or biomedical research. In the majority of cases, these agreements have no NIAID funding associated with them and collaborative activities must be undertaken with resources currently at hand in intramural laboratories or using extramural funding mechanisms. NIH intramural scientists are encouraged to collaborate with counterparts at other U.S. government agencies such as the Centers for Disease Control and Prevention, the Food and

Drug Administration, and the U.S. Army or Navy. U.S. Government scientists, however, may not compete for NIH extramural research funds. When there is mutual interest, however, NIH may negotiate interagency agreements with these and other agencies such as the State Department or the U.S. Agency for International Development that serve as a contractual mechanism to transfer funds and resources between the participating agencies. Finally, NIAID collaborates with multilateral agencies such as the World Health Organization (WHO), the Pan American Health Organization, and the Joint United Nations Program on HIV/AIDS through consultation, serving on advisory boards, and participation in technical meetings. NIAID has provided targeted funding to the WHO/World Bank/UNDP Special Program for Research and Training in Tropical Disease Research. NIAID also has a Congressional mandate to provide funding to the Global Fund to Combat AIDS, Tuberculosis, and Malaria.

NIAID: An Overview

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Figure 5. (See Color Plates). Figure 4. (See Color Plates).

Figure 6. (See Color Plates).

8

The NIAID strategy to respond globally to new or emerging infectious diseases and scientific opportunity has been first to encourage the intramural research community to turn their talent and attention to the new or underserved research area. The second step is to encourage extramural investigators working in relevant research areas to submit supplemental research proposals. The third step is to alert the more general scientific community about NIAID’s research priorities and interests in the area through notices, PAs, and RFAs in the NIH Guide for Grants and Contracts. Foreign investigators are ordinarily eligible to partner with U.S. applicants and, if they prefer, apply directly for NIAID funding. The result of these solicitations is to increase the research, the research training base, and eventually the pool of investigators in the targeted area. NIAID fulfills the need for directed activities in support of research in the targeted area through contracts to build the infrastructure and to provide research reagents and repositories. After a critical mass of individual extramural awards has been reached, NIAID usually puts out an RFA to establish multidisciplinary centers of excellence in the field. These centers of excellence provide further opportunities for research training of U.S. and foreign scientists. The centers of excellence are usually encouraged to engage in international research and/or carry out research training through the center award and/or independent research and research training awards. Examples of NIAID centers of excellence programs include the Sexually Transmitted Disease Research Centers, the Tropical Disease Research Units, the Centers for AIDS Research, the Tuberculosis Research Unit, the

K. A. Western

Regional Centers for Emerging Infectious Diseases, and the recently announced Centers for Influenza Research and Surveillance. Once the domestic centers of excellence are established, the next phase is the establishment of special programs to link the domestic network to international partners. RFAs are published to solicit applications for collaboration with one or more foreign partners. This is the time when the NIH Fogarty International Center solicits applications from U.S. institutions for international research training in the targeted area. Examples of linkage programs include the International Collaboration in Infectious Disease Research Program, the HIV Vaccine Trials Network, HIV Prevention Trials Network, the NIAID International Centers of Excellence, and the International Emerging Infectious Disease Research and Training Program. The third phase is reached when the linkage programs are mature and international partners have developed the capacity to carry out and account for their own research. NIAID develops solicitations open to foreign institutions to apply directly to NIAID in the targeted area. Examples of mechanisms to support foreign researchers include the Tropical Medicine Research Centers, the Multilateral Initiative on Malaria, the Comprehensive International Program for Research on AIDS, and the International Research in Infectious Diseases Program. Further information on NIAID and NIH international grants and funding opportunities may be found at http://grants1.nih. gov/grants; http://www.niaid.nih.gov/ncn/; and http://www. niaid.nih.gov/ncn/grants/int/default.htm.

Chapter 20 A New Highly Potent Antienteroviral Compound Lubomira Nikolaeva-Glomb, Stefan Philipov, and Angel S. Galabov

20.1

Introduction

The enteroviruses are widely spread viruses associated with diverse clinical syndromes and diseases, ranging in severity from minor febrile disorders to severe and potentially lifethreatening conditions. They may affect various organs and systems: the central nervous system, the respiratory system, the skin, the heart, the pancreas, and the eye. The enteroviruses are the most common etiological agent of viral meningitis. They may also cause encephalitis. In addition, these viruses can cause summer colds, herpangina, pleurodynia, hemorrhagic conjunctivitis, uveitis, and chronic fatigue syndrome. They are implicated in cardiac infections such as myocarditis and pericarditis that both in some cases may lead to dilated cardiomyopathy, where the singular option of recovery is the heart transplantation. Enteroviruses have also been implicated to play a role in the development of juvenile-onset (type 1) insulin-dependent diabetes mellitus (1). Antienteroviral therapy until now has had certain limitations. To date, there is no enterovirus-specific drug available for clinical use. Indeed, a great number of enterovirus inhibitors have been described so far, but only a few of them have shown effectiveness in vivo and none has been approved for clinical use yet. Thus, etiological therapy remains elusive, and there is a clear need for continued development of new and effective inhibitors of enteroviral replication.

20.2

Oxoglaucine

In a pilot study performed by our research group, a series of aporphinoid alkaloids were isolated from Glaucium flavum Crantz (yellow horn poppy) or obtained synthetically have been tested in vitro for their antiviral activity against viruses From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

belonging to several taxonomic groups, including picorna-, orthomyxo-, paramyxo-, and herpesviruses. One of the compounds, oxoglaucine, has manifested a well-pronounced inhibitory effect against the replication of poliovirus type 1 (Mahoney), of the Picornaviridae family. The antiviral effects in the preliminary screening tests have been evaluated by the semi-quantitative agar-diffusion test (2). Oxoglaucine is isolated from the epigean parts of Glaucium flavum Crantz (3) and it can also be obtained synthetically from the main plant alkaloid glaucine (4). The cytotoxic effect of oxoglaucine has been tested in two experimental procedures. The first was a microscopic evaluation of the effect of different concentrations of oxoglaucine on the morphology of the cell monolayer resulting in determination of the maximal tolerated (nontoxic) concentration (MTC). The second involved tracing the growth curve of the cell culture in the presence of different concentrations of the compound followed by determination of the concentration that reduces the number of viable cells by 50%, the cell growth inhibitory concentration 50 (CGIC50). The concentration of oxoglaucine, which produces no visible cytotoxic effect on monolayer FL cells, the MTC, is 6.4 µg/mL, and the concentration reducing their growth by 50% (CGIC50) is 4 µg/mL (Figure 20.1). The antienteroviral spectrum of oxoglaucine has been tested against 16 enteroviruses—poliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and a series of viruses belonging to human enterovirus B. The following enteroviruses have been included in the test: poliovirus type 1 (PV-1), coxsackievirus (CV)-A9, the six coxsackie B viruses (CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, and CV-B6), and echovirus (EV)-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19. The endpoint dilution method in the multi-cycle cytopathic effect (CPE)inhibition setup in FL cells and the plaque-inhibition test has been used for determining the antiviral effect. Oxoglaucine reveals a marked inhibitory effect on all of the tested viruses. Oxoglaucine concentrations that reduce virus titer by 1, 1.67, and 2 lg as compared to that of virus control 199

number of viable cells as % of the untreated control

200

L. Nikolaeva-Glomb et al.

100

50

0 0.1

1

10

100

concentration (µg / ml)

Figure 20.1. Effect of oxoglaucine on the growth of Fl cells.

Cells are seeded in a growth medium containing various concentrations of the compound. After formation of cell monolayer in the untreated control (no compound in the growth medium), cells are trypsinized and viable cells counted. The number of viable cells in each sample is compared with the number of viable cells in the control and is presented as percent of the untreated control.

of oxoglaucine. On the opposite end, the highest dose of oxoglaucine was required to inhibit the replication of CV-B1. The antiviral effects of oxoglaucine against the replication of CVs and the EVs included in the investigation are presented on Figure 20.2 and Figure 20.3, respectively. On the basis of the results presented in Table 20.1 and the cytotoxicity parameters of oxoglaucine, the selectivity index (SI) has been calculated as the ratio of MTC and the inhibitory concentration 50 (IC50). The results are shown on Table 20.2. As expected, oxoglaucine exerts the highest selectivity against CV-B4, followed by EV-9, EV-13, and EV-19 as well as by CV-B3 and CV-A9. In general, oxoglaucine reveals a broad-spectrum antienteroviral activity accompanied by high selectivity. This conclusion is supported by the results obtained in the plaque-inhibition test, which is considered the “gold standard” in experimental in vitro antienteroviral chemotherapy. The plaque-inhibition test has been carried out for poliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and the six coxsackie B viruses. The concentration that reduces the number of plaques by 50% relative to the control with no inhibitor present in the agar overlay (IC50) was determined and SI calculated. Results are presented in Table 20.3. From the tested viruses, CV-B4 is again the most sensitive one to the antiviral effect of oxoglaucine demonstrating SI approximating 400.

Table 20.1. Antienteroviral effect of oxoglaucine determined in the CPE-inhibition test.

6

IC (µg/mL) ∆ lg = 1

∆ lg = 1.67

∆ lg = 2

PV-1 (LSc-2ab) PV-1 (Mahoney) EV-2 EV-4 EV-6 EV-9 EV-13 EV-15 EV-19 CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6 CV-A9

0.0007 n.d.

0.018 n.d. 0.01 0.12 0.003 0.06 0.09 0.2 0.12 0.25 n.d. 0.07 0.03 0.04 n.d. 0.12

0.33 n.d. 0.2 0.17 0.06 0.08 0.1 0.25 0.15 0.30 n.d. 0.1 0.04 0.06 n.d. 0.15

0.03 0.04 0.03 0.14 0.03 0.17 n.d. 0.04 0.01 n.d. 0.04

n.d., not done The antiviral effect is determined in the endpoint dilution method according to the CPE-inhibition procedure and the antiviral effect is presented as the difference of titers (∆ lg) of the untreated virus control and the oxoglaucine-treated samples

(with no oxoglaucine in the maintenance medium) have been determined in the CPE-inhibition test and results are shown on Table 20.1. CV-B4 and CV-B5, followed by CV-B3, CVA9, and EV-9 were the most susceptible to the antiviral effect

4

∆ lg

Virus type

5

CAV-9 CBV-1 CBV-3 CBV-4 CBV-5

3

2

1

0

0.001

0.01

0.1

1

concentration(µg/ml)

Figure 20.2. Antiviral effect of oxoglaucine against the replication of CV-A9, CV-B1, CV-B3, CV-B4, and CV-B5 in FL cells. Monolayer FL cells in 96-well plates are inoculated with 0.1 mL virus suspension containing 100 000, 10 000, 1000, 100, 10, and 1 CCID50 (or 320 000, 32 000, 3 200, 320, 32, and 3 CCID50). After an hour for virus adsorption, the excessive virus is discarded and cells are inoculated with 0.2 mL of maintenance medium containing 0.5 lg concentrations of the tested compound. The antiviral effect is scored according to the appearance of the cytopathic effect on the 48th hour p.i., virus titer in the presence or absence of the compound is determined and the defference of titers (∆ lg) of the untreated virus control and the oxoglaucine-treated samples is calculated.

20. Antienteroviral Compound

201 Table 20.3. Antienteroviral effect of oxoglaucine determined in the plaque-inhibition test.

9

EV-2 8

EV-4 EV-6

7

EV-9 EV-13

6

EV-15 EV-19

Virus type

IC50 (µg/mL)

SI (MTC/ IC50)

0.15 0.041 0.03 0.017 0.038 0.017 0.02 0.042

42 156 213 376 168 376 320 152

PV-1 (LSc-2ab) PV-1 (Mahoney) CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6

∆ lg

5 4 3 2

Table 20.4. Direct virucidal effect of oxoglaucine. 1

Virus titer lg CCID50 /mL 0.01

0.1

1

T°C 4°C

concentration (µg / ml)

Figure 20.3. Antiviral effect of oxoglaucine against the replication of EV-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19 in FL cells. Table 20.2. Selectivity of oxoglaucine determined according to the CPE-inhibition test. SI (MTC/IC) Virus type

∆ lg = 1

∆ lg = 1.67

∆ lg = 2

PV-1 (LSc-2ab) PV-1 (Mahoney) EV-2 EV-4 EV-6 EV-9 EV-13 EV-15 EV-19 CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6 CV-A9

9 142 n.a. — 213 — 160 213 45 213 38 n.a. 160 640 — n.a. 160

355 n.a. 640 53 2 133 107 71 32 53 26 n.a. 91 213 160 n.a. 53

19 n.a. 32 38 107 80 64 26 43 21 n.a. 64 160 107 n.a. 43

n.a., not applicable

Room temperature 37°C

Oxoglaucine control Oxoglaucine control Oxoglaucine control

0 min 15 min 30 min 60 min

6h

24 h

— — — 8.0 — —

8.0 8.5 8.5 8.0 8.0 8.0

8.5 8.5 8.5 8.0 8.5 8.5

7.5 8.0 7.5 8.5 8.0 7.5

7.5 8.0 7.5 8.0 8.5 8.5

8.0 8.0 8.0 7.5 8.5 8.0

Experiments have been carried out on undiluted poliovirus type 1 (LSc-2ab; 108.25 CCID50/mL) by the virucidal quantitative suspension test in the presence of 5 µg/mL and virus samples have been titrated by the endpoint dilution method in FL cells

9

VC 0h

8

infectious virus titer (CCID50/ml)

0 0.001

1h 2h

7

3h 4h

6

5h 5

6h

4

3

2 0

1

2

3

4

5

6

7

8

hours post infection

Research on the mode of action of oxoglaucine is in progress. In a preliminary stage of the research, the direct virucidal effect of the compound has been tested in a quantitative suspension test at three temperature regimens (4°C, room temperature, and 37°C). The results obtained indicate undoubtedly that oxoglaucine possesses a virus-specific mode of antiviral action and its effect is not due to the direct inactivation of extracellular virions (Table 20.4).

Figure 20.4. Timing-of-addition study on the mode of antiviral action of oxoglaucine. Monolayer FL cells in test-tubes are inoculated with 0.1 mL of poliovirus type 1 (LSc-2ab) at a multiplicity of infection 50 and cells are incubated at 37°C. Oxoglaucine in a concentration of 1 µg/mL has been added in the maintenance medium on hours 0, 1, 2, 3, 4, 5, and 6 p.i. Virus samples are frozen and thawed on hour 3, 4, 6 and 8 p.i. followed by titration by the endpoint dilution method.

202

Initial studies on the specific mode of action of oxoglaucine have been carried out in the timing-of-addition study in the one-step replication cycle of poliovirus type 1 (LSc-2ab) at high multiplicity of infection. The study reveals that the susceptible period to the antiviral effect of oxoglaucine is the latent and lag phase of the virus replication cycle (Fig 20.4). The following conclusions may be drawn out from the results obtained so far: (i) oxogalaucine possesses a strong antiviral effect in vitro against a broad spectrum of eneteroviruses, (ii) a high selectivity ratio is observed for all tested viruses exceeding 100 in most cases, and (iii) the latent and the lag phase of the virus replication cycle is the susceptible period to the effect of oxoglaucine.

L. Nikolaeva-Glomb et al.

References 1. Galabov AS, Angelova A (2006) Antiviral agents in the prevention and treatment of virus-induced diabetes. Anti-Infective Agents in Medicinal Chemistry 5:293-307. 2. Galabov AS, Nikolaeva L, Philipov S (1995) Aporphinoid alkaloid glaucinone: a selective inhibitor of poliovirus replication. Antivir Res 26:A347. 3. Kuzmanov BA, Philipov SA, Deligiozova-Gegova IB (1992) Comparative photochemical and chemosystematic research of populations of Glaucinum flavum Crantz in Bulgaria. Fitologia 52-57. 4. Philipov S, Ivanovska N, Nikolova P (1998) Glaucine analogues as inhibitors of mouse splenocyte activity. Die Pharmazie 53:694-698.

Chapter 19 Anti-Infectious Actions of Proteolysis Inhibitor e-Aminocaproic Acid (e-ACA) V. P. Lozitsky

19.1

Introduction

For various proteins, proteolytic cleavage represents the universal mechanism of activation. The activation of proteolysis plays an important role in the pathogenesis of many diseases. So, our supposition about antiviral activity of the proteolytic inhibitors (1) has been well founded. Previous research data has made it possible to formulate the “vicious circle” concept of viral virulence. That is: the virus activates the proteolytic systems, which in turn assists in the development, generalization, and aggravation of the infectious process at the expense of influence on the etiologic and pathogenesis factors (see Scheme 19.1; refs. 1 and 2). The inhibitors of proteolysis may prevent the forming of or destroy this “vicious circle.” The antiviral action of proteolytic inhibitors was discovered on all levels from subcellular to whole organism in both the experiment stage and the clinic. Furthermore, the antiviral therapeutic and prophylactic action of the proteolytic inhibitors was demonstrated against a wide spectrum of RNA and DNA viruses, such as the influenza A and B, herpes, HIV, Newcastle disease viruses (NDVs), and the adenoviruses (3). In this study, we present results on anti-infectious action of the proteolytic inhibitor Σ-aminocaproic acid (Σ-ACA).

19.2

Materials and Methods

1. Σ-ACA manufactured by pharmaceutical company “Zdorov’ya” (Kharkiv, Ukraine) was used. 2. Patients: sick children with influenza and other ARVI, adult patients with genital herpetic infection. 3. Laboratory animals: inbred white mice, white rats.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

4. Viruses: human influenza A viruses H1N1, H2N2, H3N2; avian influenza A viruses H5N3 and H7N3; influenza B virus; NDV; adenovirus; HSV-1; and HIV. 5. Tissue and cell cultures: tissue cultures of chorioallantoic membranes (CCM) of 12- to 14-day-old chicken embryos, cell culture Hep-2. 6. Bacterial agents of emerging and nosocomial infections: vaccine strain 15 and virulent strain 29 of Francisella tularensis; strains of Vibrio cholerae: Vibrio cholerae cholerae strain 569; Vibrio cholerae El-Tor strains 754 and 878; Vibrio cholerae non-01 strain 146/11; hospital isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. 7. Anti-influenza and anti-NDV activities in vitro were studied by inhibition of virus replication in tissue cultures CCM. CCM was infected with 1000 TID50 (tissue infection dose) of virus. Samples contained Σ-ACA were called “experimental” and those without inhibitor were named “control.” Control and experimental samples were tested on viral infective titers after incubation (24 hours, 37°C for influenza A viruses and NDV, and 32°C for influenza B viruses). At least five experiments were carried out of the investigation of each compound. Anti-influenza activity is expressed in log10 TID50 and reflected suppression of viral replication in “experimental” samples to “control.” 8. Anti-influenza activity of Σ-ACA in vivo was studied in mouse models of models lethal and non-lethal experimental influenza infections (2, 4). 9. Antiherpetic action of Σ-ACA was tested using cyto-morphological method. Hep-2 cells were infected with HSV-1 strain US in dose 5 IFU/cell. The cells were incubated at 37°C during 48 hours in maintenance medium that contained Σ-ACA (experimental samples) or without its (control samples). Then, cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. The amount of infected cells with DNAcontaining virus inclusion bodies was counted by fluorescent microscopy. Anti-HSV activity of compounds was estimated as the ratio of the percentage of infected cells in treated to percentage of infected cells in untreated cell cultures. 193

194

V. P. Lozitsky

Scheme 19.1. The participation of proteolytic system during development of influenza infection and etiopathogenetic action of protease inhibitors (x-show the points of inhibitors action).

19.3

Results and Discussion

Usually Σ-ACA is used for hemostasis when fibrinolysis contributes to bleeding. Σ-ACA is a low toxic drug. The intravenous and oral LD50 values of Σ-ACA were 3.0 and 12.0 g/kg, respectively, in the mouse and 3.2 and 16.4 g/kg, respectively, in the rat. An intravenous infusion dose of 2.3 g/kg was lethal in the dog. Σ-ACA prevented the enhancement of proteolysis during the interaction of virions with cell membranes and decreased the penetration of virions into sensitive cells. It brought down the proteolytic cleavage of the HA precursor to HA-1 and HA-2, and reduced the infectious virus harvest. High levels of Σ-ACA anti-influenza efficacy were shown by us in vitro on the H1N1, H2N2, and H3N2 subtypes of influenza A human viruses, influenza B viruses (2, 4), and on H5N3 and H7N3 subtypes of avian influenza viruses (5). The obtained results showed that while both H5N3 and H7N3 avian influenza viruses are sensitive to Σ-ACA, the H5 subtype was more sensitive (Figure 19.1). Further results have shown that ε-ACA prevented enhancement of the alkaline proteases activity in lungs of mice infected with influenza virus in addition to exhibiting therapeutic and prophylactic effects (4, 6, 7). Σ-ACA intensified the production of specific antibodies, increased cell immunity, prevented vessels’ permeability and hemorrhagic phenomena, and decreased the destruction of bronchial epithelium. Mice treated by ε-ACA during infection were more protected from re-infection with influenza virus (7). ε-ACA, when used in the treatment of influenza, decreased the virus reproduction in lungs and also enhanced the humoral immune response (Figure 19.2). The antibody titers on day 21 postinfection were significantly higher in the treated animals.

On day 30 after challenge with the homologous strain A/ Hong Kong/1/68 (H3N2), the virus reproduced to low level in the lungs of untreated convalescent mice; however, no virus was detected in the lungs of mice (except one animal) that had been treated with ε-ACA during the primary infection (Figure 19.3A). A marked increase of the antibody level was found in such mice (Figure 19.3B). Upon challenge with lethal doses of the virulent strain A/Leningrad/49/32 (H1N1), the protection was significantly higher among animals treated with ε-ACA during the primary infection with sublethal virus dose. We believe that the immunomodulatory action of ε-ACA may play an important role in the increased resistance to challenge exhibited by treatment. The reproduction of influenza virus in the lungs was reduced in half 10 days after a single application of ε-ACA, and 4 weeks after 5-day prophylactic course (6). This correlated with the ability of the proteolysis inhibitor to stimulate the early production of specific serum antibodies. The favorable effect of prophylactic administration of ε-ACA was especially significant in experimental lethal influenza (Figure 19.4). A significant protection was observed from days 3 to day 14 post-infection. The prophylactic effects produced by different types of antiviral preparations, such as inactivated vaccine and ε-ACA, used separately or in combination in experimental lethal infection induced by influenza virus A/Leningrad/49/32 (H1N1) in mice were compared (8). The quantitative evaluation of the antiinfluenza effect was carried out by using the method of multifactor analysis after the optimum second-order plan based on the mathematical theory of experiment. This made it possible to determine the best combination of the preparations and their doses to establish the time of the formation of reliable protection from influenza in mice. The results of study on combined use of inactivated vaccine and ε-ACA condition for prevention of lethal experimental influenza in mice are exhibited on Figure 19.5.

19. Anti-Infectious Actions of Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA)

195

4,5 control

4

30 mg/ml E-ACA

3,5

20 mg/ml E-ACA 15 mg/ml E-ACA

- log10 TID50

3 2,5 2 1,5 1 0,5 0 1

2

Figure 19.1. The influence of E-ACA on avian influenza viruses H5N3 and H7N3 replication in tissue culture of chorio-allantoic membranes of chicken embryos. A

1,4

mice treated with E-ACA control group

1,2 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0

mice treated with E-ACA control group

-log10 TID50

-log10 TID50

A

0,8 0,6 0,4 0,2 0 31

1

2

3

4

5

33

35

37

40

44

Days of experiment

6 7 8 9 10 11 12 13 14 Days of experiment

B

1400 250

Titres of antibodies

B

Titres of antibodies

1

control group mice treated with E-ACA

200 150 100 50

1200

5

7

10

14

21

mice treated with E-ACA

800 600 400 200 0

0

control group

1000

30

31

33

35

37

40

44

Days of experiment

Days of experiment

Figure 19.2. Influence of therapeutic E-ACA usage on (A) titers of infections influenza A/Hong Kong/1/68(H3N2) in murine lungs and (B) titers of anti-hemagglutinins in their serum.

Figure 19.3. Influence of treatment with E-ACA of primary infection on (A) titers of infections influenza virus A/Hong Kong /1/68(H3N2) in murine lungs and (B) titers of anti-hemagglutinins in their serum after re-infection on day 30 of the experiment.

The Ukrainian Ministry of Public Health has allowed applying Σ-ACA as an antiviral agent for the treatment of influenza and other acute respiratory viral infections (ARVIs) in

children on the basis of our experimental results and clinical trials. Instructions for dosage and administration of ε-ACA for treatment of influenza and other ARVIs are as follows:

-Log10 LD50

196

V. P. Lozitsky

7

mice pretreated with E-ACA

6

control

5 4 3 2 1 0 21

30

42

Days of infection after beginning of E-ACA application course

Figure 19.4. Action of prophylactic usage of E-ACA on death of infected with influenza virus A/PR/8/34(H1N1) mice. 6 5

log10 LD50

4 3 2 1 0 1

2

3

4

1 - control; 2 - immunization with inactivated influenza virus (512 HAU); 3 - E-ACA (30 mg); 4 - immunization + E-ACA

Figure 19.5. Influence of E-ACA on efficacy of mice immunization with inactivated influenza virus.

●

● ●

●

ε-ACA is used orally, intravenously, nasally, or in inhalations during days 3 to 7. ε-ACA is given orally 25 to 75 mg/kg gid. Besides that, nasal instillation every 4 hours or inhalation (twice a day) of 5% ε-ACA solution are expedient. The daily dose may be up to 0.5 to 1.0 g/kg, and half of this dose may be given intravenously to individuals with toxic course of disease.

Following is an account of results of some clinical studies of efficacy of ε-ACA in the treatment of influenza and other ARVIs in children. We have determined that proteolytic activity (pH 7-6) in the blood of hospitalized patients has been statistically higher than such activity in blood of healthy children, therefore necessitating the use of the proteolytic inhibitor ε-ACAof primary importance. Including ε-ACA in the therapeutic complex for treatment of influenza and other ARVIs in children resulted in a decrease of duration of intoxication symptoms (in 2 to 3 days), fever (in 1.5 to 3 days), and catarrhal symptoms (on 1.5 to 2 days). The level of complications is reduced to 17%. The results of our studies on the anti-influenza action of ε-ACA have validated the permission to use it as prophylaxis

and treatment of influenza in both human and veterinary medicine. ε-ACA has shown significant antiviral activity not only toward avian influenza viruses but also toward NDV (9). We believe that a combined use of proteolytic and neuraminidase inhibitors may be a highly effective way for influenza prevention and treatment because while the proteolytic inhibitors would prevent the influenza viruses of entering into sensitive cells, the neuraminidase inhibitors will prevent them from exiting the infected cells. We are open for collaboration in such joint projects. Furthermore, we have determined that ε-ACA displays not only anti-influenza activity but antiherpetic efficacy as well. Thus, ε-ACA inhibits the HSV-1 replication in cell cultures, and when applied at a concentration of 13 µg/mL on cell culture Hep-2 during infection with HSV-1, it succeeded in protecting the cells by 52%. Dr. J. Drach from Michigan University had studied by our request the anti-HSV activity of this preparation on his cell models and confirmed its activity. ε-ACA also showed protective efficacy after intranasal or intraperitoneal infection of mice by HSV, as well as its therapeutic efficacy in a rabbit model of herpetic keratoconjunctivitis. Immunization of experimental animals by inactivated influenza viruses, HSV, and adenoviruses combined with ε-ACA was more effective than immunisation by each of these viruses without ε-ACA (10). The antiherpetic activity of ε-ACA may be explained in connection with the discovery of serine protease in HSV that plays an important role in the viral physiology. In further studies, we have examined the effectiveness of ε-ACA in the treatment of patients with recurrent genital herpes (11). Patients of the control group received vitamins of the B-group and the antiviral drugs Chelpinum and Megasinum. Chelpinum was administrated orally at a dose of 125 mg, 4 times daily for 7 to10 days. Megasinum was administrated topically as 3% liniment three times daily. ε-ACA was administrated orally (1 tablespoon of 5% solution) 4 times daily for 7 to 10 days. 100 mL of a 5% ε-ACA solution was administrated intravenously once a day for 5 days if the patient’s condition was serious. These patients also received 1 tablespoon of ε-ACA solution orally twice daily during the same days. Then, these patients received ε-ACA only orally. ε-ACA was also administrated topically as powder or solution for the treatment of lesions. The improvement of the patients’ conditions can be summarized as follows: ●

●

periods of time between remissions were from 1.5 to 2.5 times (2.0 ± 0.5) longer recurrences are from 2 to 4 days shorter (3.0 ± 1.0).

Improvement can be viewed as non-significant when frames of solving recurrences are lightly shortened while number of recurrences stayed the same. Twenty patients (out of 50) treated with ε-ACA (50%) had significant improvement, 15 patients (37.5%) had improvement, and five patients (12.5%) had non-significant

19. Anti-Infectious Actions of Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA)

improvement. In the control group of 30 patients, 11 (36.3%) had significant improvement, 13 (43%) had improvement and 6 (20.7%) had non-significant improvement. The overall results of this study showed that the synthetic protease inhibitor ε-ACA has higher therapeutic effectiveness during recurrent genital herpes as compared to such antiviral drugs as Chelpinum and Megasinum, thereby underlining the necessity to use it for the treatment and prevention of most largescale acute (influenza) and chronic (herpes) viral infections. In addition, our studies have shown antiviral activity of ε-ACA against adenoviruses and HIV. The possibility for blocking the transformation of the precursor of the structural core protein (polypeptide p-VII) into structural protein (polypeptide VII) by ε-ACA has been demonstrated in Hep-2 cells infected by adenovirus type 2 (12). A significant inhibition of the formation of intranuclear inclusion bodies and virus yield in the treated cells was also detected. The obtained results showed that thisrocess can be one of the targets for adenovirus reproduction inhibition. ε-ACA in a dose of 35 ég/mL inhibited the replication of HIV-1 (strain-IIIB) by 50% of C8166 cultural cells. At a concentration of 920 g/mL, ε-ACA inhibited the HIV-1 (strainIIIB)s replication by 80% (13). It is known that proteolytic inhibitors may increase efficacy of antibiotics against some microorganisms (14). We have studied the influence of ε-ACA on the sensitivity of some emerging pathogens and agents of nosocomial infections to various antibiotics (15). Thus, we have studied the antimicrobial action of 18 representatives of 10 antibiotics classes combined with addition of 5 % Σ-ACA to the nutrition medium. Two strains of Francisella tularensis [vaccine strain 15 (FT 15) and virulent strain 29 (FT 29)], and four strains of Vibrio cholerae [Vibrio cholerae cholerae strain 569 (VCC 569); Vibrio cholerae El-Tor strain 754 (VCEl 754) and strain 878 (VCEl 878): Vibrio cholerae non-01 strain 146/11 (VCNon-01 146/11)] were used as pathogens

197

of emerging infections. We have discovered that all studied infectious pathogens had been resistant to Σ-ACA when used alone without antibiotics. Furthermore, the F. tularensis FT 15 strain was resistant to carbenicillin and ofloxacin. Growth inhibition zones around the discs containing these antibiotics were totally absent. However, the addition of Σ-ACA led to the increase of FT 15 sensitivity to antibiotics. The diameter of inhibition zones was equal to 16 mm in the case of carbenicillin and to 28 mm in the case of ofloxacin. Strain FT 29 was totally non-sensitive to cefotaxim and ceftriaxone. The addition of ε-ACA resulted in FT 29 becoming highly sensitive to these antibiotics. Furthermore, at low concentration (1%) ε-ACA increased the antibacterial action of streptomycin against FT 15 and of tetracycline against FT 29. The addition of ε-ACA transformed VCNon-01 146/11 strain-resistant to cefotaxim, ceftriaxone, erythromycin, norfloxacin and tetracycline into highly sensitive one to these antibiotics (Figures 19.6 and Figures 19.7). Zones of growth inhibition for VCNon-01 146/11 due to the mentioned antibiotics combined with ε-ACA were in the 11- to 25-mm range. It has been shown that the addition of ε-ACA increased this strain’s sensitivity to kanamycin, ofloxacin, and lomefloxacin. Addition of ε-ACA caused increase in the sensitivity of the classic cholere strain VCC 569 to ampicillin, gentamicin, sisomycin, vancomycin, and kanamycin (Figure 19.8). This strain was totally resistant to erythromycin. After ε-ACA addition the growth inhibition zone diameter was increased up to 28 mm (Figure 19.6). Strain VCEl 754 became much more sensitive to penicillin antibiotics, aminoglycosides, norfloxacin, and tetracycline after combined application with ε-ACA (Figures 19.7 and Figures 19.9). It also increased the antibacterial action of gentamicin against VCEl 878. ε-ACA can enhance activity of antibiotics some agents of nosocomial infections. It increased the antibacterial action of ciprofloxacin, oxacillin, and cefuroxim against isolate 1329

Diameter of growth inhibition zone (mm)

25 28 20 15 10

11

5 0

Diameter ofgrowth inhibition zone (mm)

20 30

18

20

20

16 14 12 10

10

8 6 4 2

0

0 1

erythromycin

2

erythromycin and 5% E-ACA

Figure 19.6. The influence of E-ACA to susceptibility to erythromycin of V. cholerae cholerae 569(1) and V. cholerae non 01 146/11(2) strains.

1 tetracycline

2 tetracycline with 5% E-ACA

Figure 19.7. The influence of E-ACA on susceptibility of V. cholerae non 01 146/11 (1) and V. cholerae E1 Tor 754 (2) strains to tetracycline.

Diameter of growth inhibtion zone (mm)

198

V. P. Lozitsky

References

40 39

35 30

30

25 20 15

22

28

26

24

16

16

10 5 0 1

2

3

4

1-ampicillin; 2-sizomycin; 3-canamycin; 4-vancomycin antibiotics

antibiotics with 5% E-ACA

Diameter of growth inhibition zone (mm)

Figure 19.8. The influence of E-ACA on antibacterial action of some antibiotics in relation to V. cholerae cholerae 569 strain.

30 30

30 28

25 24

25

20

21

20

15 10

12

5 0 1 2 3 4 1-ampicillin; 2-carbenicillin; 3-sizomycin; 4-norfloxacin antibiotics

antibiotics with 5% E-ACA

Figure 19.9. The influence of E-ACA on antibacterial action of some antibiotics in relation to V. cholerae EI Tor 754 strain.

of S. aureus, of ciprofloxacin against an isolate of P. aeruginosa, and of cephazoline against an isolate of E. coli.

19.4

Conclusions

1. The results of our long-term research have demonstrated that the use of the proteolysis inhibitor Σ-ACA in human and veterinary medicine is a rational and well-warranted approach for prevention and treatment of viral and bacterial infections. 2. A further development of methods and schemes for combined usage of protease and neuraminidase inhibitors for prevention and therapy of influenza is very promising. 3. Proteolysis inhibitors represent a very promising group of drugs for future research as anti-infective agents.

Acknowledgments. This work was partially supported by the Science & Technology Center in Ukraine (STCU project #3147).

1. Lozitsky VP, Polyak RYa (1982) The role of proteolysis in reproduction of human and animal viruses and antiviral activity of proteases inhibitors. Uspekhi sovremennoi biologii 93:352–362. 2. Lozitsky VP, Fedchuk AS, Puzis LE, Buiko VP, Bubnov VV, Girlia YuI (1987) The participation of proteolysis system in realization of influenza virus virulent potential and in development of infectious process. Antiviral action of proteases inhibitors. Voprosi virusologii 32:413–419. 3. Lozitsky VP, Fedchouk AS, Girlya YuI, Puzis LE, Sudakov AYu, Buyko VP (1996) Proteolysis inhibition as the mechanism of antiviral action of various agents. Xth International Congress of Virology. Jerusalem, p. 152. 4. Lozitsky VP, Polyak RYa, Parusou VN (1979) Participation of proteolysis system in experimental infection and anti-influenza action of protease inhibitors. In Antiviral Activity and Mechanism of Action of Different Chemical Compounds. Riga, “Zinatne,” pp. 352–362. 5. Lozitsky VP, Gridina TL, Fedchuk AS, Boschenko YuA, Grigorasheva IN (2006) Antiviral action of officinal medicines e-aminocaproic acid and unithiolum toward avian influenza viruses. Odes’kyi medychnyi zhurnal 3:4–8. 6. Puzis LE, Lozitsky VP, Polyak RYa (1986) Effectiveness of prophylactic administration of epsilon-aminocaproic acid during influenza in mice. Acta Virol 30:58–62. 7. Lozitsky VP, Puzis LE, Polyak RYa (1988) Resistance of mice to reinfection after E-aminocaproic acid treatment of primary influenza virus infection. Acta Virol 32:117–123. 8. Lozitsky V, Puzis L, Razoryonov G, Polyak R (1996) Effectiveness of the combined use of inactivated vaccine (IV) and the inhibitor of proteolysis E-aminocaproic acid (E-ACA) in prevention of experimental influenza. Antiviral Res 30:53. 9. Lozitsky VP, Fedchuk AS, Mulyak SV, Sozinov VA (1994) Antiviral action of synthetic proteolysis inhibitors toward Neweastle Disease Virus. Veterenariya 1:34–35. 10. Lozitsky VP, Puzis LE,Grigorasheva IN, Tokolova SS (1985) Stimulation of antiviral immunity by synthetic fibrinolysis inhibitors. In Mekhanizmy Immunostimulyatsii Kiev, pp. 131–132. 11. Fedchouk AS, Veveritsa PG, Lozitsky VP, Girlia YuI (1999) Medical cure of recidiving herpes simplex virus infection by means of proteolysis inhibitors. Antiviral Res 41:67. 12. Nosach L, Dyachenko N, Zhovnovataya V, Lozinsky M, Lozitsky V (2002) Inhibition of proteolytic processing of adenoviral proteins by E-aminocaproic acid and ambenum in adenovirusinfected cells. Acta Biochim Polonika 49:1005–1012. 13. Lozitsky V, Ershov F, Scheglovitova O, Fedchuk A, Girlia Yu, Sudakov O, Novitsky V, Kolomiets N, Kolomiets A, Buiko V, Sozinov V, Muliak S (1995) Antiviral action of officinal medicines E-aminocaproic acid and unithiolum. Abstracts of First National Conference on Problems HIV/AIDS With Participation of Foreign Experts. Kiev, pp. 113–114. 14. Pel’kis PS, Shevchenko LI, Lozinsky MO, Kutsenko TA, Shamrai AE, Ckoroded TM (1986) Synthetic inhibitors of fibrinolysis. Kiev, Naukova Dumka, p. 172. 15. Boschenko YuA, Dronova IYu, Lozitsky VP, Pushkina VA, Yurdanova AN, Fedchuk AS, Man’kovskaya NN, Shitikova LI (2002) Ability of proteolysis inhibitor E-aminocaproic acid to amplify antimicrobial action of antibiotics against emerging infections diseases agents. International Conference on Emerging Infectious Diseases, Program and Abstracts Book. Atlanta, GA, p. 130.

Chapter 18 Antivirals for Influenza: Novel Agents and Approaches William A. Fischer, II and Frederick Hayden

18.1

Introduction

Influenza viruses are global pathogens that infect approximately 10% of the world’s population each year and cause epidemics of excess hospitalizations and deaths (http://www. who.int/mediacentre/factsheets/fs211/en/) (1). Unpredictable antigenic shifts in influenza A viruses can cause pandemic disease and in 1918 resulted in the death of more than 50 million people. Currently, the highly pathogenic avian influenza H5N1 is causing a epizootic in poultry populations, as well as sporadic human infections associated with high mortality and threatening to ignite an influenza pandemic. Annual immunization currently remains the developed world’s principle defense against the impact of seasonal influenza epidemics. Each year candidate vaccine viruses are selected on the basis of global surveillance conducted through the World Health Organization’s Global Influenza Surveillance Network. Seasonal vaccines do not provide immunity against a novel variant such as the H5N1 virus. Moreover, vaccine utility is limited by timely manufacturing of sufficient vaccine, potential for antigenic mismatches with circulating strains, and by reduced immunogenicity and efficacy in certain important target populations experiencing high morbidity and sometimes mortality from seasonal influenza (infants and young children, the elderly, and those with underlying medical conditions or compromised immune systems) (1). Because vaccine production, including manufacturing, testing, and distribution, requires at least 6 months, a rapid response to a novel virus is not possible at the present time. In addition, the global manufacturing capacity for egg-grown influenza vaccines is about 350 million doses of the standard trivalent vaccine. Various candidate human vaccines for H5N1 appear safe and immunogenic, but initial studies indicate the need for two doses and the use of high levels of hemagglutinin (HA) From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

antigen and/or adjuvants (2–4). In addition, the virus continues to evolve antigenically, so that multiple vaccine candidate strains are necessary (http://www.who.int/csr/disease/avian_ influenza/guidelines/h5n1virus/en/index.html). Historically, antiviral drugs for influenza have been perceived as an adjunct to vaccination. Given the current lack of an approved H5N1 vaccine and uncertainties regarding vaccine availability for a pandemic virus in general, antivirals have emerged as an important pharmacologic response to both sporadic human H5N1 infections and to a possible pandemic event. The range of antiviral use strategies includes individual case management, short-term chemoprophylaxis of at-risk contacts, long-term prophylaxis of selected populations, and potentially geographically targeted mass chemoprophylaxis to extinguish or delay an emerging pandemic virus (4a–9). Currently, two classes of antiviral drugs are licensed for use against influenza: adamantanes and neuraminidase (NA) inhibitors (10, 11). Adamantanes, specifically amantadine and rimantadine, block the M2 transmembrane ion channel that is integral to the uncoating of the ribonucleoprotein complex early in influenza replication (12–14). Despite documented efficacy as a prophylactic agent in seasonal and pandemic influenza, the utility of the adamantanes is limited by adverse central nervous system and gastrointestinal effects, a spectrum of activity limited to influenza A, and the emergence of drug-resistant isolates (10). Recent studies have documented the presence of a particular mutation in the M2 protein (Ser31Asn) that confers high-level resistance to adamantanes in increasing proportions of community H3N2 isolates. Increased resistance was first documented in isolates from China and Hong Kong in 2003 and reached more than 90% of such isolates in North America in 2005–2006 (15, 16). An increasing prevalence of this resistance mutation has also been reported recently in community H1N1 viruses. Furthermore, all clade 1 and some clade 2 H5N1 viruses harbor this same, or less often other mutations, that confer resistance to both amantadine and rimantadine (17). Such observations make this antiviral class unreliable as a therapeutic 179

W. A. Fischer and F. Hayden

180 Table 18.1. Representative studies of the in vitro activity of antiviral agents against differing strains of influenza. Study Virus Ref. 56a H1N1 H3N2 B Ref. 57 H1N1 H3N2 H2N2 H6N2 B Ref. 56b

Oseltamivir carboxylate EC50 (ug/mL)

Zanamivir IC50 (nM)

Peramivir

Number of strains tested

IC50 (nM)

EC50 (ug/mL)

5 6 8

0.69–2.24 0.27–0.45 5.33–18.33

0.73–1.05 1.85–3.13 2.00–3.10

0.26–0.43 0.47–0.87 1.08–1.95

6 4 4 1 8

0.69–2.2 0.21–0.56 0.01–1.5 0.84 6.4–24.3

0.3–0.8 0.68–2.3 0.76–1.8 1.07 1.5–17.0

0.09–0.81 0.14–0.83 0.17–1.4 1.08 0.6–10.8

0.22 to > 100 < 0.01–0.65 0.2–0.22 0.03–1.3

IC50 (nM)

EC50 (ug/mL)

A-315675 EC50 (ug/mL)

T-705 EC50 (ug/mL)

H1N1

5

0.17 to >100

0.09–21

H3N2 H5N1 B Ref. 62 H1N1

12 2 5

< 0.01–0.50 0.22–0.26 0.11–3.0

8

≤ 0.01–0.7

≤ 0.015–1.25

H3N2 B Ref. 67 H1N1 H2N2 H3N2

4 9

≤ 0.01–5.1 ≤ 0.01–0.125

≤ 0.03–5.1 ≤ 0.01

5 4 4

0.029–0.2 0.013–0.3 0.078–0.48

B

3

0.002–0.0096 0.00017–0.68 0.00049– 0.003 0.0063–0.031

C

3

> 100

0.03–0.57

< 0.01–0.19 0.01–0.02 0.06–3.2

0.039–0.089

EC50 values determined by cell culture assays; IC50 values determined by neuraminidase inhibition assays

or prophylactic agent in both seasonal influenza and in H5N1 infections in the absence of local susceptibility data. NA inhibitors, zanamivir and oseltamivir, comprise the principle class for pharmacotherapy of influenza infections today. Products of rational drug design, NA inhibitors target the active enzymatic site of NA, a highly conserved region in influenza A and B viruses and inhibit NA’s cleavage of terminal sialic acid residues on receptors for viral HA (Table 18.1; ref. 18). Oral oseltamivir and orally inhaled zanamivir are highly effective as prophylactic agents against human influenza A and B infections (18–23). When used as early treatment of uncomplicated influenza, both reduce lower respiratory complications, and oseltamivir also reduces hospitalizations and appears to reduce all-cause mortality after influenza infection (21, 24–28). In contrast to the adamantanes, emergence of community isolates of influenza resistant to the NA inhibitors has been low (29–31). Emergence of oseltamivir resistance has been documented in less than 1 to 4% of adults and in 5.5 to 18% of children during or shortly after treatment for human influenza (32–34). These drugs are generally well-tolerated, but inhaled zanamivir has been associated with serious bronchospasm and oseltamivir with gastrointestinal, cutaneous, and possibly neuropsychiatric side effects. Despite activity against the virus in vitro and in animal models (35), the therapeutic value of oseltamivir in human H5N1

disease is unproven and the optimal dose regimen is uncertain (4a, 36). Resistance has already been documented in three H5N1 patients treated with oseltamivir and appeared to be associated with prolonged viral replication and fatal outcome in two (37, 38). Inhaled zanamivir is unstudied in human H5N1 infections and there are serious concerns about the tolerability and antiviral efficacy of inhaled zanamivir in H5N1 patients with progressive viral pneumonia, in part because it may not reach sites of replication in affected alveoli, distal airways, or extra-pulmonary sites (4a, 15, 17). In general, there is a need for a parenterally administered antiviral agent that provides reliable delivery of drug to affected sites in seriously ill influenza patients. Given the paucity and limitations of currently available agents, the purpose of this chapter is to review the properties of investigational antiviral agents for influenza viruses. We selectively discuss mechanisms of antiviral action and resistance, pre-clinical activity, human pharmacology, and, when available, data about tolerability and antiviral efficacy in humans. Our focus is on interventions that are nearing or are presently in early clinical development, particularly ones that may provide new options for influenza management in terms of improved pharmacology, alternative routes of delivery, or novel antiviral mechanisms of action that provide the potential for activity against viruses resistant to other classes and for use in combination therapies (39–42).

18. Antivirals for Influenza: Novel Agents and Approaches

The reader is referred to a recent review that discusses potential influenza antiviral targets and a number of novel agents that have shown activity in pre-clinical testing (43).

18.2 18.2.1

NA Inhibitors Intravenous Zanamivir

Zanamivir (5-acetamido-4-guanidino-6-(1,2,3-trihydroxypropyl)-5,6-dihydro-4H-pyran-2-carboxylic acid) is a potent NA inhibitor in vitro and is approved in many countries for both treatment and prophylaxis of influenza infections by oral inhalation (15, 18, 44). Controlled clinical trials leading to its regulatory approval demonstrated the importance of drug delivery to the tracheobronchial tree, as contrasted with intranasal application alone (15, 45, 46). Of note, the commercial device currently used for zanamivir administration requires a cooperative patient and may not be appropriate for younger children, cognitively impaired persons, or very frail persons. Regarding potential pandemic threat strains of influenza, topically applied zanamivir protected mice against lethal challenge with H5N1 and chickens from H7N7 (47). Despite the ability to generate strains of influenza resistant to zanamivir in the laboratory (48), only one clinical case of resistance in an immunocompromised patient has been reported during treatment (49). In vitro characterization of virus with the mutation (Arg152) causing resistance to zanamavir demonstrated attenuation of the virus due to altered NA activity (50). Of particular importance, zanamivir retains inhibitory activity against most oseltamivir-resistant variants, including the His274Tyr mutation that confers high-level oseltamivir resistance in N1 NAs (Table 18.2; refs. 38, 51, and 52). Consequently, systemic zanamivir has the potential to be useful in treating patients with severe influenza infections due to oseltamivir-resistant variants, including H5N1. Initial human studies examined the pharmacokinetics of zanamivir by differing routes of administration. Although all routes of administration were well tolerated, oral bioavailability was only approximately 2%. Repeated inhalation of 10 mg of the dried powder formulation led to maximal serum concentration (Cmax) of 39 to 54 ng/mL at 1 to 2 hours post-dosing with an elimination half-life of 4 to 5 hours (53, 54). In comparison, intravenous zanamivir at doses of 600 mg resulted in a Cmax of 32,000 to 39,000 ng/mL achieved in 30 minutes and led to levels in the respiratory tract many-fold higher than required

181

to inhibit viral NA (54). In comparison, oral oseltamivir at a standard dose of 75 mg twice daily gives a carboxylate Cmax of about 350 ng/mL (54a). The plasma elimination half-life of intravenous zanamivir averages about 2 hours. In a small, randomized, placebo-controlled, double-blind study, intravenous zanamivir prophylaxis (600 mg iv bid × 5 days starting 4 hours before viral inoculation) prevented influenza seroconversion (86% efficacy) and viral shedding (100% efficacy) in subjects experimentally infected with A/ Texas/36/91 (55). In this trial, intravenous zanamavir also demonstrated significant reductions in clinical measurements of disease, including fever, respiratory illness, cough, and myalgias compared to placebo (55). Zanamavir was detected in nasal washes on day 2 and 4, consistent with effective respiratory tract distribution after intravenous administration. Additional phase I studies of intravenous zanamivir are in progress at present, and it is hoped that intravenous zanamivir will progress to phase II clinical trials in the near future.

18.2.2

Peramivir

Peramivir (ethyl (5S,3R,4R)-4-(acetylamino)-5-amino-3(ethylpropoxy)cyclohex-1-enecarboxylate) is a cyclopentane derivative that targets the active site of NA in both influenza A and B strains. It has a positively charged guanidino group, a negatively charged carboxylate group, and lipophilic side chains that lead to some differences in antiviral spectrum and pharmacology compared to available NA inhibitors (47, 56). In vitro studies indicate that peramivir is as active as oseltamivir against human influenza A viruses and even more effective against influenza B viruses (57). Peramivir also demonstrated greater activity against H2N2 and H3N2 viruses and comparable activity against Trfluenza B Viruses when compared with zanamivir in vitro (Table 18.1; ref. 57). For avian strains, all nine NA subtypes were susceptible in enzyme inhibition assays with IC50 values ranging from 0.9 to 4.3 nM compared with zanamivir 2.2 to 30.1 nM and oseltamivir carboxylate 1.9 to 69.2 nM (57). Studies in lethal murine models have demonstrated the in vivo efficacy of oral peramivir against human and avian strains of influenza. Complete protection against the H5N1 and H9N2 strains related to human cases in Hong Kong in 1997 and 1999, respectively, was achieved with peramivir 1 or 10 mg/kg/day; oral peramivir was not significantly different from oral oseltamivir (47).

Table 18.2. NA inhibitor resistance profiles. Susceptibility in the NAI assay (fold change) NA mutation

NA type/subtype

Oseltamivir

Zanamivir

Peramivir

A-315675

E119V

A/N2

R (>50)

S (1)

S (1)

S (1)

R292K

A/N2

R (>1000)

S (4–25)

R (40–80)

S (8)

H274Y

A/N1

R (>700)

S (1)

R (40–100)

S (3)

R152K

B

R (>30–750)

R (10–100)

R (>400)

R (150)

Data taken from refs. 51 and 52 R= resistant, S = susceptible. The fold change compared to value for wile-type NA is indicated

W. A. Fischer and F. Hayden

182

Peramivir remains active against some strains resistant to oseltamivir and/or zanamivir. However, it shows substantial loss of inhibitory activity for certain oseltamivir-resistant variants including the Arg292Lys in N2 and His274Tyr in N1 Ns (Table 18.2). Attempts at selecting influenza strains resistant to peramivir in vitro have resulted in variants with mutations at Lys189Glu, a change that appears to be associated with reduced fitness. Parallel efforts designed to select resistant strains in mice were unsuccessful in at least one study (58), and resistance was not detected in the context of experimental human infections (59). Several randomized, double-blind, placebo-controlled trials evaluated oral peramivir’s activity as a prophylactic and therapeutic agent. In volunteers inoculated with an H1N1 virus (A/Texas/36/91), oral peramivir treatment initiated at 24 hours after infection showed dose-related antiviral effects; peramivir 400 mg q24h resulted in a 73% reduction in viral shedding compared with placebo (59). Similarly, subjects inoculated with an influenza B virus and treated with 800 mg q24h of peramivir had 61% decreased viral shedding. However, peramivir prophylaxis did not demonstrate a significant decrease in viral shedding at doses less than to 400 mg q day compared with placebo (59). Furthermore, oral peramivir failed to significantly reduce the time to relief of symptoms in phase III human clinical trials, likely due to its poor oral bioavailability (less than or equal to 3%), and thus is no longer being developed (59a, 60). The gastrointestinal tolerability of oral peramivir appears better than oseltamivir (59) and no doselimiting end-organ toxicities have been recognized to date. Parenterally delivered peramivir overcomes the limitation of low oral bioavailability and has demonstrated promising results in animal models (60). One-time dosing is effective in animal models because of the prolonged plasma T1/2elim and also the prolonged time of dissociation between the NA and the drug (measured as Kioff). A single intramuscular injection of 10 mg/kg of peramivir was comparable to a 5-day course of oral oseltamivir in preventing mortality from H3N2 and H1N1 in lethal murine influenza models (60). A single intramuscular injection of 30 mg/kg was comparable to oral oseltamivir in a murine model of A/Vietnam/1203/04 (H5N1) infection, and repeated intramuscular doses of 30 mg/kg/day were active in a ferret infected with this virus (61). In recently completed human studies, intravenous peramivir was well-tolerated up to doses of 8 mg/kg/day for as long as 10 days and demonstrated predictable, dose-dependent pharmacokinetics (J Beigel, unpublished observations). Intravenous doses of 4 mg/kg provide mean Cmax of 20,492 ng/mL and are associated with a prolonged plasma T1/2elim of about 20 hours (J. Alexander, unpublished observations). Intramuscular peramivir at doses of 300 mg also provided drug exposure, based on plasma levels over time, comparable to an intravenous dose of 4 mg/kg. The high plasma concentrations and prolonged plasma T1/2elim of peramivir offers the possibility of oncedaily intravenous dosing in seriously ill patients and possibly single-dose intramuscular therapy in outpatients. Phase II studies of intravenous peramivir in patients hospitalized with

serious influenza and of intramuscular peramivir in outpatients were initiated during the 2006–2007 season.

18.2.3

A-315675

A-315675 (D-proline, 5-[(1R,2S)-1-(acetylamino)-2-methoxy2-methylpentyl]-4-(1Z)-1-propenyl-, (4S,5R)-) is a novel pyrrolidine-based NA inhibitor with documented in vitro and in vivo activity against influenza A and B (Table 18.1; ref. 62). In enzymatic ligand-binding assays, A-315675 had superior potency against influenza B and A/N2 NAs (Ki of 0.14–0.31 nM and 0.19, respectively) compared with oseltamivir carboxylate (Ki of 1.1–2.1 nM and 1.3, respectively) and comparable potency against N1 and N9 enzymes (62). A-315675’s potency in the binding assays can be attributed to a delayed dissociation of the inhibitor from the NA (Kioff of A-315675 is 18 times slower than that of oseltamivir) with a T1/2 of approximately 10 to 12 hours, compared with 30 to 60 minutes seen with oseltamivir carboxylate for representative NAs (62). In cell culture assays, A-315675 exhibited greater antiviral activity than oseltamivir against the majority of clinical influenza B strains and greater than or equal activity against a wide range of N1- and N2-containing viruses (Table 18.1) (62). One important feature of A-315675’s antiviral spectrum is inhibitory activity against most oseltamivir-resistant variants (Table 18.2). In particular, it shows minimal or no loss of activity against the most common oseltamivir-resistant variants in N1and N2-containing viruses. Oral A-322278, the prodrug of A315675 is comparably active to oseltamivir in murine models of H2N2 infection, although both agents are associated with resistance emergence in immunocompromised mice (62a). This feature and the compound’s oral bioavailability make it an interesting development candidate. However, it has not yet progressed to human testing.

18.2.4

Long-acting NA Inhibitors

The term long-acting NA inhibitors (LANI) refers to several chemically diverse molecules that have been designed to maximize lung retention times after topical application to the respiratory tract. One of these molecules, CS-8958 (or R-125489) undergoes enzymatic activation in the respiratory tract. Others that are multivalent zanamivir constructs, also known as Flunet and exemplified by “compound 8,” that transcend typical monovalent binding and show improved antiviral potency and compound retention in the lung (63, 64).

18.2.4.1

Multivalent LANIs

Compound 8, a dimeric derivative of zanamivir connected via a 7-carbamate group to a linker of 16 atoms, exhibited potency (EC50s, 0.07–0.1 ng/mL) at least 100 times greater than zanamivir (EC50s, 17–40 ng/mL) in cytopathic effect inhibition assays (63). In plaque reduction assays, this derivative demonstrated 1,000-fold more inhibitory activity (EC50, 55 pM) than zanamivir (EC50s, 11,000–50,000 pM; ref. 63). The increased potency of this dimeric zanamivir derivative is believed to be

18. Antivirals for Influenza: Novel Agents and Approaches

secondary to its multivalency, which is postulated to promotes inter-NA and inter-virion binding. Enzymatically, compound 8 manifested a Kioff six times longer than zanamavir (6 × 10–4 and 1.1 × 10–4, respectively), demonstrating increased binding of the drug to the active site in addition to significant compound retention in the lung (63). Rats sacrificed 168 hours post-dosing exhibited a 100-fold greater concentration of compound 8 compared with zanamivir (63). Remarkably, one intranasal dose 7 days prior to an infectious challenge in the murine model led to a significant decrease in the amount of virus remaining in the lungs 24 hours post-challenge compared to zanamivir and at a fraction of the dose (63). In lethal murine models, early polyvalent zanamivir compounds based on a dextran polymer backbone demonstrated animal protection over a 2-week period following administration of a single dose of 12.5 mg/kg (S. Tucker, 15th International Conference on Antiviral Research, 2002; S. Tucker, personal communication). Several smaller multimers, including dimeric zanamivir compounds like compound 8, were found to share long lung retention profiles ranging from 10- to more than 100- fold longer than seen with zanamivir (S. Tucker, personal communication, ref. 64). Such observations raise the possibility of a one-dose treatment or once weekly prophylaxis. A topically applied zanamivir dimer is expected to enter initial human testing in 2008.

18.2.4.2

CS-8958

CS-8958 is a LANI with single-dose efficacy as both a prophylactic and therapeutic agent in animal models. Through hydrophobic interactions, the long hydrophobic acyl chain allows CS-8958 to colocalize with airway epithelial cells for an extended duration. Once in the airways it is cleaved by esterases into an active antiviral hydrophilic molecule that is retained locally (65). In a murine model, 100% of mice treated with 0.5 µmol/kg and then infected with A/PR/8/34 (H1N1), A/Aich/2/68 (H3N2), or B/Hong Kong/5/72 4 days later survived compared with 20% of zanamavir-treated mice and 0% of mice receiving saline as a placebo (65). In studies of prophylactic efficacy, almost all mice given a single dose of 0.4 µmol/kg CS-8958 24 hours prior to infectious challenge survived, compared with less than 10% of mice protected with a similar dose of zanamivir (66). This molecule has progressed through single-dose tolerability studies in healthy volunteers and is anticipated to move into phase II dose-ranging efficacy studies in natural influenza in Japan soon (Biota Annual Report 2006).

18.3 18.3.1

Nucleoside Analogs T-705

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) represents a novel therapeutic agent with an expanded antiviral spectrum including not only influenza A and B viruses, but also influenza C and several other RNA viruses (67). T-705 is a purine analog that undergoes intracellular metabolism to a nucleo-

183

side triphosphate that selectively inhibits influenza viral RNA polymerase (68). Antiviral inhibition by T-705 can be reversed through addition of purines and purine nucleosides but not pyrimidines (68). The monophosphate inhibits cellular IMP dehydrogenase to a limited extent, but this probably does not contribute to its anti-influenza activity as more selective inhibitors of this cellular target do not inhibit influenza replication in vitro (69). T-705’s novel antiviral mechanism of action results in inhibitory action against influenza strains resistant to the amantadanes or NA inhibitors and offers the potential for combination antiviral therapy. In plaque reduction assays T-705 demonstrated less potency than oseltamivir against most strains of influenza A and B strains (Table 18.1). However, T-705 is active against influenza C (EC50, 0.095 µg/mL), whereas oseltamivir is not (EC50 >100 µg/mL; ref. 67). T-705 has inhibitory effects for poliovirus, rhinovirus, and RSV replication in vitro at 10- to 100-fold higher concentrations than for influenza. No T-705-resistant influenza viruses have been reported to date, although further selection studies are in progress. In time-of-removal experiments, production of progeny virus was not observed after removal of T-705 from cell culture media in contrast to NA inhibitors (67). Whether this effect might play a role in limiting the development of T-705 resistance during clinical use, particularly in the setting of non-compliance or suntherapeutic drug exposure, remains to be determined. Murine models have demonstrated dose-dependent inhibitory effects with complete survival from A/PR/8/34 infection at 200 mg/kg/day T-705 orally and 85.7% protection at 100 mg/kg/day (67). Furthermore, lung virus titers decreased in a dose-dependent fashion with undetectable viral lung levels in 50% of mice treated with 100 mg/kg/day and 80% treated with 200 mg/kg/day (67). Significant survival benefit was seen in mice treated orally with 200 to 400 mg/kg/day T-705 (100% survival in both groups) compared with 200 and 400 mg/kg/day oseltamivir (7 and 21% survival, respectively) after challenge with A/PR/8/34 at more than 1000-fold LD50 (70). More recently, oral T-705 has shown protective effects against H5N1 infections in mice, even with once-daily dosing and when dosing was initiated upto 60 hours after infection (70a). T-705 represents an orally bioavailable, broad spectrum anti-influenza agent that appears to target viral RNA polymerase and thus has a novel mechanism of action. This agent to entered phase I human testing in 2007.

18.3.2

Viramidine and Ribavirin

Ribavirin has long been recognized as an inhibitor of influenza A and B virus replication in vitro, and oral ribavirin has been approved for influenza treatment in some countries. Only high doses have shown evidence of clinical benefit in uncomplicated influenza (71). Intravenous or aerosolized ribavirin has been used in severely ill or immunocompromised patients (72), and aerosolized ribavirin showed modest clinical benefits in children hospitalized with influenza (73). No influenza resistance to ribavirin has been recognized.

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Systemic ribavirin use has been limited by a narrow therapeutic index with documented adverse events including haemolytic anemia, electrolyte disturbances and potential teratogenesis. However, intravenous ribavirin is an option for study in combination therapy with NA inhibitors for severe influenza (42). Viramidine, a carboxamidine analog of ribavirin, is a second purine analog with documented activity against influenza A and B and was better tolerated than ribavirin in phase I human trials (74). Viramidine is thought to act as a prodrug of ribavirin and requires conversion to its active form by adenosine deaminase (74). Viramidine exhibits less cellular cytotoxicity but also decreased anti-influenza potency in vitro compared to ribavirin (EC50s, 2.1–32 µg/mL and 0.6–5.5 µg/mL, respectively; refs. 75 and 76). Overall, the selectivity index as measured by the ratio of CC50 (concentration of drug which causes 50% decrease in cell growth)/EC50 remained lower for viramidine than ribavirin (75). Viramidine inhibits H5N1 virus replication in vitro (75). In animal studies, treatment with viramidine via oral gavage conferred 100% survival among mice infected with A/NWS/33 at doses of 125 to 500 mg/kg/day, A/Victoria/3/75 at 62.5 to 250 mg/kg/day, B/Hong Kong/5/72 at 125 mg/kg/day, and B/Sichuan/379/99 at 80 mg/kd/day (75). When initiation of therapy was delayed to 48 hours post-infection, both ribavirin and viramidine remained effective (75). Clinical trials in chronic hepatitis C have confirmed the superior tolerability of viramidine over ribavirin but also found less efficacy at the doses tested. Further evaluations of its safety profiles and dosing requirements are needed before testing in human influenza.

18.4

HA and Attachment Inhibitors

Viral attachment to a host cell, an integral step in viral pathogenesis, has recently emerged as a potential site of targeted antiviral activity. Cell surface receptors bearing terminal sialic acid residues serve as primary sites for influenza binding. The preferred receptors for avian and equine influenza viruses vary from those for human viruses by the type of linkage connecting sialic residues to the penultimate galactose of carbohydrate side chains, alpha 2,3-linked and alpha 2,6-linked, respectively. In vitro studies demonstrate decreased influenza infection by over 90% when these sialic acid receptors are removed (77). Furthermore, sialic acid polyacrylamide conjugates, or sialylglycopolymers, have been shown to be inhibitory for influenza virus replication in vitro and in murine models (78). Topical delivery to the respiratory tract is required for these agents, and significant hurdles exist with respect to safe and effective delivery in humans.

18.4.1

DAS 181

DAS 181 also termed Fludase is a recombinant fusion protein consisting of a human epithelium-anchoring domain,

called amphiregulin, linked to a bacterial salidase derived from Actinomycosis viscosus (77). The construct is expressed in Escherichia coli and has a molecular weight of about 44.8 kDa. The exosialidase mediates the removal of sialic acids from glycoconjugates (77). In cell protection assays and viral replication assays, it demonstrated significant viral inhibition (EC50 values of 0.1–15.6 nM) and cell protection with multiple strains of influenza A and B (77). The presence of the epithelial binding domain was found to increase inhibitory effects (77). Additionally, pre-treatment of cells 24 hours prior to viral exposure yielded results that mirrored those with viral exposure immediately following cell treatment. Murine studies have demonstrated increased survival and improved lung function when DAS 181 was administered topically to the nose as prophylaxis before influenza challenge (77). Additionally, when DAS 181 was used as a therapeutic agent (25–30 units/treatment intranasally), a significant survival benefit was noted, even when initiation of treatments was delayed to 48 hours post-infection with A/NWS/33 virus (77). A precursor molecule, designated DAS178, was evaluated in the ferret model of influenza; intranasal dosing demonstrated decreased viral shedding and increased protection against nasal inflammatory responses compared with control (77). In this study, only 3 of 12 animals shed virus on day 1 compared with 8 of 8 in the vehicle-treated group. Intranasal DAS 181 at doses of 1 mg/kg/d was recently found to be protective against lethal H5N1 infections in mice and showed therapeutic effects upto 72 hours post infection (77a). No serious safety problems or enhancement of bacterial infection have been found in limited pre-clinical studies to date (77). DAS 181 represents a novel mechanism of antiviral activity that targets virus receptors. It has demonstrated efficacy as both a prophylactic and therapeutic agent in vivo and is anticipated to entry initial human phase I testing in 2007.

18.4.2

Cyanovirin-N

Cyanovirin-NA is a protein with potent anti-human immunodeficiency virus (HIV) activity that also shows inhibitory activity against influenza A and B viruses. Cyanovirin-NA inhibits HIV by blocking high-mannose oligosaccharides on the HIV glycoproteins gp120 and gp41. For influenza, incubation with whole virus lysates suggested viral HA as the likely target of cyanovirin-N. Almost all strains of influenza A and B, including NA inhibitor-resistant strains, were inhibited in vitro by cyanovirin-NA with EC50 values ranging from 0.005 to 0.2 µg/mL for influenza A and 0.02 to 1.3 µg/mL for influenza B viruses (79). Two strains, however, PR/8/34 and NWS/33, displayed high levels of natural resistance to cyanovirin-NA (79), probably due to differences in glycosylation patterns in these highly passaged laboratory strains of influenza. Cyanovirin-N’s activity against a number of influenza A and B strains via an anti-HA mechanism is encouraging but in vivo studies are needed.

18. Antivirals for Influenza: Novel Agents and Approaches

18.4.3

Entry Blocker (EB)

EB is a novel 20-amino-acid peptide, derived from the signal sequence of human fibroblast growth factor 4, which inhibits influenza virus attachment and replication (80). EB is believed to inhibit viral attachment by binding directly to viral HA. Pre-treatment of H5N1 virus with EB decreased cell death in vitro with a mean EC50 of 4.5 µM. Inhibition of the hemagglutinating activity of other influenza A and B viruses occurrs at 3 to 10 µM EB. However, the peptide shows cellular cytotoxicity at concentrations of about 50 µM, so that its therapeutic margin is narrow in vitro. When EB 2 mM was delivered intranasally with H5N1 virus, 100% of mice survived and lung titers were decreased. In contrast, daily EB administration starting at 6 hours after virus inoculation failed to prevent mortality and demonstrated non-significant effects on lung virus titers (80). EB shows initial promise as a lead molecule for a topically applied viral attachment inhibitor, but further confirmatory testing is needed.

18.5

Protease Inhibitors

HA is translated as a single protein, HA0, and requires cleavage by a protease for activation into its infectious conformation. The liberation and conformational alignment of two protein domains, HA1 and HA2, from this cleavage step are essential for viral fusion activity and further replication (81–83). Both endogenous and exogenous protease inhibitors, most notably aprotinin (82, 83), inhibit influenza replication, and agents like ambroxol that stimulate endogenous inhibitor secretion are active in murine models (81). Aprotonin and camostat mesilate were shown to inhibit influenza A (mean EC50 values of 170 and 2.2 µg/ml, respectively) and B in vitro (mean EC50s, 260 and 5.8 µg/ml, respectively) at levels significantly below their CC50 (84). No studies to date have evaluated whether a therapeutic level of camostat mesilate is achievable with an oral formulation, as doses used for pancreatitis in the past have not been sufficient. Animal studies demonstrated a 50% protection rate against lethal doses of influenza when treated with inhalational aprotinin at 6 µg/day (85). One human study reported that inhaled aprotinin decreased indices of disease in 52 human subjects during natural influenza infection (83). The inhibition of the influenza protease is yet another potential target of viral activity that to date has been relatively unexplored.

18.6

Serotherapy

Passive immunoprophylaxis has been shown to be efficacious against a variety of viral illnesses including hepatitis A, hepatitis B, rabies, and RSV (86, 87). Passive immunotherapy with anti-HA antibodies is effective in various animal models of

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influenza, including immunocompromised mice (88–90). A retrospective meta-analysis evaluating the use of convalescent blood products for pneumonia patients during the1918 influenza pandemic found a clinically important reduction in casefatality rates between treated (16% or 54 of 336) and untreated groups (37% or 452 of 1219; ref. 91). Early administration (< 4 days after onset) appeared to be important for benefit. Several different antibody preparations have been reported to show anti-H5 activity in animal models. Fab fragments derived from horses immunized with an H5N1 vaccine were shown to provide dose-dependent protection against lethal challenge in a murine model (92). However, such equine products are limited by potential for immune reactions, particularly with repeated dosing. Use of convalescent or postimmunization blood products has not been rigorously studied in H5N1 disease to date. Humanized monoclonal antibodies to HA have been shown to be inhibitory for H5N1 viruses in vitro and in lethal murine models (87). These chimeric antibodies, the most effective of which is called VN04-2-huG1, are composed of variable domains derived from mice infected with H5N1 fused with constant domains of the human kappa light chain and IgG1 heavy chain. VN04-2-huG1 shows neutralizing activity in vitro for a clade 1 H5N1 virus and is believed to be directed against epitopes on the 140 loop of H. When given to mice 24 hours prior to lethal virus inoculation, doses of 5 to 10 mg/kg protected mice from disease (87). Additionally, when VN04-2-huG1I was given one day following challenge, 80% of mice treated with 1 mg/kg and all of those receiving 5 or 10 mg/kg survived; treatment with 10 mg/kg beginning 3 days following inoculation also conferred complete survival (87). In another approach, B cells isolated from convalescent H5N1 patients in Vietnam have been immortalized to produce virus-neutralizing antibodies that are protective in vitro and in vivo against infection with H5N1 virus (92a). Two such human monoclonal antibodies, 3F3 and 5F12, not only neutralized the homologous strain of H5N1 from clade 1, but also exhibited activity against viruses from clade 2. However these antibodies were not active against a H3N2 virus, suggesting a H5-specific protective profile (92a). When used as therapeutic agents, 5F12, and to a lesser extent 3F3, protected mice from a lethal challenge with A/Vietnam/1203/04 even when treatment was initiated 18 hours later. Mice treated with either monoclonal antibody manifested 10-100-fold lower titres of virus in the lung and undetectable viral levels in the brain (92a). Passive immunotherapy with these monoclonal antibodies not only blocks initial adhesion of viral particles but also reduces viral burden in lungs and subsequent dissemination to other organs. However, passive immunotherapy may be limited by cost, production, and antigenic variability. The constraints of providing large scale immunotherapy in the setting of a pandemic are likely to be substantial. The specificity of monoclonal antibodies and the potential for escape mutants to emerge will

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likely require use of panels of monoclonal antibodies. However, this type of approach offers new possibilities for both prevention and treatment of severe influenza that could move rapidly into clinical development.

efficacy, resistance patterns, safety, and delivery mechanisms and routes are needed.

18.7

Interferons are critical mediators of host cell defense against viral infection and are currently used as the standard of care in some viral infections like pegylated α interferon in the treatment of chronic hepatitis C. Interferons are inhibitory for influenza viruses in vitro but very limited contemporary information is available for human influenza. Several clinical trials evaluating the use of intranasal interferon failed to demonstrate prophylactic benefit against seasonal influenza (103–105). In a community-based, placebo-controlled trial during an H3N2 outbreak, intranasal interferon at 1.5, 3, or 6 MU twice a day for 28 days displayed no protective benefit over placebo (105). A second trial evaluating prophylaxis with intranasal α-2b interferon also failed to significantly protect the treated group from those receiving placebo (106). Intranasal interferon are clearly ineffective against natural human influenza and use longer than 1 to 2 weeks causes dose-dependent respiratory mucosal irritation, consisting primarily of nasal stuffiness and epistaxis. Whether alternative routes of administration, specifically systemic or perhaps inhalational, would be safe and effective for treatment of influenza remains to be determined. While interferon responses are brisk in uncomplicated human influenza, limited evidence suggests that they might be depressed (107) or possibly suboptimal (108) in highly pathogenic H5N1 infections. Absent lung interferon levels were reported in patients dying of influenza viral pneumonia by the discoverer of the interferon system, Aleck Isaacs (109), and a recent primate study with the reconstituted 1918 virus also demonstrated a failure of interferon responses (110). If such observations are confirmed in H5N1-infected patients, systemic administration of interferon would warrant consideration for study as part of an antiviral treatment regimen.

RNA Interference

RNA interference represents an innovative approach to drug design and virus inhibition. Originally described in Caenorhabditis elegans, RNA interference is a mechanism by which double stranded RNA modulates sequence specific degradation of mRNA in order to control gene expression (93, 94). This process is directed by the dsRNA-specific endonuclease called Dicer-RDE-1 and it can be reproduced by artificially introducing synthetic duplex strands of RNA (21–25 nt in length) into a cell (95, 96). Treatment with siRNA targeting sequences coding for influenza nucleocapsid protein (NP) and a component of the RNA transcriptase complex (PA) both pre- and post-infection demonstrated inhibitory activity in vitro (96). Because of the central role of these protein products in viral RNA synthesis, inhibition was not limited to the mRNA for NP or PA, but involved all viral RNAs without significantly altering RNA transcription levels of the host cell. Other sequences including the matrix 1 protein have been targeted in vitro with mixed results (97–99). In animal models, the same siRNA fragments targeting NP delivered by inhalation and intravenous modes led to a significant reductions of lung virus titers in infected mice (9, 11, 21, and 56-fold, when challenged with H7N7, H5N1, H9N2, and H1N1 viruses, respectively) and protected mice from a lethal challenge with clinically relevant strains, including H5N1 (100). Additional in vivo experiments demonstrated a 1000fold decrease in lung titer when mice were treated with 120 µg of siRNA to NP intravenously 5 hours post H1N1 challenge (98, 101). The importance of target sequence homology of the siRNA for the infecting strain of influenza as was seen with failure to decrease lung virus titers for a B strain with only 50 to 70% homology (100). Phosphorodiamidate morpholino oligomers (PMOs) are single-stranded anti-sense agents that cause steric interference with mRNA transcription and/or translation. One study found that pre-treatment with micromolar concentrations of PMOs targeting the PB1 transition start region and the 3' terminal region of NP vRNA significantly decreased viral titers in a cell culture assays against a wide range of influenza viruses including H1N1, H3N2, H3N8, H7N7, and H5N1 (102). Similar to other studies using RNA interference, the efficacy of the PMOs is limited by sequence homology, as oligomers with more than 1 basepair discordance demonstrated a significant loss of inhibitory activity. PMOs also appear to have a narrow therapeutic index in vitro (102). In general, siRNA and related technologies remain in their infancy with regard to its human applicability and further pre-clinical studies of

18.8

18.9

Interferons

Host Cellular Targets

Influenza infection provokes a multitude of intracellular signal transduction events that lead to the activation of type I interferons (α and β), interleukins, and other pro-inflammatory cytokines and chemokines that contribute to host antiviral responses. One synthetic human Toll-like receptor 7/8 receptor agonist and cytokine inducer, designated 3M-001, showed antiviral activity after intranasal delivery in a rat influenza model that correlated with the compound’s ability to stimulate type I interferons and other cytokines (111). Intranasal application of liposomal poly ICLC, a TLR3 agonist, or of synthetic lipid A mimetics that stimulate TLR4 receptors are also protective in murine models (111a, 111b). However, drugs that inhibit innate immune responses like gemfibrizol have also been reported to be beneficial in such models (111c).

18. Antivirals for Influenza: Novel Agents and Approaches

Viruses have evolved gene products to interfere with these antiviral responses, and the NS1 protein of influenza that appears to be central in suppressing the induction and effects of type I interferons (112–114). However, influenza viruses also possess the capability to exploit these signaling cascades to benefit replication. Two signaling pathways have been recently identified as necessary to influenza viral replication, specifically the IKK-nuclear factor-κB (NF-κB) and Raf/MEK/ERK cascades (115, 116). Consequently, targeting these host cell pathways is another potential arena for influenza chemotherapy. For example, an inhibitor of the upstream MAPK/ERK kinase, designated U1026, has been shown to impair influenza viral growth by inhibiting the export of viral ribonucleoprotein complexes without apparent host cell cytotoxicity (117, 118). Influenza infection also activates NF-κB, which promotes the gene expression of pro-inflammatory and antiviral cytokines such as IFN-α/β and tumor necrosis factor (TNF)-α. Interestingly, recent studies have demonstrated decreased viral titers when influenza viruses have infected cells with impaired NF-κB signaling (118), suggesting that this pathway can be manipulated by the influenza virus into a virus-supporting cascade. It has been proposed that the NF-kB pathway is responsible for promoting the expression of TNF-related apoptosis-inducing ligand and Fas, which induce caspase activation and thus facilitate the release of RNP complexes from the nucleus via a caspase-mediated nuclear pore complex disruption (116). The NF-κB inhibitor SC75741 shows dose-related effects on influenza replication in vitro and on survival in experimentally infected mice (O. Planz, VIRGIL Annual International Symposium on Antiviral Drug Resistance, Lyon, May 23, 2007). Targeting intracellular signaling cascades as opposed to viral gene products represents a novel approach to antiinfluenza chemotherapy but one in which safety concerns will need close attention, as modulating the host antiviral and inflammatory responses to achieve the optimal balance will be challenging in different stages and severities of influenza infection. While short-term use of an agent directed against host responses (i.e., for treatment of a severe infection) might be acceptable, longer-term use for chemoprophylaxis would likely be more problematic.

18.10

Combination Chemotherapy

Antiviral combination therapy, usually targeting different steps in the viral replication cycle, is an important approach that has been used in efforts to enhance antiviral efficacy, reduce emergence of resistant strains, and possibly decrease dosage and the toxicity of individual agents. This strategy has been applied with clinical success in HIV infections with highly active antiretroviral therapy and in chronic hepatitis C infections. The first study in influenza reported that interferons and amantadine combinations showed enhanced activity in vitro (119), and subsequent pre-clinical reports

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showed that effectiveness of various combinations of M2 inhibitors, ribavirin, interferons and NA inhibitors compared to monotherapy (39–42, 119–125). Studies evaluating oseltamivir and ribavirin in vivo found that certain combinations were superior to single agents alone in a lethal mouse model of influenza B infection, although not in influenza A (124). In cell culture studies, the combination of oseltamivir and rimantadine was associated with prevention of resistant variants emerging during serial passage (125). The only controlled clinical trial tested a combination of aerosolized zanamivir with rimantadine compared to rimantadine with placebo in patients hospitalized with serious influenza (126). This study was under-enrolled but was notable for the finding that the only M2 inhibitor-resistant variants were observed in the rimantadine monotherapy group. In a lethal murine model due to an amantadine-sensitive A(H5N1) virus, survival was improved from 30% with oseltamivir alone (10 mg/kg/day) to 90% when combined with amantadine (30 mg/kg/day), and greater reductions in lung and visceral viral titers were found with the combination compared to either monotherapy (127). Survival was not augmented by the combination compared to oseltamivir alone when mice were challenged with an amantadine-resistant strain of A(H5N1). No mutations in the HA, NA, and M2 proteins were detected with combination therapy. The high frequencies of pre-existing resistance to M2 inhibitors in circulating A(H3N2) and A(H1N1) strains, as well as in many A(H5N1) viruses, limits the applicability of this particular combination at present (16, 128). However, antiviral combination therapy or therapy with combinations of antivirals and immunomodulators represent important approaches, and further animal model studies that evaluate different combinations of chemotherapeutic agents are needed to identify the optimal ones for clinical testing.

Acknowledgments. The authors thank Dr. Nikki Shindo, Global Influenza Program, World Health Organization (WHO) for her helpful comments on the manuscript and for facilitating Dr. Fischer’s visit to the WHO. We also thank Drs. Robert Sidwell and Dale Barnard, Utah State University, Logan; John Beigel, National Institutes of Health, Bethesda; Yousuke Furuta, Toyama Pharmaceuticals, Japan; Jane Ryan, Biota, Melbourne; James Alexander, Biocryst, Birmingham, AL; Michael Ossi, GlaxoSmithKline, Research Triangle Park; Jackie Katz, Centers for Disease Control and Prevention, Atlanta, GA for sharing unpublished information. We also thank Diane Ramm, University of Virginia, for assistance in manuscript preparation.

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Chapter 1 Biotools for Determining the Genetics of Susceptibility to Infectious Diseases and Expediting Research Translation Into Effective Countermeasures Malak Kotb, Robert W. Williams, Nourtan Fathey, Mohamed Nooh, Sarah Rowe, Rita Kansal, and Ramy Aziz

1.1

Introduction

Infectious diseases, like most human diseases, are affected by complex polymorphic and nonpolymorphic interactive traits that influence host–pathogen interactions and modulate disease phenotype. It is well established that host genetic variability strongly affects susceptibility to infectious diseases and can significantly potentiate the severity of their clinical manifestations. The same individual could be highly susceptible to a particular infection yet completely resistant to another—ultimately these complex genetic variations ensure that some of us will be selected to survive catastrophic biological threats and help protect our species from extinction. As a result of global environmental, social and political changes, we are facing real danger that could result from major natural, deliberate, or accidental biological threats. The best means of protection against these impending threats is to be better prepared. To do so, we need to gain a deeper understanding of how our genotypes modulate our susceptibility and reaction to specific infectious agents, because this information helps us to better understand disease mechanism. Our research has been focused on linking specific genotypes to susceptibility phenotypes and delineating pathways and molecular events that modulate host resistance or susceptibility to specific infectious pathogens. Inasmuch as it is quite difficult to conduct certain infectious disease studies in humans, there has been a critical need for small animal models for infectious diseases. Appreciating the limitations of the existing models, we have developed several novel and complementary mouse models that can be used to gain a better understanding of complex disease mechanisms and reveal the interactive network(s) that lead to eradication of the infection or to serious pathology caused by our overzealous response to it. From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

Recombinant inbred (RI) mouse strains are a powerful tool for identifying quantitative trait loci (QTL) and interactive gene networks modulating infectious disease phenotypes. Data generated using the RI reference population provides a roadmap for the disease that helps focus hypotheses and expedite the process of discovery and forward research translation. Potential diagnostics, therapeutics, and vaccines suggested from the RI mice studies can also be tested in our fully humanized mouse model, where the mouse immune system has been replaced with a human immune system. Together, these models provide valuable preclinical information and allow the screening for vaccine efficacy or adverse effects, to focus and expedite the translation of research into effective countermeasures in major biological threats.

1.1.1 A Genetically Diverse, Genomically Well Defined Reference Mouse Panel Afford an Ideal Model for a Systems Biology Approach to Infectious Diseases Traditionally, most experimental models of infectious diseases have involved inbred rodents, including the most common 10 strains of inbred mice (i.e., A/J, BALB/c, CBA, C3H/He, C57BL/6J, DBA/2, NZB, and AKR). Whereas these models have been invaluable to scientists, their downfall is their limited genetic variability, where certain phenotypes may be suppressed or grossly exaggerated. A good analogy would be like conducting a clinical trial using the same eight people every time and expecting to generalize the results to the rest of the human population. Clearly, this is neither optimal nor representative of the variation seen in humans. For this reason, several groups have been generating panels of genetically diverse mice to study the genetics of susceptibility to various diseases. Of these, the RI mice are ideal for many reasons (1, 2). The RI strains are generated by crossing two inbred strains followed by ≥ 20 consecutive generations mating among siblings (1–4) (Figure 1.1). These RI mouse strains are a powerful tool for identifying QTL and interactive gene networks 13

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Figure 1.1. Derivation of BXD RI strains of mice. (A) RI strains are derived by systemic inbreeding beginning with the F2 generation of the cross of two pre-existing inbred progenitor strains (B6 and DBA/2J). Multiple independent strains are then derived without selection. Once inbred, such a set of RI strains can be thought of as a stable segregated population. (B) The RI strains are typed with respect to the numerous genetic differences that distinguish the progenitor strains. Each locus has a particular pattern of inheritance called the strain distribution pattern (SDP). Comparisons are made between SDPs. A significant excess of parental genotypes, with respect to two SDPs, signals the possibility of genetic linkage. From: Mountz JD, Van Zant GE, Zhang HG, Grizzle WE, Ahmed R, Williams RW, et al (2001) Genetic dissection of age-related changes of immune function in mice. Scand J Immunol 54:10–20 (See Color Plates).

modulating infectious disease phenotypes. The panel we have in Memphis is the advanced RI (ARI) mice derived from the parental C57BL/6J and DBA/2 strains, which are known to differ considerably in their susceptibility to a number of infectious agents. These ARI BXD strains contain roughly twice as many recombinations as standard RI strains (4, 5). The BXD ARI strains can therefore be used to map QTLs with twice the positional precision as can be achieved with the original BXDs (1, 2). Figure 1.1 illustrates the schema used for generating those strains. We currently have 80 BXD strains that are being extensively phenotyped and genotyped. Each BXD strain is genetically distinct from other strains, but all members of a given BXD strain are inbred (i.e., genetically identical). Thus, studies can be repeated on the same strain (individual) at different times, for as many times, and with a large number of biological replicas, thereby providing strong statistical power for the data. Another important feature of our BXD strains is that both parental strains have been sequenced and this greatly facilitates the identification of genes within mapped QTLs.

Prior to using the ARI mice for mapping and reverse genetics studies, we spend considerable time optimizing and standardizing the infection model. Once this has been accomplished, we basically infect mice from the ARI panel with the same dose of pathogen and measure different phenotypes (e.g., survival, weight loss, pathogen load in blood and dissemination to peripheral organs, etc.). The ARI mice are then ranked relative to each other for a given phenotype. These relative phenotype values are then analyzed in the context of the mouse genotype using WebQTL tools available on www.genenetwork.com, which provides the QTL mapping for phenotypes of interest. The bioinformatics tools allow us to inspect the single nucleotide polymorphism density within the mapped loci and to examine the genes within the loci in order to narrow down the number of candidate genes that should be further interrogated. The tools also allow us to identify interactive loci, through which we can discover interactive pathways modulating the measured phenotype. Data generated using the ARI reference population reveal polygenic and pleiotropic networks modulating disease

1. Genetics of Infectious Disease Susceptibility

phenotype and thereby providing a disease roadmap that helps focus hypotheses and expedite the process of forward systems discovery and research translation. The studies described in this chapter illustrate the utility of these mice in infectious disease studies.

1.1.2 Studies on the Genetics of Susceptibility to Invasive Group A Streptococcal (GAS) Sepsis Illustrate the Utility of RI Mice in Infectious Disease Research Severe forms of invasive GAS infections associated with high morbidity and mortality were prevalent during the 1918 flu pandemic, then virtually disappeared from 1920 to the 1980s, when suddenly severe invasive disease reemerged in many parts of the world, causing panic and leading the media to dub it “the flesh-eating disease” (6–10). The bacteria are considered an ideal model for studying the effect of host genetics on the infection outcome, because the same bacteria can cause a wide spectrum of diseases in different individuals. These diseases range from mild sore throat to deadly diseases, such as streptococcal toxic shock (STSS), necrotizing fasciitis (NF), rheumatic fever and rheumatic heart disease (RHD), glomerulonephritis, and neurological disorders. We and others have identified specific immunogenetic polymorphisms that predispose to particular forms of GAS diseases and determine the level of risk for the severe forms of these illnesses, including RHD, NF and STSS (11–13). Our STSS susceptibility studies have been ongoing since 1992 in collaboration with Dr. Donald E. Low and the Ontario Streptococcal Study Group. GAS are the richest known bacteria in superantigens (SAgs), with more than 13 identified SAgs to date (SpeA-C, SpeF-M, SSA, and SmeZ 1-24), with different GAS strains having different SAg repertoires. SAgs trigger excessive activation of T cells and MHC II-expressing cells, and cause massive release of inflammatory cytokines (e.g., TNF-β and IFN-γ). Responses of different individual to the same SAg can also vary quite drastically (11–13). Besides the SAgs, GAS possess many surface and secreted proteins that interact with the immune system (immune cells and complement proteins), e.g., M protein, C5a peptidase, SIC, and many streptodornases, which are involved in degrading neutrophil extracellular traps (14). In the first phase of our studies, we focused on genetic elements that may potentiate the host response to GAS SAgs. We identified specific HLA-II alleles and haplotypes that confer very strong resistance to STSS, and others that predispose to it . We validated our association studies, biologically, through both in vitro studies with human PBMC (different HLA types) and in HLA-tg mice carrying alleles of interest. The role of HLA-II variation in STSS susceptibility is logical because the GAS SAgs, which are pivotal mediators of STSS, utilize the HLA-II molecules as receptors through which they interact with TCRVβ elements and elicit potent inflammatory

15

responses leading to STSS in genetically predisposed high responders (Figures 1.2 and 1.3). Thus, we hypothesize that other host genetic elements might also modulate susceptibility to severe GAS sepsis and STSS, notably in earlier stages of infection controlled by the innate immune response of the host, and we are interested in finding pathways and networks rather than individual genes that are modulated by immune cells in response to GAS. To discover additional genetic variations and pathways that modulate the outcome of GAS sepsis, we turned to the ARI BXD mice described earlier. The data included in the following paragraph underscore the utility of these mice in the discovery process. Our initial studies showed that DBA/2J mice are more susceptible to severe GAS sepsis than C57BL/6J. Initially, we used approximately 300 mice from 20 BXD strains to optimize the infection dose and identify confounding nongenetic factors that need to be adjusted or included as significant covariates in the final analysis. An optimal dose of 1–3 × 107 CFU/100 µL per mouse of a virulent M1T1 GAS clinical isolate and BXD strains ages 40 to 120 days were used in this intravenous model of GAS sepsis. As shown in Figure 1.4, several BXD strains showed phenotypes outside the ranges of the parental strains, with several significantly more susceptible or resistant than their ancestors. In nine ensuing experiments, about 360 mice from 34 strains (32 BXDs and two ancestors) were infected intravenously with the optimal dose of the bacteria and survival was monitored every eight hours for seven days. Mice were weighed every 12 hours and weight loss was calculated. Bacterial load in blood (CFU/mL) was determined for all mice at 24 hours, and a bacteremia index was determined and corrected for covariates. All mice developed bacteremia, but with considerable differences in severity and survival rates (Figure 1.4).

Low protective Abs Increase risk for bacterial invasion T cell Group A streptococci Superantigen infection

HLA allelic variation modulate sepsis severity

TCR HLA class II (DRB1*15/DQB1*0602) Lower cytokine response protects against STSS

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Figure 1.2. HLA class II allelic variation modulate susceptibility to severe streptococcal sepsis (See Color Plates).

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Figure 1.4. A bar chart showing the mean values of corrected relative survival indices (cRSI) for 20 BXD strains, arranged in ascending order. Parental strains (C57Bl/6J and DBA/2J) are shown on the two extremities of the x-axis. Error bars represent the standard errors of the means. The total number of animals (n) used per strain is indicated. Data from Aziz RK, Kansal R, Abdeltawab NF, Rowe SL, Su Y, Carrigan D, et al (2007) Susceptibility to severe Streptococcal sepsis: use of a large set of isogenic mouse lines to study genetic and environmental factors. Genes and Immunity 8:404–415.

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Figure 1.3. Validation of HLA class II association with severe streptococcus sepsis using HLA-transgenic mice. (A) In vivo differential susceptibility of HLA-tg mice to M1T1 GAS sepsis. (5 × 106 CFU) live M1T1 bacteria in 250 µL PBS were injected intravenously into DQ6 (Σ) or DR4/DQ8 (□) mice. Mice survival was monitored twice daily and death was recorded over seven days. (B) IFN and TNF levels in plasma of HLA-tg mice, 24 hours postinfection. (C) Bacterial load in the blood, liver, and spleen of DQ6 (Σ) or DR4/DQ8 ( ) mice intravenously infected with 5 × 106 CFU bacteria. ∗p < 0.05; ∗∗p < 0.01. From: Nooh MM, El-Gengehi N, Kansal R, David CS, Kotb M (2007) HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis. J Immunol 178:3076–3083.

Survival was recorded in day/day fraction post infection, and for each experiment the data was normalized to calculate a relative survival index (RSI). RSI for each strain and for each experiment were corrected for variables (mainly age) to generate a corrected index, cRSI. Using the Data Desk statistical program, we conducted multiple regression analyses for individual as well as for all nine combined experiments. Age was confirmed as a significant determinant of survival and bacterial spread, but the strongest factor influencing survival, as expected, was the genetic background of BXD strains (p ≤ 0.0001). These studies allowed us to map a strong QTL-modulating sepsis severity to a locus on chromosome 2. We are currently fine-tuning the mapping using additional BXD strains and interrogating genes of interest within the mapped QTL. We believe it will be quite informative to acquire systems information on GAS in the BXD strains in vitro and in vivo. We plan to compare the data to human in vitro responses and patient’s acute and convalescent plasma. This will provide a comprehensive mouse to human in vitro and in vivo correlation using a very well characterized set of samples.

References 1. Bailey DW (1971) Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11:325–327.

1. Genetics of Infectious Disease Susceptibility 2. Taylor B (1978) Recombinant inbred strains: using gene mapping. In Origins of Inbred Mice (IIIHC M, ed.), pp 423–438Academic Press New York. . 3. Chesler EJ, Lu L, Shou S, Qu Y, Gu J, Wang J, Hsu HC, Mount JD, Baldwin NE, Langston MA, Threadgill DW, Manley KF, Williams RW (2005) Complex trait analysis of gene expression uncovers polygenic and pleiotropic networks that modulate nervous system function. Nat Genet 37:233–242. 4. Peirce JL, Lu L, Gu J, Silver LM, Williams RW (2004) A new set of BXD recombinant inbred lins from advanced intercross populations in mice. BMC Genet 5:7. 5. Shifman S, Bell JT, Copley RR, Taylor M, Williams RW, Mott R, Flint J (2006) A high-resolution single nucleotide polymorphism genetic map of the mouse genome. PLoS Biol 4:e395. 6. Cunningham MW (2000) Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470–511. 7. Cone LA, Woodard DR, Schlievert PM, Tomory GS (1987) Clinical and bacteriologic observations of a toxic shock-like syndrome due to streptococcus pyogenes. N Engl J Med 317:146–149. 8. Low DE (1997) The reemergence of severe group A streptococcal disease: an evolutionary perspective. In Emerging Infections I (Hughes WMSa, ed.), American Society for Microbiology Press Washington, D.C.

17 9. Stevens DL, Tanner MH, Winship J, Swarts R, Ries KM, Schlievert PM, Kaplan E (1989) Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 321:1–7. 10. Davies HD, McGeer A, Schwartz B, Green K, Cann D, Simor AE, Low DE (1996) Invasive group A streptococcal infections in Ontario, Canada. Ontario Group A Streptococcal Study Group. N Engl J Med 335:547–554. 11. Kotb M, Norrby-Teglund A, McGeer A, Green K, Low DE (2003) Association of human leukocyte antigen with outcomes of infectious diseases: the streptococcal experience. Scand J Infect Dis 35:665–669. 12. Kotb M, Norrby-Teglund A, McGeer A, El-Sherbini H, Dorak MT, Khurshid A, Green K, Peeples J, Wade J, Thomson G, Schwartz B, Low DE (2002) An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat Med 8:1398–1404. 13. Kotb M (1995) Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 8:411–426. 14. Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V (2005) DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Current Biology 16:396–400.

Chapter 8 Clinical Mycobacterium tuberculosis Strains Differ in their Intracellular Growth in Human Macrophages Sue A.Theus, M. Donald Cave, and Kathleen D. Eisenach

8.1

Introduction

Tuberculosis (TB) remains a global public health crisis despite being a curable disease (1). Access to the complete genomic sequence of the Mycobacterium tuberculosis (MTB) laboratory strain H37Rv and clinical isolate CDC1551 has facilitated investigations into the pathogenicity of MTB. However, mycobacterial factors that contribute to the virulence of MTB or modulate the interaction of this pathogen with the human host are only beginning to be elucidated. Despite previous studies suggesting that MTB has an extremely low mutation rate, there are compelling reasons to suspect that strain-specific attributes contribute directly to virulence and disease outcome. Our studies of infections in THP-1 cells with epidemiologically distinct clinical MTB strains provide strong evidence that specific clinical MTB strains may be differentially pathogenic (2, 3). Clinical strains associated with TB outbreaks grow significantly faster in human macrophages than do non-outbreak strains. The rapid growth demonstrated by strains associated with outbreaks was highly correlated with rapid production of interleukin (IL)-10 and suppression of tumor necrosis factor (TNF)-α. These results suggest that the enhanced capacity of MTB to grow rapidly in human macrophages is a marker of virulence, and virulence of certain strains may be attributed to downregulation of the Th1type immune response. The ability to combine genomic information with pathogenesis studies employing diverse clinical strains will enable us to continue to unravel the molecular basis of MTB virulence and identify strain-specific attributes that influence clinical presentation, outcome (treatment failure, relapse, and development of drug resistance), and transmission of infection. Ultimately, having the ability to identify relevant strain characteristics could From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

potentially impact how we treat and control the spread of TB and elucidate whether strain virulence will impact the protection afforded by a new candidate vaccine.

8.2

TB Infection and Disease

MTB as a successful intracellular pathogen has evolved sophisticated strategies to infect and persist in host macrophages (4). In approximately 90% of immunocompetent people, the infection is asymptomatic and infecting bacilli are either killed or remain viable but latent. In the infected individuals who develop active disease, bacilli appear to evade or subvert the host’s protective cellular immune responses. Infection is established, bacilli continue to replicate in host tissues, and clinical symptoms are seen. Development of active TB is likely determined by multiple factors. First, evidence suggests that host genetic factors are involved in determining susceptibility or resistance of an individual to TB (5, 6). Also, naturally occurring mutations in the human genes for interferon (IFN)-γ receptor and IL-12 receptor have been shown to be associated with increased susceptibility to TB infection (7–10). Second, MTB itself may undergo genetic changes, modifying virulence (11). Third, the ability of a particular MTB strain to elicit a strong or weak host immune response may be important in determining development of disease. For example, the MTB clinical isolate CDC1551 induces an unusually high frequency of seroconversion in exposed persons, yet the rates of active disease are not unusually high (12).

8.3 8.3.1

Strain-specific MTB Pathogenesis Murine Models of Virulence

Few studies have investigated the differentially pathogenic nature of specific clinical MTB isolates; thus, the role of strain variability in outcome of infection remains uncertain. 77

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Lopez et al. investigated the influence of strain diversity on the course of experimental disease in BALB/c mice using MTB strains of major genotype families (defined on the basis of IS6110 restriction fragment-length polymorphism [RFLP] analysis and spoligotyping) found worldwide (13). Infection with the Beijing genotype induced accelerated bacterial multiplication, early and massive pneumonia, and death. Conversely, infection with the Canetti genotype induced a slowly progressive disease characterized by delayed bacterial multiplication, limited pneumonia, steadily increasing granulomas, and virtually no mortality. Similarly, Manca et al. examined the differential response of B6D2/F1 mice to infection with various outbreak clinical isolates (14, 15). One isolate associated with an unusually high proportion of active TB cases in Texas, HN878, was found to be hypervirulent as demonstrated by very early death of infected immunocompetent mice compared to infection with other isolates. Isolate HN878 failed to induce MTB-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, lower levels of TNF-α. IL-6, IL-12, and IFN-γ mRNA levels were observed in lungs of HN878 infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. These results suggest that the hypervirulence of HN878 may be due to failure to stimulate Th1-type immunity. Conversely, other outbreak strains, CDC1551 and NHN5, induced very high cytokine levels in the lungs, levels associated with long-term survival in the mice. Subsequently, isolate HN878 was found to be a Beijing family strain and had the same IS6110 RFLP pattern as strain 210; CDC1551 and NHN5 are non-Beijing strains.

8.3.2

Macrophage Models of Virulence

To examine strain-specific pathogenesis, we determined intracellular growth rates for clinical MTB strains, as well as the virulent MTB strain H37Rv in IFN-γ-activated THP-1 cells (2). Macrophages infected for seven days showed that H37Rv and clinical isolate 282 grew significantly more rapidly than another clinical isolate 284. Isolates 282 and 284 were from an epidemiologic study of TB transmission in homeless shelters in Los Angeles, CA (16). IS6110 genotyping showed that 27% of the TB cases in Los Angeles were caused by strain 210 (isolate 282 being a 210 strain), whereas isolate 284 accounted for only one TB case. Subsequently, it was found that strain 210 is a member of the Beijing family and is associated with a high proportion of TB cases worldwide (17). Zhang et al. have reported that strain 210 grows significantly faster in human blood monocytes (18). The significance of the rapid-growth phenotype is further supported by the recent finding that the principal sigma factor sigA, which may regulate expression of virulence genes, is upregulated in macrophage-grown isolates of strain 210 relative to other clinical strains (19). We also tested 26 strains from TB cases in IS6110 RFLP clusters that have

persisted in Arkansas three years or more and six strains from TB cases with unique RFLP patterns (caused TB in only one patient; ref. 3). The persistent clustered strains had significantly faster growth rates in THP-1 cells than strains with unique RFLP patterns. Together, these findings indicate that clinical strains differ in their ability to grow intracellularly and those with enhanced intracellular growth capacity are at an advantage for either transmission or establishment of disease in humans. TNF-α, a proinflammatory cytokine produced primarily by monocytes and macrophages, is essential for protection against acute MTB infection (14, 15, 20). Studies have shown that avirulent or less virulent mycobacteria induce significantly more TNF-α production by macrophages than virulent mycobacteria. For example, the attenuated M. bovis BCG induced more TNF-α production than virulent M. leprae, and the nonpathogenic M. smegmatis elicited higher levels of TNF-α compared with virulent strains of M. avium and MTB (21–23). Thus, there appears to be an inverse correlation between mycobacterial virulence and TNF-α production. Our results concur with these findings (i.e., strains considered less virulent on the basis of slow intracellular growth rates induced significantly higher levels of TNF-α than more virulent strains in THP-1 infections; ref. 3). Most likely, the course of infection with the slow-growth phenotype is modulated by the rapid and robust TNF-α response, which restricts mycobacterial replication. In contrast, the rapid-growth phenotype suppresses TNF-α secretion, likely through induction of high levels of IL-10. In contrast to TNF-α, IL-10 is generally considered to be primarily anti-inflammatory. Not only has IL-10 been shown to suppress the Th1-type immune response to MTB infection, but also it downregulates the release of TNF-α from macrophages; its inhibitory effect depends on concentration (24, 25). We observed that strains considered highly virulent based on rapid intracellular growth rates induced significantly higher levels of IL-10 within the first 24 hours of THP-1 cell infection than do strains of less virulence (3). This suggests that a cell signaling pathway is blocked during the early interaction of highly virulent strains and macrophages, resulting in a rapid anti-inflammatory response from the infected cells, providing an increased chance of survival. Two reports linking strain diversity to innate immune responses provide additional evidence that clinical isolates with different epidemiologic parameters behave differently in macrophage models of virulence. In one study, significant differences were observed in the cytokine profiles triggered in RAW murine macrophage cells: a spectrum of high to low TNF-α levels was observed, with the lowest level being in infections with the highly transmissible isolates (26). In the other study, a strain associated with the largest recorded outbreak in United Kingdom was shown to induce less TNF-α and more IL-10 from human monocytes (MNs) than CDC1551 and H37Rv, suggesting that the high attack rate was related to the strain’s ability to skew the innate response toward a nonprotective phenotype (27).

8. M. tuberculosis: Intracellular Growth in Human Macrophages

8.4 Virulence Assessment of Household Transmitted Isolates To further demonstrate the validity of the macrophage model system for identifying strains with virulence potential, we examined another group of isolates thought to have an advantage for transmission (28). These isolates were from households in which there was evidence of TB transmission. For comparison, another group of isolates from households in which MTB infection was not transmitted was selected. In addition to using THP-1 cell cultures, growth rates and cytokine secretion in MNs were determined. Strains were isolated from patients enrolled in the National Institutes of Health TB Research Unit Household Contact Study conducted in Kampala, Uganda (29). The strains tested consisted of three pairs from matched household (HH) with infection (PPD+) and no infection (PPD), three pairs from matched HH with co-prevalent disease and no disease, and three pairs from matched HH with incident disease or no disease. For the infection set, a case HH was defined as a one in which at least one HH contact of the index case was PPD+ at baseline visit; control HH were free of PPD+ contacts. For the co-prevalent and incident sets, a case HH was defined as a HH in which a contact of the index case had disease with the same strain as the index case at baseline visit or develops disease with the same strain within six months post-baseline, respectively. Households were matched on the basis of clinical characteristics of the index cases (degree of smear positivity, duration of cough) and HH descriptions (number of residents, number of rooms). Fifteen strains had unique IS6110 patterns and two strains shared the same pattern; copy number ranged from 11 to 19. In matchedpair analyses of log growth ratios observed for the incident and co-prevalent strain pairs combined, there was an overall statistically significant difference in mean log growth ratio (day 7/day 0) between the case and control HH strains after adjusting for pairs effects for both culture systems (p < 0.001). The size of the difference between log growth ratios between the matched cases and controls was not identical across pairs. Growth rates obtained in THP-1 cell infections were comparable to those in primary MNs. Because the THP-1 and MN models both demonstrated differences in intracellular growth between MTB isolates from HH with co-prevalent disease and their matched HH, these six paired isolates were used to evaluate the correlation between TNF-α and IL-10 responses and intracellular growth. In the THP-1 model system, peak TNF-α levels for each isolate were observed 48 hours following infection. In each pair of MTB isolates, the organism from the transmission HH induced significantly less TNF-α than did the corresponding matched HH isolate. In MN cultures, peak concentrations of TNF-α were observed 24 hours after infection. Comparisons of the results of infection of MN from each of 10 donors with paired organisms indicated that two of the three co-prevalent transmission isolates tended to induce less TNF-α than the isolates from matched HH in which co-prevalent disease was not observed.

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Due to donor-to-donor variability, these results were not statistically significant. The third co-prevalent transmission isolate tended to induce more TNF-α than its matched isolate, although this finding was again not statistically significant. TNF-α data from 48 hours after infection showed similar patterns, but again the differences observed were not statistically significant. In the THP-1 model system, the kinetics of IL-10 production by isolates from transmission and matched HH were consistently distinct. Specifically, transmission isolates demonstrated rapid induction of IL-10 with peak levels observed 24 hours after infection, whereas matched isolates displayed more gradual induction of IL-10, resulting in peak levels at day 5. In contrast, induction of IL-10 within primary MN did not result in such distinct contrasts between isolates from transmission and matched HH. Indeed, in the comparisons of both two co-prevalent pairs, isolates from transmission HH induced less IL-10 than isolates from matched HH without co-prevalent disease, although these differences were not statistically significant at any of the time points studied. For the third pair of isolates, differences in IL-10 induction were also non-significant at all time points, although the isolate associated with co-prevalent disease did induce somewhat more IL-10 than did the isolate from its matched HH in which co-prevalent disease was not observed. Thus, the two infection models yielded different results in terms of the relationship between intracellular growth and cytokine profiles, as assessed in studies of the CP transmission and matched isolates. The results from the THP-1 cultures coincide with those reported for clinical strains associated with outbreaks, i.e., rapid production of IL-10 and suppression of TNF-α in the THP-1 model is highly correlated with the rapid growth phenotype (3). In the MN model, none of the comparisons of cytokine production were statistically significant, as is frequently observed given the wide variability of cytokine responses of donor cells. However, this was a second study in which intracellular growth of clinical MTB isolates correlated with epidemiologic evidence of strain virulence.

8.5 Virulence Assessment of Strains of the Beijing Family In the third assessment, isolates representing the Beijing strain family were tested in THP-1 macrophages. Because members of the Beijing family are widely distributed around the world and have been responsible for several large outbreaks, it has been suggested that this genotype may have a selective advantage over other MTB strains (30). One such example is isolate HN878, a 210 strain (designated 210 for having 21 copies of IS6110) and a member of the Beijing family, which caused outbreaks in Texas over a three-year period. Infection with HN878 induces a weak Th-1-associated cytokine response in the lungs of infected mice and results in early death. Similarly, isolate 282, also a strain 210 genotype and member

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of the Beijing family, was responsible for the majority of TB cases in Los Angeles homeless shelters (2, 16). As described earlier, isolate 282 grows rapidly in macrophages and modulates the macrophage response. To determine if the Beijing genotype has a growth advantage, growth rates of 14 strains belonging to the Beijing family were measured in the activated THP-1 cell culture system (30). Strains were genetically diverse on the basis of having many different IS6110 RFLP patterns, with IS6110 copy number ranging from 9 to 22, and variable number of single nucleotide polymorphisms (31). These strains demonstrated a full range of growth phenotypes, and again we observed the strong correlation between rapid growth and suppression of TNF-α. Four strains grew significantly slower, and three (one of which was a 210 genotype) grew significantly faster than the other strains. We tested an additional 11 isolates that were strain 210 genotype (several being variants; +1, –1, or shift 1 IS6110 band) and they all grew as rapidly as the other fast growing strains. The growth advantage of strain 210 is consistent with strain 210 having been present for many years in different geographic locations. Furthermore, these results indicate that strains of the Beijing family vary phenotypically and few are as virulent as the hypervirulent strain 210.

8.6

Summary

We have shown that the IFN-γ-activated THP-1 cell culture system is a reliable model for distinguishing clinical isolates on the basis of intracellular growth rates and cytokine production. Result.s with three groups of clinically and epidemiologically characterized strains demonstrate that strains at an advantage for either transmission or establishment of disease in humans can be readily distinguished from other clinical strains on the basis of these phenotypic differences. This model will be useful to elucidate differences in virulence among MTB isolates. Furthermore, isolates identified in this manner should be of interest for further studies aimed at clarifying virulence mechanisms of MTB.

References 1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC (1999) Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country [consensus statement]. WHO Global Surveillance and Monitoring Project. JAMA 282:677–686. 2. Theus SA, Cave MD, Eisenach KD (2004) Activated THP-1 Cells: an attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates. Infect Immun 72:1169–1173. 3. Theus SA, Cave MD, Eisenach KD (2005) Intracellular macrophage growth rates and cytokine profiles of Mycobacterium tuberculosis strains with different transmission dynamics. J Infect Dis 191:453–460. 4. Russell DG (2001) Mycobacterium tuberculosis: here today, and here tomorrow. Nat Rev Mol Cell Biol 8:569–577.

5. Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV (1998) Assessment of the interleukin 1 gene cluster and other candidate gene polymorphisms in host susceptibility to tuberculosis. Tuber Lung Dis 79:83–89. 6. Bellamy R, Beyers N, McAdam KP, Ruwende C, et al (2000) Genetic susceptibility to tuberculosis in Africans: a genomewide scan. Proc Natl Acad Sci USA 97:8005–8009. 7. Altare F, Ensser A, Breiman A, Reichenbach J, et al (2001) Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. J Infect Dis 184:231–236. 8. de Jong R, Altare F, Haagen IA, Elferink DG, et al (1998) Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 280:1435–1438. 9. Park GY, Im YH, Ahn CH, Park JW, et al (2004) Functional and genetic assessment of IFN-gamma receptor in patients with clinical tuberculosis. Int J Tuberc Lung Dis 8:1221–1227. 10. Fraser DA, Bulat-Kardum L, Knezevic J, Babarovic P, et al (2003) Interferon-gamma receptor-1 gene polymorphism in tuberculosis patients from Croatia. Scand J Immunol 57:480–484. 11. Sreevatsan S, Pan X, Stockbauer KE, Connell ND, et al (1997) Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 94:9869–9874. 12. Valway SE, Sanchez MP, Shinnick TF, Orme I, et al (1998)An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N Engl J Med 338:633–639. 13. Lopez B, Aguilar D, Orozco H, Burger M, et al (2003) A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 133:30–37. 14. Manca C, Tsenova L, Bergtold A, Freeman S, et al (2001) Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α /β. Proc Natl Acad Sci USA 98:5752–5757. 15. Manca C, Tsenova L, Barry CE, Bergtold A, et al (1999) Mycobacterium tuberculosis CDC1551 induces a more vigorous host immune response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 162:6740–6746. 16. Barnes PF, Yang Z, Preston-Martin S, Pogoda JM, et al (1997) Patterns of tuberculosis transmission in Central Los Angeles. JAMA 278:1159–1163. 17. Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D (2002) Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 8:843–849. 18. Zhang M, Gong J, Yang Z, Samten B, Cave MD, Barnes PF (1999) Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis 179:1213–1217. 19. Wu S, Howard ST, Lakey DL, Kipinis A, et al (2004) The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo. Mol Microbiol 51:1551–1562. 20. Kisich KO, Higgins M, Diamond G, Heifets L (2002) Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect Immun 70:4591–4599. 21. Oliveira MM, Charlab R, Pessolani MC (2001) Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-α secretion in human monocytic THP-1 cells. Mem Inst Oswaldo Cruz 96:973–978.

8. M. tuberculosis: Intracellular Growth in Human Macrophages 22. Roach SK, Lee SB, Schorey JS (2005) Differential activation of the transcription factor cyclic AMP response element binding protein (CREB) in macrophages following infection with pathogenic and nonpathogenic mycobacteria and role for CREB in tumor necrosis factor alpha production. Infect Immun 73:514–522. 23. Yadav M, Roach SK, Schorey JS (2004) Increased mitogen-activated protein kinase activity and TNF-α production associated with Mycobacterium smegmatis- but not Mycobacterium aviuminfected macrophages requires prolonged stimulation of the calmodulin/calmodulin kinase and cyclic AMP/protein kinase A pathways. J Immunol 172:5588–5597. 24. Moore KW, de Waal MR, Coffman RL, O’Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683–765. 25. Sharma S, Bose M (2001) Role of cytokines in immune response to pulmonary tuberculosis. Asian Pac J Allergy Immunol 19:213–219. 26. Stefanidou M, Griffin R, Ponce de Leon A, Sifuentes-Osornio, J, et al (2005) Comparative study of induction of early cytokines by diverse clinical strains of Mycobacterium tuberculosis. Abstract 3074, Keystone Symposium; Tuberculosis: Integrating Host and Pathogen Biology.

81 27. Newton SM, Smith RJ, Wilkinson KA, Nicol MP, et al (2006) A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion. Proc Natl Acad Sci USA 103:15,594–15,598. 28. Theus SA, Cave DM, Eisenach K, Walrath J, Lee H, Mackay W, Whalen C, Silver RF (2006) Differences in the growth of paired Ugandan isolates of Mycobacterium tuberculosis within human mononuclear phagocytes correlate with epidemiological evidence of strain virulence. Infect Immun 74:6865–6876. 29. Guwatudde D, Nakakeeto M, Jones-Lopez EC, Maganda A, Chiunda A, Mugerwa RD, Ellner JJ, Bukenya G, Whalen CC (2003) Tuberculosis in household contacts of infectious cases in Kampala, Uganda. Am J Epidemiol 158:887–898. 30. Theus S, Eisenach K, Fomukong N, Silver RF, Cave MD (2006) Beijing Family Mycobacterium tuberculosis strains differ in their intracellular growth in THP-1 macrophages. Int J Tuberc Lung Dis 10:1087–1093. 31. Filliol I, Motiwala AS, Cavatore M, Qi M, et al (2006) Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 188:759–772.

Chapter 14 Control of Notifiable Avian Influenza Infections in Poultry Ilaria Capua and Stefano Marangon

14.1

Introduction

Avian influenza (AI) represents one of the greatest public health concerns that has emerged from the animal reservoir in recent times. Over the past 5 years there has been a sharp increase in the number of outbreaks of AI in poultry compared with the previous 40 years. It has been calculated that the impact of AI on the poultry industry has increased 100-fold with 23 million birds affected in a 40-year period between 1959 and 1998 and more than 200 million from 1999 to 2004 (1). In fact, from the late 1990s, AI infections have assumed a completely different profile both in the veterinary and medical scientific communities. In recent times, some outbreaks have maintained the characteristic of minor relevance, while others, such as the Italian outbreak of 1999–2000, the Dutch outbreak of 2003, the Canadian outbreak of 2004, and the ongoing Eurasian epidemics, have led to devastating consequences for the poultry industry, negative repercussions on public opinion, and, in some cases, created significant human health issues, including the risk of generating a new pandemic virus for humans via the avian–human link. Influenza viruses are segmented, negative-strand RNA viruses that are placed in the family Orthomyxoviridae in three genera: Influenzavirus A, B, and C. Only influenza A viruses have been reported to cause natural infections of birds. Type A influenza viruses are further divided into subtypes based on the antigenic characteristics of the surface glycoproteins hemagglutinin (H) and neuraminidase (N). At present, 16 H subtypes (H1–H16) and nine N subtypes (N1–N9) have been identified. Each virus has one H and one N antigen, apparently in any combination; all subtypes and the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of the severity of the disease they cause. The very virulent viruses cause highly pathoFrom: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

genic AI (HPAI), a systemic infection, in which flock mortality in some susceptible species may be as high as 100%. These viruses have been restricted to strains belonging to the H5 and H7 subtypes, exhibiting a multi-basic cleavage site at the precursor of the H molecule, or exhibiting a cleavage site motif similar to other HPAI viruses (2). HPAI is a dead-end infection in certain domestic birds, e.g., chickens and turkeys, and has a variable clinical behavior in domestic waterfowl and in wild birds, in which it may or may not cause clinical signs and mortality. To date, the potential role of wild birds and waterfowl as reservoirs of infection has been described only for the Asian HPAI H5N1 virus. The ecological and epidemiological implications of this unprecedented situation are not predictable. To the contrary, viruses belonging to all subtypes (H1–H16) lacking the multi-basic cleavage site, are perpetuated in nature in wild bird populations. Feral birds, particularly waterfowl, represent the natural hosts for these viruses and are therefore considered an ever-present source of viruses. Following introduction into domestic bird populations, these viruses cause low pathogenicity AI (LPAI). This is a localized infection, resulting in a mild disease consisting primarily of respiratory disease, depression, and egg production problems in laying birds. Current theories suggest that HPAI viruses emerge from H5 and H7 LPAI progenitors by mutation or recombination (3–5), although there must be more than one mechanism by which this occurs. This is supported by phylogenetic studies of H7 subtype viruses, which indicate that HPAI viruses do not constitute a separate phylogenetic lineage or lineages, but appear to arise from non-pathogenic strains (6, 7) and the in vitro selection of mutants virulent for chickens from an avirulent H7 virus (8). It appears that such mutations occur only after the viruses have moved from their natural wild bird host to poultry. However, the mutation to virulence is unpredictable and may occur very soon after introduction to poultry, or after the LPAI virus has circulated for several months in domestic birds. This hypothesis is further strongly supported by a study by Munster et al., who demonstrated that there is minor genetic and antigenic diversity between H5 and H7 123

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LPAI viruses found in wild birds and those having caused HPAI outbreaks in domestic poultry in Europe (9). The scientific evidence collected in recent years leads to the logical conclusion that not only HPAI viruses must be controlled in domestic populations but also LPAI viruses of the H5 and H7 subtypes, as they represent HPAI precursors. For this reason, the World Organization for Animal Health (OIE) has adopted the definition “Notifiable Avian Influenza” (NAI) to define all viruses of the H5 or H7 subtype, regardless of their virulence for chickens, and all viruses which have an intravenous pathogenicity index of 1.2 or higher.

14.2

Prevention of AI

The primary introduction of AI viruses in domestic poultry occurs either through direct or indirect contact with infected birds. This may occur through movement of infected poultry, contaminated equipment, fomites, or vehicles and penetration of contaminated infectious organic material. Airborne transmission has not been demonstrated to date. For these reasons, it is clear that if biosecurity measures are implemented at the farm level AI infections can be prevented. Outbreaks that involve significant numbers of animals are characterized by the penetration of infection into the commercial circuit. This includes industrially reared poultry but also all other poultry that is traded, including those from semiintensive and backyard farms. Concepts of disease prevention that are applied to industrially raised poultry in theory should not differ from management strategies that should be applied to smaller holdings. In practice, however, things differ significantly as very basic biosecurity measures, such as preventing the introduction of animals of different origin into a flock and avoiding the contact between poultry and feral waterfowl, are sufficiently well respected in the industrial system, but find very little compliance in the semi-industrial or rural environment. For this reason, in certain parts of the world, particularly where mixed species are reared together and traded through the live-bird market system, rural poultry may become a never-ending source of virus, perpetuating virus circulation and resulting in the establishment of an endemic situation. Biosecurity (encompassing bioexclusion and biocontainment) represents the first and most important means of prevention. It follows that if biosecurity measures of a high standard are implemented and maintained, they can be a firewall against the penetration and perpetuation in the industrial circuit. However, breaches in biosecurity systems do occur. On one hand, the occurrence and extent of the breach should be evaluated and corrective measures should follow, but on the other they indicate the need for the establishment of warning systems to aid diagnosis at an

early phase of infection and the urge to implement additional control tools for AI.

14.3

Vaccination for AI

Between December 1999 and April 2003, more than 50 million birds died or were depopulated following HPAI infection in the European Union alone (1), causing significant economic losses to the private and public sectors. The pre-emptive slaughter and destruction of great numbers of animals is also questionable from an ethical point of view. This would suggest that the strategies and control measures utilized to combat the disease at the European level require improvement both from a disease control and animal welfare perspective. Until recent times, infections caused by NAI viruses of the H5 and H7 subtype occurred rarely and vaccination was discouraged as stamping out was the recommended control option. Primarily for this reason, the knowledge of AI vaccinology has not grown at the same rate as that generated for other infectious diseases of animals. Data on experimental and field research in the field of AI vaccinology are currently being generated but there are still areas of uncertainty concerning the rather complex task of vaccinating poultry in different farming and ecological environments. The issue of disease control in developing countries has been addressed on several occasions following the spread of H5N1 in Southeast Asia. Guidelines on disease prevention and control have been issued as joint OIE/Food and Agriculture Organization of United Nations/World Health Organization recommendations in the recent meetings in Rome (February 3–4, 2004), Bangkok (February 26–28, 2004), and Ho-Chi Min City (February, 23–25 2005; ref. 10). These recommendations, however, need to be put into practice in a variety of different field situations, and the applicability of one system rather than another in a given situation must be evaluated bearing in mind the benefits of a successful result but also the drawbacks of a failure. Vaccination has been shown to be a powerful tool to support eradication programmes if used in conjunction with other control methods. Vaccination has been shown to increase resistance to field challenge, reduce shedding levels in vaccinated birds and reduce transmission dynamics (11–13). Both these occurrences contribute to controlling AI. However, previous experiences have indicated that to be successful in controlling and ultimately in eradicating the infection vaccination programmes must be part of a wider control strategy, which includes monitoring the evolution of infection and biosecurity. To eradicate AI, the vaccination system must allow the detection of field exposure in the vaccinated flock. This can be achieved both by using conventional inactivated vaccines and recombinant vector vaccines. Conventional inactivated vaccines containing the same viral subtype as the field virus allow the detection of field exposure by regularly testing unvaccinated sentinels left in the flock.

14. Avian Influenza Infections in Poultry

This system is applicable in the field but is rather impracticable, especially for the identification of sentinel birds in premises that contain floor-raised birds. An encouraging system based on the detection of anti-NS1 antibodies has been recently developed, and is in a concept applicable with all inactivated vaccines provided they have the same H subtype as the field virus (14). This system is based on the fact that the NS1 protein is synthesized only during active viral replication and is therefore not significantly present in inactivated vaccines. Birds that are vaccinated with such vaccines will develop antibodies to the NS1 only following field exposure. Full and field validation under different circumstances of this system is still in progress and should be made available before this system is recommended (15). To date, the only system that enables the detection of field exposure in a vaccinated population that has been used successfully and has resulted in eradication is a DIVA (Differentiating Infected from Vaccinated Animals) system based on heterologous vaccination. This system was developed to support the eradication programs against several introductions of LPAI viruses of the H7 subtype (1, 11). Briefly, a vaccine containing a virus possessing the same H but a different N to the field virus is used. This vaccination strategy enables the detection of field exposure in a vaccinated population through the detection of antibodies to the N antigen of the field virus. For the sake of clarity, a vaccine containing an H7N3 virus can be used against a field virus of the H7N1 subtype. Antibodies to H7 are cross-protective, thus ensuring clinical protection, increased resistance to challenge and reduction of shedding, while antibodies to the N of the field virus (in this case N1) can be used as a natural marker of infection. Experimental data on the quantification of the effect of vaccination on transmission within a flock using this system have been generated, indicating that the reproduction ratio can be reduced to <1 by one week following vaccination (12). Such a reproduction ratio is indicative of minor rather than major spread of infection. In simple terms, such vaccination interventions will significantly reduce (although not prevent) secondary outbreaks although this will very much depend on the immune status of contact birds and flock. Promising results have also been obtained with vaccines generated by reverse genetics (16). These vaccines are expected to have performances similar to conventional inactivated vaccines; however, to date no data are available on their efficacy under field conditions. Recombinant fowlpox vaccines expressing the H protein of the field virus have also been reported to be efficacious in reducing shedding levels and in providing clinical protection. They enable the detection of field exposure as vaccinated unexposed animals do not have antibodies to any of the other viral proteins. Any test developed to detect antibodies to the nucleoprotein, matrix, NS1, or N of the field virus can be used to identify field-exposed birds in a vaccinated population. However, there is some uncertainty about the performances of these vaccines in relation to the immune status of

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the host to the vector virus (17). Recent encouraging studies indicate that vaccination of day-old chicks with maternal antibodies against fowlpox has been successful. Data on the performances of such vaccines in a population that has been once or repeatedly field-exposed to fowlpox are currently lacking. Another aspect that must be carefully considered is the host issue. These vaccines are likely to induce protective immunity only in birds that are susceptible to infection with the vector virus. Regardless of the vaccine and companion test used, it is imperative that the occurrence of infection is mapped within the vaccinated population. This is primarily to monitor the evolution of infection and to manage field exposed flocks appropriately. The latter represent a means by which infectious virus may continue to circulate in the immune population and for this reason, vaccination can only be seen as part of a control strategy based on biosecurity, monitoring, controlled marketing, and stamping out. A vaccination campaign which is not managed appropriately is most likely going to result in the virus becoming endemic. Inadequate biosecurity or vaccination practices can lead to transmission between flocks and selection of variants that exhibit antigenic drift. Antigenic drift of H5N2 viruses belonging to the Mexico lineage resulting in less homology to the vaccine strain has been described recently (18). It clearly appears that the extensive prophylactic use of vaccine in Mexico has resulted in the emergence of antigenic variants that escape the immune response induced by the vaccine. Mexico has been practicing vaccination since the HPAI outbreak in 1994 without applying the DIVA principle. Although no HPAI virus has been reported following the implementation of the vaccination campaign, LPAI viruses continue to circulate. Conversely, a similar approach in Pakistan following the HPAI H7N3 outbreaks in 1995 has resulted in the isolation of HPAI H7N3 virus in 2004— approximately 10 years later (19).

14.4

Emergency Vaccination

Recent outbreaks occurring in developed countries, notwithstanding an efficient veterinary infrastructure and modern diagnostic systems, have resulted in the culling of millions of birds. Since 2000, AI epidemics in densely populated poultry areas (DPPAs) have resulted in 16,000,000 dead birds in Italy (H7N1), 5,000,000 dead birds in the US in 2002 (H7N2), 30,000,000 dead birds in the Netherlands in 2003, and 17,000,000 depopulated in Canada in 2004. In all these episodes it appears that the biosecurity measures implemented at the farm level were insufficient to prevent massive spread of AI. Emergency vaccination for AI has become an acceptable tool to combat the spread of AI, in conjunction with other measures, and therefore could represent an alternative to pre-emptive culling in reducing the susceptibility of healthy

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flocks at risk, by reducing the transmission rate. The effectiveness of such a policy depends on variables such as the density of poultry flocks in the area, the level of biosecurity and integration of the industry, and on the characteristics of the virus strain involved. In addition, practical and logistical problems such as vaccine availability and adequate and speedy administration must be kept in mind. The dimension of the vaccination zone in case of a ring vaccination depends not only on the transmission rate but also on the initial spread during the high-risk period and on the functional interconnections of the infected zone. For this reason, the concept of compartmentalization may be more appropriate than zoning for AI management strategies. The risk of virus spread is not limited to adjacent farms, but the virus can spread over a longer distance through fomites and vehicles. It is generally accepted that AI cannot be controlled when interventions strategies are based on geography only. Another issue of relevance is that of the time interval necessary to obtain protective immunity. It is estimated that a minimum of 7 to 10 days are necessary for the initial development of the immune response, and more than two weeks may be necessary to have protective antibody levels. This implies that the decision-making process must be fast-tracked and vaccine must be available for immediate use. In the face of an emergency, however, uncontrolled movement of vaccination crews may result in the spread of infection rather than controlling its spread. For this reason contingency plans that include decision-making patterns under different scenarios should be formulated. It also appears rather clear that even when vaccination is considered a valid option, it is not possible to lay down general conditions for vaccination programs that can be applied worldwide. Although the industrial system often has overlapping areas, there are major differences in animal densities, species reared, husbandry systems, and genetic profile. Analyzing transmission dynamics and identifying critical points for virus spread from past and future outbreaks should provide data that is required to design appropriate vaccination programmes in different situations. Pivotal work on emergency vaccination has been carried out in Italy, and the application of the DIVA vaccination strategy has resulted in the approval of the use of vaccination as an additional tool for the eradication of two epidemics of LPAI (H7N1 and H7N3) without massive pre-emptive killing of animals. Vaccination was used to complement restriction measures already in place and was integrated with an intensive monitoring program, targeted at identifying viral circulation in the area (1) and culling of infected birds. In 2000, for the first time, heterologous vaccination was used in the field against an H7 virus as a “natural marker vaccine” and subsequently in Hong Kong vaccination using a DIVA strategy contributed to preventing further spreading of HPAI to neighbouring farms in the face of an outbreak of HPAI H5N1 (13). Although the use of a DIVA system enabled the continuation of international trade of poultry products (20), vaccination

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for NAI is a new concept and several countries are reluctant to even consider it as a possibility. Governmental authorities ultimately make the decision as to whether vaccination should be used in a given country or not, and their reluctance is probably driven both by legislative and scientific uncertainties, coupled with doubts about how this practice will be used in the field as well as other considerations such as defining an exit strategy. In countries where there are no DPPAs, control by rapid diagnosis and stamping out may be the most appropriate route to the eradication of AI. Given the current situation, however, it would be wise both for public and private entities to take into account all the possible control options, including the possibility of vaccinating poultry at high risk of exposure. This should result in the preparation of contingency manuals in times of peace that should include sourcing vaccine beforehand. The decision on whether to use vaccination in the face of an emergency should then be made on the basis of the characteristics of the outbreak and of the poultry industry in the area. With reference to trade implications, the new OIE Terrestrial Code Chapter on AI (21) enables the continuation of trade in the presence of vaccination, provided the exporting country is able to produce surveillance and other data that confirm that NAI is not present in the compartment from which the exports are derived. This is the result of extensive work done by OIE experts and by the OIE Central Bureau on the issue of reducing the impact of animal diseases through the use of vaccination and is contained in a recommendation document issued as a result of an International Conference held in Buenos Aires (April 14–17, 2004) that strongly supports the use of vaccines for the control of OIE listed diseases A diseases (22).

14.5 Vaccination Versus Pre-emptive Culling The financial losses due to AI epidemics can be huge for both the commercial and the public sectors, especially once AI viruses are introduced in areas that have high bird densities. In these areas, the high density of poultry farms, the organization of the poultry production sector, and the difficulties in applying rigorous biosecurity measures increase the risk of major AI epidemics. Epidemics in such areas have proved difficult to control despite the enforcement of draconian eradication measures based on the depopulation of farms that are infected, suspected of being infected, suspected of being contaminated or located in areas at risk of infection. Unpublished field evidence from the current situation in Asia indicates that despite the enforcement of massive stamping out and depopulation measures, both LPAI and HPAI viruses may persist undetected in domestic reservoirs or potentially in the wild, re-emerge and rapidly spread after repopulation of poultry farms in previously affected areas. Management of outbreaks by a stamping out and pre-emptive culling policy alone can lead to very high costs and economical losses for the

14. Avian Influenza Infections in Poultry

public sector, the industry, and ultimately for the consumers. Such policies need to be carefully balanced against the trading advantages of rapid eradication and the potentially lower costs of alternative measures. There is no doubt that the enforcement of heightened biosecurity and stamping out measures on AI-affected farms can be effectively applied in areas with a low poultry density, especially if the sites first infected are promptly detected and adequately managed. If this is the case, the depopulation of infected premises can allow the rapid eradication of the disease at acceptable costs for both the producers and the public. Taking into account the high risk of major AI epidemics, once AI viruses are introduced in areas with high poultry densities and the reluctance of national governments or international bodies to actively discourage the formation of DPPAs, alternatives to the application of stamping out alone, which will inevitably lead to pre-emptive culling in cases of LPAI or HPAI outbreaks in DPPAs, should be pursued.

14.6 Prophylactic Vaccination Prophylactic vaccination for NAI viruses of the H5 and H7 subtypes had never been considered until recently. This is primarily due to the fact that it is only recently that situations which may find a cost-effective solution in this policy have been pinpointed and identified. The rationale behind the use of prophylactic vaccination is that it should be able to generate a level of protective immunity in the target population. The immune response may be boosted if there is evidence of the introduction of a field virus to avoid a situation where low level immunity confuses and interferes with diagnosis. Prophylactic vaccination should increase the resistance of birds and, in the case of virus introduction, reduce levels of viral shedding, provided the same levels of biosecurity are maintained. It should be perceived as a tool to maximize biosecurity measures when a high risk of exposure exists. Ultimately, it should result in preventing the index case, or alternatively in reducing the number of secondary outbreaks and thus minimising the negative aspects of animal welfare and potential economic losses in areas where the density of the poultry population will otherwise result in uncontrollable spread without pre-emptive culling. Prophylactic vaccination should only be considered when there is circumstantial evidence that a country/area/compartment is at risk of infection. Risk of infection may be subdivided into two categories: (1) high risk of infection with either H5 or H7 subtype (e.g., from migratory birds) or (2) risk of infection with a known subtype (e.g., live bird markets in the United States, countries with high exposure to H5N1). In the first case a bivalent (H5 and H7) vaccination program could be implemented. Italy has recently implemented such a program in the DPPA at risk of infection (23). In the second case, a monovalent (either H5 or H7) program would be sufficient.

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The choice of the vaccine is crucial to the outcome of prophylactic vaccination campaigns. Ideally, vaccines that enable the detection of field exposure with any AI virus should be used. Ideal candidates would be vaccines that enable the identification of field exposed flocks through the detection of antibodies to an antigen that is common to all type A influenza viruses such as NP, M, or possibly NS1. Such a strategy would be able to detect the introduction of any subtype of AI. The DIVA system using heterologous N has some limitations in its application for prophylaxis or in epidemiological situations where there is the risk of introduction of multiple AI subtypes. The main limitation is that as there is no active viral circulation in category 1 above, or in case of risk of multiple introductions, it is impossible to identify a vaccine strain that has a different N. An approach to resolving this difficulty is to use seed vaccine strains of the H5 and H7 subtypes exhibiting rare N subtypes such as N5 or N8. The selection criteria of vaccine strains will greatly reduce the chance that an AI virus of a similar N subtype is introduced. In any case, for surveillance purposes, unvaccinated sentinels should be present in the flock. Moreover, prophylactic vaccination should not mean vaccination forever. Prophylactic vaccination should be carried out as long as the risk of infection exists, and can be used in a targeted manner for limited periods of time. This means a detailed exit strategy should be formulated before preventative vaccination is undertaken. What appears to be lacking in some situations are guidelines that define an appropriate territorial approach. These guidelines may be derived from general guidelines on surveillance for epizootic diseases but must be adapted to the local situations and must be targeted towards a well defined and pursuable objective. In addition, due to the recent exposure of a vast variety of avian species to HPAI, it is imperative that specific research programs are developed to evaluate the efficacy of vaccination in these species and to develop and validate novel vaccination concepts that enable the DIVA system.

14.7

Conclusions

The control of AI infections in poultry is a target the scientific veterinary community must achieve to manage the pandemic potential, preserve a profit of the poultry industry, and guarantee food security to developing countries. Although biosecurity is recognized as an excellent means of preventing infection, in certain situations it is very difficult to sustain the biosecurity standards necessary to prevent infection. Vaccination has the potential of being a powerful tool to support eradication programs by increasing the resistance of birds to field challenge and by reducing the amount and duration of virus shed in the environment. The development of vaccination strategies encompassing monitoring of infection in the field is crucial for the success of such efforts.

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In view of the zoonotic potential it would appear important that certain AI strains, such as the Asian HPAI H5N1 virus (and probably the H9N2), are eliminated from poultry and not just contained by the use of vaccination, as has been the strategy with other poultry viruses, especially Newcastle disease virus which remains endemic in many parts of the world. Additionally, the application of control programmes encompassing vaccination may result in the generation of strains that have progressively drifted away from the original antigenic profile (18). At present, it is unclear how the immunological pressure generated by the variety of seed strains contained in available and planned veterinary vaccines will affect the antigenic properties of isolates. The results of these two driving forces in the genetic and antigenic profile require careful monitoring of viral strains and a close collaboration between the parties involved in the crisis management. The monitoring effort should aim at the collection and characterization of strains in order to identify genetic mutations and antigenic properties. Information should be collated and made available to the international scientific community, so that those involved in both animal and human health are fully informed of the current situation. Efforts to bring about control and eradication internationally will have to take into account the extremely complex situation, especially in any given geographical location: the characteristics of the poultry producing sector in its entirety, the eco-epidemiological situation, the response capacity of the veterinary infrastructure, and the availability of adequate resources. These features must be integrated with the social environment, including those linked to the rearing of birds for recreational and farming purposes. It is possible that in some areas control and eradication will never be achieved and great changes in the way poultry are reared, and they and their products marketed will be necessary. For this reason, international organizations that govern trade regulations and animal disease control should establish a set of guidelines so that control programs may be “accredited” and consequently internationally recognized. Such a policy would appear to have several practical advantages, ultimately resulting in an improved crisis management. These include rapid approval of established control programs, constant update on the field situation, feedback of information on successes and failures, harmonization of protocols and systems, and public availability of control and eradication programs. In this way, even inexperienced countries can maximize the outcome of other experiences to combat this infection in an educated manner—thus avoiding wastage of resources and time. There are areas in which knowledge needs to be improved in a timely manner, such as specific studies on the efficacy of vaccination in a variety of different avian species, bearing in mind the diverse farming systems employed in developed and developing countries. The outcome of such efforts should be made available to the international community because decision makers are currently lacking sufficient information to

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make educated choices. An enormous effort is required from national governments and funding bodies to make resources available to develop research programs aiming at the development of improved control measures that can be applied under different local conditions. It is imperative that transversal research programs on AI control, encompassing veterinary and agricultural science, are developed and sustained to maximize the global effort to combat this disease.

References 1. Capua I, Alexander DJ (2006) The challenge of avian influenza to the Veterinary community. Avian Pathol 35:189–205. 2. World Organization for Animal Health, Terrestrial Animal Health Code. (2005) 14th chapter 2.7.12.1 on avian influenza 2005. Available at: www.oie.int/eng/novmes/mcode/en_chapter_2.7.12.htm. 3. Garcia M, Crawford JM, Latimer JW, Rivera-Cruz E, Perdue ML (1996) Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico. J Gen Virol 77:1493–1504. 4. Perdue M, Crawford J, Garcia M, Latimer J, Swayne DE. (1998) occurence and Possible mechanisms of cleavage site insertions in the avian influenza hemagglutinin gene. In Proceedings of the 4th International Symposium on Avian Influenza, Athens, Georgia. U.S. Animal Health Association 182–193. 5. Suarez DL, Senne DA Banks J, Brown IH, Essen SC, Lee et al CW, (2004) Recombination resulting in virulence shift in avian influenza outbreak, Chile. Emerg Infect Dis 10:693–699. 6. Banks J, Speidel EC, McCauley JW, Alexander DJ (2000) Phylogenetic analysis of H7 haemagglutinin subtype influenza A viruses. Arch Virol 145:1047–1058. 7. Rohm C, Horimoto T, Kawaoka Y, Suss J, Webster RG (1995) Do hemagglutinin genes of highly pathogenic avian influenza viruses constitute unique phylogenetic lineages? Virology 209:664–670. 8. Li SQ, Orlich M, Rott R (1990) Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes. J Virol 64, 3297–3303. 9. Munster VJ, Wallensten A, Baas C, Rimmelzwaan GF, Schutten M, Olsen et al B, (2005) Mallards and highly pathogenic avian influenza ancestral viruses, northern Europe. Emerg Infect Dis 11:1545–1551. 10. World Organization for Animal Health/Food and Agriculture Organization of the United Nations. (2005) Recommendations of the second FAO/OIE regional meeting on Avian Influenza control in Asia OIE/FAO, Ho-Chi Min City 23-25/02/2005, 2005. Avilable at: http//www.fao.org/AG/AGAInfo/subjects/documents/si/AI_2nd_RegMtg_HoChiMinhCity_Rep.pdf. 11. Capua I, Terregino C, Cattoli G, Toffan A (2004) Increased resistance of vaccinated turkeys to experimental infection with an H7N3 low-pathogenicity avian influenza virus. Avian Pathol 33:158–163. 12. Van Der Goot JA, Koch G, De Jong MC, Van Boven M (2005) Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens. PNAS 102:18,141–18,146. 13. Ellis TM, Leung CY, Chow MK, Bissett LA, Wong W, Guan et al Y, (2004) Vaccination of chickens against H5N1 avian influenza

14. Avian Influenza Infections in Poultry

14.

15.

16.

17.

in the face of an outbreak interrupts virus transmission. Avian Pathol 33:405–412. Tumpey TM, Alvarez R, Swayne DE, Suarez DL (2005) Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus. J Clin Microbiol 43: 676 – 683 . Dundon W, Milani A, Cattoli G, Capua I (2006) Progressive truncation of the Non Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry. Protein appearance of serum antibodies against the avian influenza non structural 1 protein in experimentally infected chickens and turkeys. Virus Research 119:171–176. Tian G, Zhang S, Li Y, Bu Z, Liu P, Zhou J, Li C, Shi J, Yu et al K, (2005) Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics. Virology 341:153–162. Swayne DE, Beck JR, Kinney N (2000) Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine. Avian Dis 44:132–137.

129 18. Lee CW, Senne DA, Suarez DL (2004) Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol 78:8372–8381. 19. Naeem K, Siddique N (2005) Use of strategic vaccination for the control of avian influenza in pakistan. In Proceedings of OIE/ FAO International Scientific Conference on Avian Influenza, Paris (France), April 7–8, 2005. 20. CEC (2001) Commission Decision 2001/847/EC of 30 November amending for the third time decision 2000/721/EC to modify the Italian avian influenza vaccination programme and current trade restrictions for fresh meat originating from vaccinated turkeys. Official Journal of the European Commission 315:61–63.. 21. World Organization for Animal Health (2004) OIE International Conference on the Control of Infectious Aniaml Diseases by Vaccination. Buenos Aires, Argentina, April 14-17.2004. Available at: www.oie.int/eng/press/Rec_Concl_argentine_04.pdf 22. Capua I, Cattoli G, Marangon S (2004) DIVA—a vaccination strategy enabling the detection of field exposure to avian influenza. Development Biology (Basel) 119:229–233. 23. Marangon S, Capua I (2006) Control of AI in Italy: from stamping out to emergency and prophylactic vaccination. Development Biology (Basel) 124:109–115.

Chapter 25 Development of Immunodiagnostic Kits and Vaccines for Bacterial Infections Valentina A. Feodorova and Onega V. Ulianova

25.1

Introduction

In many respects, efficacy of epidemic surveillance of infectious diseases depends on the quality of diagnostic kits used for detection of pathogenic bacteria in clinical or environmental specimens. Opportune administration of effective vaccines, which significantly decrease human morbidity and mortality from numerous dangerous diseases, is also critical. Threat of bioterrorism, high risk of anthropogenic disasters, emergence of a number of causative agents of new spreading diseases affecting humans and animals, recurrence of “old” diseases such as anthrax and plague, and isolation of multi-antibiotic-resistant bacterial strains with atypical properties dictate the pressing need to improve the present methods and possible design of technological innovations in laboratory diagnostics and prophylaxis of infectious diseases. Although development and application of some known methods of molecular diagnostics have progressed in the past few years, ELISA, which is fast, simple, reproducible, and cheap, remains applicable and irreplaceable for every diagnostic laboratory. In this paper, some general principles of obtaining different kinds of antibodies (Abs) than the main active components of ELISA diagnostic kits will be presented, along with a discussion of the advantages or disadvantages of the Abs based on our recent studies using Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Vibrio cholerae 01 and 0139, Chlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae. Special emphasis will be given to possible vaccine strategies that rely on molecular understanding of plague immunogenesis and pathogenesis. The accumulated information can provide a powerful tool for monitoring infectious agents and combating dangerous bacterial diseases.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

25.1 Immunoreagents for Immunodiagnostic Kits The main reagent of an immunodiagnostic test-system is an Ab, which is highly specific to the corresponding antigen and can easily detect it in any simple or complex mixture of antigens, bacterial cells, clinical, or environmental specimens. Originally, more than 100 years ago, a standard immunological technique was developed to obtain polyclonal absorbed antisera from large animals (rabbit, horse, sheep). Such Abs were successfully used in a slide agglutination test and hemagglutination or immunofluorescent assays, but appeared to be of little use for highly sensitive immunoglobulin kits such as ELISA, dot-ELISA, and immunoblot. Subsequently, some advanced techniques for obtaining of other kinds of Abs were developed.

25.1.1 Polyclonal, Monoclonal, and Anti-idiotypic Abs 25.1.1.1

Polyclonal Abs (PAbs)

PAbs obtained after immunization of animal biomodels with a chemically isolated antigen have some advantages over conventional absorbed antisera (Table 25.1). They have been recently demonstrated when the PAbs to some V. cholerae antigens using both rabbit and murine biomodels were obtained (1–3). These PAbs were used for development of ELISA diagnostic kits for detection of V. cholerae 0139 (based on PAbs to V. cholerae 0139 lipopolysaccharides [LPS]) and V. cholerae 01 (based on PAbs to V. cholerae 01 Inaba or Ogawa LPSs) or for differentiation of V. cholerae 01 and non-01 (based on PAbs to V. cholerae 0139 lipid A). All the tests were highly specific and detected the strains of the relevant serogroup with a sensitivity of 9.4 × 104 – 7.5 × 105. They demonstrated no cross-reactions with any other bacteria, in contrast to the same tests based on the Abs to heatstable O-antigens (Table 25.2), in which some cross-reactive heat-stable proteins were found. 241

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Table 25.1. Comparative characteristics of different types of Abs Types of Abs PAbs

Advantages ● ● ●

● ●

Disadvantages

Oligospecificity No need to remove cross-reactive agglutinins High titers of specific homologous Abs in antisera of the immunized animals Smaller quantity needed in an assay Cost lower than that of antisera to heat-stable antigens

●

●

●

● ● ●

MAbs

●

● ● ●

●

●

●

Anti-Id Abs

●

●

●

●

●

● ●

Recognize a single epitope of high diagnostic value of any bacterial antigen No need of purified antigen for development of MAbs No need of absorption Application of original methods of screening allows development of MAbs with desired specificity Can be obtained in a limited amount from an immortal hybridoma All of Ab molecules are identical in structure (i.e., isotype, antigen-binding properties including affinity, avidity, etc.) Once generated, a hybridoma can secrete specific immunoglobulins continuously, providing an everlasting source of MAbs in vivo or in vitro

●

Can be obtained to any antigen, a single epitope/receptor, haptens, etc., when it is difficult or impossible to purify them Suitable for development of immunodiagnostic kits for detection of Abs to any poorly immunogenic and low-weight substances, haptens, viruses, etc., or Abs against them Possess all immunochemical, immunobiological, and antigen-binding properties of original antigen Can be obtained in unlimited amount from antisera or ascitic fluids of biomodels Highly homogenous due to application of monospecific polyclonal or absolutely specific monoclonal Abs in contrast to different series of antigens isolated by same chemical method Good sensibilizing activity because of protein nature Suitable for development of any modifications of highly sensitive immunodiagnostic kits (direct, indirect, sandwich, competitive, etc.)

●

●

●

●

●

● ●

●

Each antiserum is different from all other antisera, even if raised in a genetically identical animal using the identical preparation of antigen and same immunization schedule Produced in limited volumes insufficient for large-scale experiments or clinical tests Long-term immunization of animals needed to induce high titers of specific Abs Permanent need for preparation of antigen for immunization Unexpected cross-reactions Time-consuming and expensive Need for expensive equipment, reagents, and highly qualified personnel to obtain and maintain hybridomas Long and technically difficult generation procedure with unpredictable results Difficulties of quick generation of MAbs suitable for detection of novel pathogens because of the need for well-characterized antigen for screening Because a MAb is directed to a single epitope, its application for detection of bacterial pathogens is limited because of frequent origination of microorganisms with atypical properties, i.e., multidrug-resistant ones, with different plasmid composition, etc., resulting in changes of the epitope composition of their antigens Costs for generation of MAbs cannot be recovered in case of small-scale production of test-systems Difficult to devise/develop optimal immunization schedule to induce Ab2b, bearing the “innate image” of the antigen itself Limited volume of series of anti-Id Abs obtained Necessity to use inbred animal models only to exclude any removal cross-reactive anti-Id Abs of other kinds (Ab2a, Ab2g, Ab2e) Limited application for diagnostic kits for detection of specific Abs in antisera of patients with chronic and other diseases accompanied generation and persistence of Ab1 in a body for a long time

Table 25.2. Comparative specificity of ELISA based on PAbs to LPSs, heat-stable O-antigens, or lipid A of V. cholerae O1 and O139 Percentage of positive reactions with PAbs to the antigens of V. cholerae (%)

Bacterial strains V. cholerae O1 V. cholerae non-O1 V. cholerae O139 V. cholerae O22 Enterobacteriaceae

O139

O1

Number of strains

LPS

Heat-stable O-antigen

Lipid A

LPS

Heat-stable O-antigen

36

0

40

0

100

100

14

0

75

30

0

35

22

100

100

100

0

50

1

0

100

0

0

100

29

0

15

10

0

20

25.1.1.2

Monoclonal Abs (MAbs)

MAbs with a single and known specificity and homogeneous structure are generated using a hybridoma technique developed in 1975 by Köhler and Milstein (4). MAbs have many advantages over PAbs (Table 25.1) and are now used as diagnostic probes in most serological assays and as therapeutic and urgent prophylactic agents. For this, MAbs to genus-, species-, and serogroup-specific epitopes of the surface-located bacterial antigens are selected. Combinations of different screening systems based on Ab specificity in ELISA, dot-ELISA, and immunofluorescence test (IFT), or on characteristic immunobiological activities of the antigens can be used. MAbs to Y. pestis Pla were selected in IFT and dot-ELISA with

25. Immunodiagnostic Kits and Vaccines

a panel of 114 wild and recombinant Y. pestis pPst + or pPst – strains cultured at 28 and 37°C and in reaction of inhibition of fibrinolytic and coagulase activities. This resulted in a panel of the MAbs with unique desirable properties suitable for detection and identification of the pathogenic Yersinia strains independently of cultivation temperature by means of the developed kits. These monoclonal test systems have great advantages over monoclonal diagnostic kits based on the MAb to Y. pestis F1 in which wild virulent Y. pestis F1 – strains cannot be detected as well as Y. pestis F1+ cultured at lower temperatures (5) . Applicability and value of a similar test system for diagnostics of human pneumonic plague were recently confirmed (6). MAb to specific Y. pestis LPS epitope was successfully used for detection of Y. pestis strains with different plasmid composition including plasmid-less ones (7) . Among the rest of the MAbs obtained, the great promise for immunodiagnostic kits demonstrated the MAbs to Y. pseudotuberculosis, Y. enterocolitica, and V. cholerae 01 based on production of species- and serogroup-specific heat-stable proteins ( 2, 8, 9 ). Another application of the MAbs was their use in different ELISA modifications for investigation of molecular organization of the antigens up to a single epitope in immunobiological studies. This revealed bifunctional properties of Y. pestis Pla, newly identified genes involved in Y. pestis F1 biosynthesis, to develop a basis for a possible new classification of Y. pseudotuberculosis , Y. enterocolitica, and V. cholerae based on production of species- and serogroup-specific heatstable proteins. The MAbs were used as effective immunobiological tools for studying molecular mechanisms of intracellular and extracellular resistance to pathogenic Yersinia or plague pathogenesis (2, 8 , 10– 13).

25.1.1.3

Anti-idiotypic (Anti-Id) Abs

Anti-Id Abs are also of great importance for the development of immunodiagnostic kits. They are obtained by immunization of biomodels by monospecific Abs (that is, Abs to a single antigen [14] ), or MAbs specific for the antigen or its epitope. Anti-Id Abs are the precise protein copy of the antigen itself bearing a so-called “innate image” of the original antigen (15). They have many advantages over the other kinds of Abs (Table 25.1) for immunodiagnostic kits. Some of these advantages were demonstrated when the diagnostic value of the anti-Id Ab to Y. pestis LPS was compared with the antigen itself in experimental ELISA with antisera from volunteers once or repeatedly immunized with a live plague vaccine EV line NIIEG (16) . No difference in the amount of positive and negative reactions was registered. The anti-Id Abs against biovar-specific V. cholerae O1 epitopes, which have not been yet isolated and characterized, were also generated. The ELISA based on the anti-Id Abs will be useful for identification and differentiation of V. cholerae strains of the relevant biovar (17).

243

25.1.2 Abs With Desired Specificity Obtained Using Other Immunological Approaches (Directed Immunogenesis) Some well-known basic principles of induction of adaptive immune response and immunological tolerance to the most basic nonspecific antigens of a bacterial cell with Ab production only to specific ones can serve a basis for significant improvement and simplification of a technology for obtaining of strictly specific Abs. Currently, two main methods to obtain these Abs (D-Abs) through induction of an immunological tolerance to nonspecific antigens/epitopes have been elaborated.

25.1.2.1 The Use of Inbred Biomodels With Certain Genotypes Providing Induction of Abs to Some Desired Antigens/Epitopes of a Complex Immunizing Agent and Tolerance to the Others This approach was first used for induction of species-specific Abs to N. gonorrhoeae, T. vaginalis, and C. trachomatis (18, 19). BALB/c mice, which are responsive to carbohydrate antigens and tolerant to proteins, were used. In all cases, suspensions of killed bacteria or group-specific chlamydial glycoprotein antigen were used for immunization. This resulted in D-Abs to the C. trachomatis LPS-specific molecule components recognized in ELISA, the relevant homologous antigens or bacteria in specimens of patients with skin and sexually transmitted diseases (20). In comparison with commercial immunodiagnostic kits based on PAbs or MAbs (ELISA, hemagglutination, or immunofluorescent tests) and polymerase chain reaction for C. trachomatis, our test systems based on D-Abs in each case demonstrated higher specificity and sensitivity (Table 25.3).

25.1.2.2 Inoculation of Cross-Reactive Immunizing Agents to Newborn Mice Following Injection of the Antigen to 6- to 8-Week-Old Mice It is known that the immune system of newborn mice recognizes the majority of inoculated antigens as their own, but not foreign ones (15). If the complex antigen injected in adult mice (6 to 8 weeks and older) contains the epitopes used for treatment of the animals in the neonatal period, the relevant epitopes will not be reactive for the immune system. Thus, the Ab response will be induced to all the other antigenic determinants but not those previously inoculated in the newborn mice. This phenomenon was used to obtain the murine specific Abs against Y. pseudotuberculosis II, III, and IV serogroups (14). Newborn mice were immunized according to optimal schedules by killed bacterial cells of Salmonella typhi, Salmonella paratyphi, and Y. pestis, which are usually used for absorption of rabbit pseudotuberculosis antisera to remove cross-reactive agglutinins with the injection of Y. pseudotuberculosis cells of a certain serogroup 6 to 8 weeks later (14) All the D-Abs used for ELISA kits demonstrated high specificity, detected the Y. pseudotuberculosis strains of the relevant serogroup, and gave no cross-reactions with the bacteria of other serogroups used in the analyses.

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V. A. Feodorova and O. V. Ulianova Table 25.3. Comparative efficacy of immunofluorescent test, PCR, and dot-ELISA for detection of C. trachomatis in clinical specimens from patients with suspected diagnosis of urogenital chlamidiosis Reaction in Immunofluorescent test Positive

PCR

Negative

Positive

Dot-ELISA Negative

Positive

Negative

NS* % M ± m

NS* % M ± m

NS* % M ± m

NS* % M ± m

NS* % M ± m

NS* % M ± m

86

9

28

67

86

9

9.5 ± 3.4

9.5 ± 3.4

29.8 ± 4.7

70.5 ± 4.7

90.5 ± 3.4

9.5 ± 3.4

NS*, number of specimens; PCR, polymerase chain reaction.

In summary, advantages of the D-Abs obtained by directed immunogenesis are: significant decrease of the amount of antigens—up to a few µg for the first immunization of newborn mice and 50 to 100 µg for the following 1 to 2 injections for 6- to 8-week-old mice ● significant reduction of the immunization schedule—up to 8 to 10 weeks (a single inoculation in the neonatal period and 1 to 2 injections 6 to 8 weeks later) ● high specificity of the Abs due to directed Ab response to the specific antigens ● no need to remove cross-reactive Abs ● complex rather than purified antigens and bacterial suspensions are suitable for induction of Abs with desirable specificity ● suitability for very quick development of diagnostic kits for laboratory diagnosis of newly emerging dangerous diseases caused by bacterial agents with a weakly studied antigenic structure ● significant decrease of the cost of the Ab-based diagnostic kits ●

25.1.3 Murine Immune Ascitic Fluids as a Source for Obtaining Different Kinds of Abs To obtain specific Abs in large amounts sufficient for development of immunodiagnostic kits, a convenient approach was developed in detail (1–3, 11, 12, 14, 17–20). It is based on the use of small inbred biomodels (usually BALB/c mice) immunized by any bacterial antigenic substances (antigens, bacterial cells, etc.) or Abs, followed by inoculation of syngenic myeloma cell lines inducing a production of ascitic fluids containing specific Abs. In this way, all of the abovementioned types of Abs can be produced. The advantages of the Abs from murine immune ascitic fluids are: small amounts of antigens (not more 150 to 200 µg per mouse for a cycle) are required to get high titers of specific Abs ● quick production of sufficient amount of Abs (45 to 60 days from the first immunization) ● one mouse yields from 5 to 20 or 25 mL of ascitic fluid (containing about 10 to 15 mg/mL of specific Abs) in case of repeated collections ●

1 mL of the ascitic fluid or purified Abs is sufficient for thousands of assays ● because the products are monospecific (i.e., they contain Abs to the only one antigen epitope), they do not need any absorption ● Abs from each inbred mouse are the same homogeneity, high specificity, and affinity allow use of the Abs in highly sensitive immunodiagnostic kits such as ELISA, dot-ELISA, and immunoblot ● use of small animals in contrast to large ones (horses, sheep, rabbits) during a relatively short period (1 to 2 months instead of 3 to 5 years) leads to a significant decrease in cost of the diagnostic kits based on the Abs ●

25.2 Prospects for New Vaccine Development An important question is whether an ideal effective vaccine can be developed, and how? In other words, how could the human immune system be manipulated to achieve the highest level of immunity? Current knowledge about the main requirements for an effective vaccine can be summarized as follows. An ideal effective vaccine must: be protective, that is, induce high and long-term immunity against a disease, preferably after a single inoculation ● lack the need for regular “booster” vaccinations ● be safe; it must not cause illness or death ● induce neutralizing Abs ● provide immediate neutralization of bacteria by specific Abs immediately after entering a body ● induce the relevant type of immune response (mucosal, cutaneous, etc.) ● be free of side effects ● induce a vigorous protective immunity to only correct protective antigens or their epitopes ● lack harmfully high levels of induced Abs ● suppress unwanted immune response (autoimmunity, allergy) ● be applicable both pre-exposure and in emergent cases, and including combination with antibiotics ● confer immunity against a single or several infectious diseases ●

25. Immunodiagnostic Kits and Vaccines

245

Clearly, realization of these requirements must be based on a detailed understanding of microbial pathogenicity, analysis of the protective host response to pathogenic organisms, and molecular mechanisms of resistance of bacteria to evade or overcome the host immune system and to establish infection (21, 22). Some new rational approaches that look promising enough for application to induce enhanced immunity against other bacterial infections are being considered using the plague model, because there are no effective licensed vaccines except the live plague attenuated vaccine EV line NIIEG (16).

25.2.1 Vaccines Against Pathogens With Extracellular and Intracellular Life Cycles To induce an enhanced immunity to experimental plague, the behavior of Y. pestis bacteria of vaccine and virulent strains in in vitro conditions simulating the main stages of their interactions with susceptible mammals were studied in detail. Two main types of resistance to phagocytosis after entry into a body were found in Y. pestis (Table 25.4). One of them, intracellular type (capsular-dependent), is developed during the first hours after inoculation of bacteria grown at the temperature of the plague vector, when they are recognized and captured by phagocytes (23). This type provides protection of the bacteria from the phagocyte killing systems. It is expressed by the pLCR-encoded proteins (Yersinia outer membrane proteins [Yops]) produced mainly in intracellular conditions, although these substances can be detected on the bacterial surface in small amounts as receptors or protein precursors outside a phagocyte as well (12, 24–26). Thus, the bacteria inside a phagocyte are not fully available for the Abs to the majority of these antigens, which cannot sufficiently neutralize plague bacteria in extracellular conditions. Probably, pLCR-encoded proteins demonstrated low protective properties (12) and limited promise for vaccine

development except LcrV, which was reported to provide active immunity in mice (27). However, some Yops can be processed and presented with generation of antigen-presenting cells and induction of the relevant Abs because specific anti-LcrV or anti-Yops Abs were detected in antisera of mice treated with different Y. pestis strains or Yop-containing preparation (12, 28, 29). The other type of resistance, extracellular type (capsular-independent one), is developed in vivo some hours after bacteria have entered a body or, in case of transmission from ill human or animal to human, inoculation of the microbes cultured at host temperature. It allows the survival of Y. pestis bacteria outside phagocytic cells, extensive extracellular multiplication, and rapid spread into the bloodstream (10). This is accompanied by suppression of the normal inflammatory response (28, 30) due to modification in production of Y. pestis antigens and limited antigens synthesized extracellularly (10). These antigens used for immunization induced a good immunity to experimental plague (about 90% of survivors) because of their high neutralizing activity, significantly reducing the level of the Abs induced by about 2,000 times. Importantly, induction of Abs to the extracellularly produced antigens can be achieved through pre-cultivation of some vaccine strains without any chemical purification procedure, which sometimes provides alteration of antigenic and protective activity. Indeed, immunization of biomodels with the killed bacteria pre-cultured at host temperature or in the presence of an adequate concentration of calcium has allowed enhancement of immunity to experimental plague due to direct immune response to these antigens, which cannot be synthesized when the bacteria are grown in routine bacterial media (31). There is, then, a simple method of enhancing immunogeneity of some known vaccine strains by pre-cultivation in conditions simulating the extracellular

Table 25.4. Characteristic features of intracellular and extracellular types of resistance to phagocytosis in Y. pestis Intracellular type Develops during the first 2 to 3 hours after inoculation of bacteria grown at 28°C (a temperature of flea “plague vector”) ● Bacteria are recognized and captured by phagocytes ● Enables their survival inside phagocytic cells ● Expressed by the yop regulon or pLcr-encoded proteins (Yersinia outer membrane proteins [Yops]) produced in only intracellular conditions (20 mM Mg2+, 37°C) ● Yops are not produced by the bacteria extracellularly in sufficient amounts ● Abs to some Yops are weakly or not at all protective because they cannot neutralize plague bacteria outside a phagocyte ● Yops possess antigenic activity inducing detectable level of specific Abs in antisera of vaccinees or convalescents ●

Extracellular type ●

● ● ● ●

● ●

●

●

Developed by the microbes in vivo 2 to 3 hours after entering a body or inoculation of the bacteria cultured at 37°C (host temperature) Developed independently of bacterium–host phagocyte interaction Bacteria are not recognized and captured by phagocytes Accompanied by downregulation of species-specific epitopes Accompanied by unmasking of cross-reactive antigens or epitopes that mimic some human tissues and blood components No immune response in and inflammation at site of infection at early stage Enables survival of bacteria outside phagocytic cells, extensive extracellular multiplication, and spread into circulation Limited number of antigens synthesized extracellularly, which are neutralized by protective Abs Induces a good immunity to experimental plague (about 90% of survivors) followed by significant reducing of the level of Abs induced

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V. A. Feodorova and O. V. Ulianova

25.2.2 Vaccines With Decreased Reactogenicity and Increased Immunogeneity

a

1200 1000

Specific titre

environment. This approach can be suitable for the majority of pathogenic bacteria. It is safe, provides induction of neutralizing Abs to the limited amount of correct antigens that partially reduce some side effects, and blocks bacterial dissemination in a body. Further, detailed characterization of the antigens makes it possible to develop new and effective chemical, subunit, or recombinant vaccines because the availability of complete bacterial genome sequences.

25.2.2.1 Organisms With Mutation in the Genes of Lipid A: Toxic Part of Bacterial LPS

25.2.2.2

Anti-idiotypic Vaccines

An alternative approach to development of not reactogenic antigen-free vaccine is based on the use of anti-Id Ab bearing the “innate image” of a complementary determining region of the protective epitope of the antigen (15). The advantages of anti-idiotypic vaccines are: immunoglobulin nature no reversion to virulence ● no material of the pathogen ● antigen-free ● directed immune response to the protective epitope ● reduction of unwanted immune response against non-protective or toxic components of an antigen or a bacterial cell ● identical immunobiological properties to the original antigen ● no need of the antigen purification ● can be obtained using specific monoclonal Ab to any antigen including those poorly presented on the bacterial surface ● induce remarkable immune response ● can be obtained in unlimited amounts ● ●

Several anti-Id Ab vaccines against some protective proteins encoded by Y. pestis pLCR expressed on the bacterial surface extracellularly in a small amount were constructed (12, 33). In preliminary results, the relevant MAbs were

600 400 200

IV III II

0 0

5

6

I

7

8

9

10

Time of taking broth culture samples, h

b

1200 1000

Specific titre

The most important problem of the majority of vaccine strains is marked local and systemic reactions in vaccinees. These reactions are relatively common and usually well known, and they are described in detail for licensed vaccines. High level of their reactogenicity is conditioned by toxic activity of the LPS molecule. The question remains: Is it possible to reduce the relevant side effects and, if so, how will this influence the immunity? For this, some Y. pestis mutants with a deletion-insertion in the lpxM gene in the attenuated strain Y. pestis EV line NIIEG have been constructed (32). The mutants cannot synthesize the most toxic hexa-acylated lipid A molecule. Instead, they produce a penta-acylated lipid A lacking a single fatty acyl chain C12 group when grown at 25°C. The DlpxM mutation caused a significant increase of the protective efficacy and marked decrease of reactogenicity of the mutants.

800

800 600 400 200

IV III

0 5

II 6

7

8

I 9

10

Time of taking broth culture samples, h Figure 25.1. Changes of specific activity of O-antigen in the native samples of broth culture during apparatus cultivation of the V. cholerae O1 strains M-41 (a) and 569B (b) in the dot-ELISA with: I—a diagnostic absorbed cholera antiserum Inaba; II—a diagnostic absorbed cholera antiserum Ogawa; III—murine Abs to V. cholerae Inaba Oantigen; IV—murine Abs to V. cholerae Ogawa O-antigen (See Color Plates).

obtained (12). The relevant F(ab’)2 fragments were isolated and used for immunization of mice. Complementarity of the anti-Id Abs to the antigens themselves was confirmed using all main criteria—immunochemical, functional, immunological, and immunobiological. Comparative analysis of immunogeneity of the anti-Id Ab vaccines demonstrated their capability to induce an enhanced immunity against experimental plague (33).

25. Immunodiagnostic Kits and Vaccines

247

a 140

Specific titer

120 100 80 60 40

B10B2 B8B2 A8G3 MAb A2G3

20 0 5

6

7

8

9

Time of taking broth culture samples, h

b 300

Specific titer

250

(34), have been developed. They were compared with commercial agglutination tests based on absorbed cholera serotype-specific antisera in the dot-ELISA with the use of the similar absorbed reagents. Polyclonal and monoclonal ELISA kits were more effective during all main stages of production of the vaccine and allowed early (in the first hours), quick, and precise detection of antigens in native specimens of broth cultures, semifinished products, and ready-made drug of the vaccine (2, 35). A significant difference in antigen production between two vaccine V. cholerae strains was found, which was not detected when absorbed antiserum was used. Thus, similar reactions in ELISA by both Inaba and Ogawa antisera with V. cholerae Inaba strain were registered (Figure 25.1). Application of a panel of MAbs to species- and serotype-specific cholera epitopes gave more precise information about peculiarities of production of protective epitopes of the antigens (Figure 25.2). These data, rapidly obtained during the manufacturing process, allow correction of cultivation conditions of the vaccine strains for optimal biosynthesis of the protective antigens. Such a versatile monitoring system will finally lead to improvement of the quality of the chemical cholera vaccine.

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Time of taking broth culture samples, h Figure 25.2. Changes of specific titers of immunoreactive epitopes of the V. cholerae O-antigen during apparatus cultivation of the V. cholerae O1 strains M-41 (a) and 569B (b) in dot-ELISA with MAbs (See Color Plates).

25.3 Methods for the Control of Biosynthesis of Protective Antigens for Vaccines During Their Manufacture Generally, the main aim of apparatus cultivation of vaccine strains is to obtain the largest yield of bacterial cells—a source of live, killed, or chemical vaccines. It is very important to optimize the cultivation conditions for adequate production of the protective antigens up to a single protective epitope. Effective methods for control of immunoreactivity of the desired antigens play a crucial role. Indeed, some dot-ELISA kits based on PAbs and MAbs to V. cholerae O-antigens Inaba and Ogawa, which are necessary components of the cholera bivalent chemical tableted vaccine manufactured in Russia

Acknowledgments. We thank Drs. NY Teryoshkina, NA Syrova, AB Golova, LN Pan’kina, EP Savostina, SV Dentovskaya, and RZ Shaikhutdinova for active participation in the experiments and help in preparing of the manuscript; and Drs. AP Anisimov and LV Sayapina and Professors ZL Devdariani, YA Popov, and IA Dyatlov for helpful discussion. This work was supported by grants (No. 04-04-48279a, 01-04-48048, 02-04-06637, and 03-04-48067) from the Russian Foundation for Basic Research.

References 1. Feodorova VA, Gromova OV, Devdariani ZL, Dzhaparidze MN, Teryoshkina NY (2001) Immunochemical characterisation of Vibrio cholerae O139 O antigens and production of a diagnostic antiserum without absorption. J Med Microbiol 50:499–508. 2. Syrova NA, Feodorova VA, Dyatlov IA (2005) Developement of different ELISA kits for control of biosynthesis of the O-antigen in tableted bivalent chemical cholera vaccine and its semifinished products. In Panorama (Onischenko GG, Kutyrev VV, Alekseev VV, eds.), pp. 295–297. Russian Federation, Volgograd. 3. Feodorova VA (2004) Theoretical and experimental aspects of studying of protein and carbohydrate antigens of Yersinia pestis and Vibrio cholerae O139 with the use of polyclonal and monoclonal antibodies. D.Sci. Thesis. Russia State Research Antiplague Institute ‘Microbe’, Saratov, Russia. 4. Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497. 5. Feodorova VA, Devdariani ZL (2000) Development, characterisation and diagnostic application of monoclonal antibodies against Yersinia pestis fibrinolysin and coagulase. J Med Microbiol 49:261–269.

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6. Mahesh S, Shukla J, Tuteja U, Batra HV (2005) Molecular detection of Yersinia pestis isolates of Indian origin by using Pla specific monoclonal antibodies. Comp Immunol Microbiol Infect Dis 28:131–144. 7. Devdariani ZL, Verenkov MS, Feodorova VA, Solodovnikov NS, Belov LG (1993) Identification of Yersinia pestis with varied plasmid composition using monoclonal and polyclonal fluorescent immunoglobulins. FEMS Immunol Med Microbiol 6:31–36. 8. Feodorova VA, Samelija JG, Devdariani ZL (2003) Heat-stable serogroup-specific proteins of Yersinia pseudotuberculosis. J Med Microbiol 52:389–395. 9. Utkin DV, Devdariani ZL, Feodorova VA, Drozdov IG (2004) Development and characteristics of the monoclonal antibodies to some serogroup-specific epitopes of Yersinia enterocolitica serogroup. Biotechnology 1:91–96. 10. Feodorova VA, Golova AB (2005) Antigenic and phenotypic modifications of Yersinia pestis under calcium and glucose concentrations simulating the mammalian bloodstream environment. J Med Microbiol 54:435–441. 11. Feodorova VA, Devdariani ZL (2001) New genes involved in Yersinia pestis Fraction I biosynthesis. J Med Microbiol 50:969–978. 12. Feodorova VA, Devdariani ZL (2001) Immunogeneity and structural organisation of some pLCR-coded proteins of Yersinia pestis. J Med Microbiol 50:13–22. 13. Feodorova VA, Devdariani ZL (2002) The interaction of Yersinia pestis with erythrocytes. J Med Microbiol 51:150–158. 14. Feodorova VA, Samelija JG, Devdariani ZL (2003) Development of a method for obtaining of the Yersinia pseudotuberculosis species- and serogroup-specific antibodies using the directed immunogenesis. Biotechnology 3:49–57. 15. Janeway CA, Travers P, Shlomchik M, Walport M (eds.) (1997) Immunobiology. The Immune System in Health and Disease. Current Biol. Ltd., London, England. 16. Feodorova VA, Devdariani ZL (2006) Development of an experimental immunoglobulin test-system based on anti-idiotypic immunoglobulins for the determination of specific plague antibodies in the sera of persons inoculated with live plague EV NIIEG. Klin Lab Diagn 4:54–56. 17. Feodorova VA, Devdariani ZL, Syrova NA, et al (2005) Antiidiotypic antibodies to Vibrio cholerae O1 biovar-specific epitopes. In Diagnotics, Treatment and Prophylaxis of Dangerous Diseases. Biotechnology. Veterinary (Pimenov EV, Darmov IV, eds.) pp. 76–77. NIIM MO RF, Russian Federation, Kirov. 18. Feodorova VA, Bannikova VA, Eliseev Y, Grashkin VA (2005) Obtaining of preparative amount of murine diagnostic anti-Chlamydia antibodies using direct immunogenesis. Biotechnology 2:82–90. 19. Grashkin VA (2000) Efficacy of application of different ELISA test-systems for diagnostics of gonorrhoeae and trichomoniasis. Russia State Research Anti-plague Institute ‘Microbe’, Saratov, Russia. 20. Feodorova VA, Bannikova VA, Alikberov ShA, Eliseev Y, Grashkin VA (2007) Comparative efficiency of detection of urogenital chlamydiosis agent by immunofluorescent, PCR and dot-ELISA methods. Klin Lab Diagn 7:30–37.

V. A. Feodorova and O. V. Ulianova 21. Beukema EL, Brown MP, Hayball JD (2006) The potential role of fowlpox virus in rational vaccine design. Expert Rev Vaccines 5:565–577. 22. Keitel WA (2006) Recombinant protective antigen 102 (rPA102): profile of a second-generation anthrax vaccine. Expert Rev Vaccines 5:417–430. 23. Feodorova VA, Petrova AV, Motin VL, (2006) Comparative study of growth of Yersinia pestis and Yersinia pseudotuberculosis at in vitro conditions simulating the phagolysosomal environment. Proceedings of the 9th International Symposium on Yersinia, Lexington, KY, pp. 56–57. 24. Bolin I, Forsberg A, Norlander L, Skurnik M, Wolf-Watz H (1988) Identification and mapping of the temperature - inducible, plasmid-encoded proteins of Yersinia spp. Infect Immun 56:343–348. 25. Feodorova VA, Petrova AV, Devdariani ZL (2005) Influence of cultivation conditions on the expression of Yersinia pestis YopE. Zh Mikrobiol Epidemiol Immunobiol 4:3–7. 26. Charnetzky WT, Shuford WW (1985) Survival and growth of Yersinia pestis within macrophages and an effect of the loss of the 47-megadalton plasmid on growth in macrophages. Infect Immun 47:234–241. 27. Motin VL, Nedialkov YA, Brubaker RR (1996) V antigen-polyhistidine fusion peptide: binding to LcrH and active immunity against plague. Infect Immun 64:4313–4318. 28. Braciale VL, Nash M, Sinha N, Zudina VL, Motin VL (2007) Correlates of immunity elicited by live Yersinia pestis vaccine. In NIAID: frontiers of Research (Georgiev V st, ed.). Humana Press, Totowa, NJ. 29. Benner GE, Andrews GP, Byrne WR, et al (1999) Immune response to Yersinia outer proteins and other Yersinia pestis antigens after experimental plague infection in mice. Infect Immun 67:1922–1928. 30. Nakajima R, Motin VL, Brubaker RR (1995) Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization. Infect Immun 63:3021–3029. 31. Feodorova VA, Ulianova OV (in press) Identification and protective activity of the antigens produced by Yersinia pestis in the conditions simulating mammalian extracellular environment. J Med Microbiol . 32. Anisimov AP, Shaikhutdinova RZ, Pan’kina LN, et al (2007) Effect of deletion of the lpxM gene in Yersinia pestis on lipopolysaccharide structure, virulence, and vaccine efficacy of a live attenuated strain. J Med Microbiol 56:443–453. 33. Feodorova VA, Devdariani ZL (2006) Prospects of development of experimental anti-idiotypic vaccines against plague. Immunology 27:144–148. 34. Dzhaparidze MN, Naumov AV, Nikitina GP, et al (1991) Chemical vaccine, prepared from Vibrio cholerae hypertoxigenic strains KM-76 Inaba and KM-68 Ogawa, for oral administration. Zh Mikrobiol Epidemiol Immunobiol 4:31–33. 35. Fedorova VA, Syrova NA, Gromova OV, et al (2000) Synthesis of protective antigens during submerged cultivation of Vibrio cholerae. Zh Mikrobiol Epidemiol Immunobiol 6:72–74.

Chapter 16 Development of Prophylactics and Therapeutics Against the Smallpox and Monkeypox Biothreat Agents Mark Buller, Lauren Handley, and Scott Parker

16.1

Introduction

With the global eradication of smallpox in 1979, the causative agent, variola virus (VARV), no longer circulates in human populations; however, there is concern that clandestine stocks of VARV exist. The reintroduction of aerosolized VARV (or perhaps monkeypox virus [MPXV]) into human populations would result in high levels of mortality. The attractiveness of VARV as a bioweapon, and to a certain extent MPXV, is its inherent ability to spread from person-to-person. A natural threat also exists, as cases of human monkeypox are increasing in Africa, and for the first time human monkeypox was observed in the Western hemisphere during an outbreak in 2003. The threat posed by the intentional release of VARV or MPXV, or an MPXV epizoonosis will require a capacity to rapidly diagnose the disease and intervene therapeutically with antivirals. Intervention is likely to take place during the diagnosis of the first wave of cases approximately 10 to 15 days post-infection (p.i.). Preimmunization of “at-risk populations” with vaccines will likely not be practical, and circumstacial evidence suggests that the therapeutic use of vaccines is ineffective 4 days p.i. with VARV. Instead, immunization will be used to prevent further spread of the pathogen by treating at-risk populations. This chapter describes this threat and the processes for development of new orthopoxvirus vaccines and antivirals to meet it.

16.2

Human Poxvirus Diseases

Ten poxviruses have been documented to infect humans. All poxvirus diseases are zoonoses except for molluscum contagiosum virus (MCV) and VARV (1). With rare exception, these zoonotic poxviruses fail to establish a human chain of transmission. Most human poxvirus infections occur through minor From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

abrasions in the skin. MPXV (orthopoxvirus), orf (parapoxvirus), and MCV cause the most frequent human poxvirus infections worldwide. In addition to MPXV and VARV, two other orthopoxviruses can cause human infections: vaccinia virus (VACV) and cowpox virus (CPXV). Attenuated VACV is used as a vaccine for orthopoxvirus, and is currently used to immunize military recruits and laboratory workers. Natural human infections with VACV are limited to individuals exposed to VACV and a vaccinia-like virus that infects buffalos and dairy cattle in Asia and Africa (buffalopox virus) and cattle in Brazil (Cantagalo virus; refs. 2 and 3). As a vaccine, VACV is not completely avirulent and has caused a large number of vaccine-related complications. CPXV infections have also been acquired zoonotically from cows and rodents, but the domestic cat is responsible for the majority of human CPXV infections. Between 1969 and 1993 there were approximately 45 human CPXV cases in Britain, 3 published case histories from Germany, and 2 each from Belgium, Sweden, and France (4). Human CPXV infections are acquired through the introduction of the virus into minor abrasions in the skin, usually directly from an animal source. VACV and CPXV cause limited numbers of infections with low mortality. Conversely, unlike the relatively benign symptoms experienced following VACV and CPXV infections, MPXV and VARV have the potential to cause mortality and severe morbidity in humans.

16.2.1

Smallpox

16.2.1.1

History

Smallpox was named to differentiate it from great-pox now known as syphilis (5). Smallpox is estimated to have infected approximately 400 million people in the 20th century alone. Historically, smallpox has had a close association with humans. The origin of VARV remains unknown, but the dubious accolade probably goes to Egypt or India. Before the 15th century, smallpox was generally confined to the Eurasian landmass. However, European colonialists introduced smallpox to the Americas, central and southern Africa, and Australia 145

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between the 15th and 18th centuries with devastating consequences, as indigenous populations were decimated with case fatality rates approaching 90%. Smallpox enabled a handful of conquistadors, such as Cortez and Pizarro, to subjugate large parts of Central and South America to Spanish rule, thereby permanently altering the future of these regions. This was not an isolated pattern. By the end of the 19th century, a milder and less lethal form of smallpox became apparent. This virus was named variola minor to distinguish it from variola major (classic smallpox). It was first documented in South Africa during 1904, but it had been clinically apparent in the United States since 1896. Originally described as Amass (alastrim in South America), this virus eventually became recognized in Brazil during the 1960s and in Botswana, Ethiopia, and Somalia during the 1970s. It is believed to have originated in several places throughout the globe as the virus adapted to humans.

16.2.1.2

Clinical Disease

The case-fatality rates for variola major (classic or ordinary smallpox) were 16 to 30% and 1% for variola minor. Clinically, smallpox in an unvaccinated person has a 7- to 19-day incubation period from the time infection is established within the respiratory tract until the first symptoms of fever, malaise, headache and backache occur, culminating in the start of the characteristic rash (Figure 16.1; refs. 5 and 6). The rash starts with papules that sequentially transform into vesicles and then pustules; a majority of these lesions are located on the head and limbs (often confluent) compared to the trunk. The rash is typically centrifugal (head and limbs), but centripetal (trunk)

rashes have been reported. Lesions range from 0.5 to 1 cm in diameter and can spread over the entire body. Once pustules have dried, scabs will form that eventually desquamate during the following 2- to 3-week period. The resultant feature of these cutaneous lesions is the formation of the classic pock scars, which are apparent on the skin of surviving patients. Two clinical variations of classic smallpox have been identified. Flat-type smallpox is a rare form of the disease (about 6% in unvaccinated people) and it is characterized by lesions that remain level with the skin. It was more frequently observed in children and usually resulted in death. Another variation of the disease is hemorrhagic smallpox (<2% in unvaccinated people), which occurred mainly in adults. Although a rare form of the disease, it also had a high mortality rate and is characterized by hemorrhages into the skin and/or mucous membranes early in the course of illness. Subconjuctival hemorrhages were most common as well as bleeding from the gums and other parts of the body.

16.2.1.3

Person-to-Person Transmission

As shown in Figure 16.1, the peak period of infectiousness is shortly after the onset of rash. The transmission of virus from person to person and spread through the body is diagramed in greater detail (Figure 16.2). The relative infectiousness of variola major and variola minor as measured by secondary attack rates was determined to be 58 and 61%, respectively (5). Because variola minor is characterized with smaller and fewer lesions that evolved more rapidly, the oropharyngeal secretions of persons with variola minor likely contained less

Figure 16.1. Clinical manifestations of smallpox. Panel A shows the initial phases of infection and the clinical manifestations, which include temperature spikes, the development of skin lesions, and period of infectiousness. Reproduced with permission from ref. 133 and Dr. David Heymann, World Health Organization. Panel B indicates the distinctly different options for prevention and treatment of disease (See Color Plates).

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A more sinister case is thought to have occurred in 1971 in the Aral Sea when a trawler deck-hand was infected approximately 15 km south of a Soviet bioweapons testing facility on Vozrozhdeniye island (8). The Aralsk outbreak, as it became known, occurred a year prior to the signing of the Biological and Toxic Weapons Convention by the Soviet Union.

16.2.1.4

Figure 16.2. Smallpox-systemic spread and transmission. A schematic of stages in the natural life-cycle of VARV. Step 1: Release of the virions from lesions in the oropharyngeal mucosa and exit via the exhaled respiratory gases. Step 2: Virions are transmitted by large droplet aerosols. Step 3: Seeding of the new host’s respiratory mucosa is initiated. Step 4: Replication creates a foci of infection and production of unique host-specific proteins to neutralize the immune response. Step 5: Primary viremia denotes successful virus replication and spread from the initial site(s) of infection to lymphoid tissues and internal organs. Step 6: Secondary viremia occurs when the virus moves from the infected lymphoid tissues and internal organs to the cornified and mucosal epithelium to cause the exanthem and enanthem, respectively. Transmissibility is dependent on the number of lesions in the host oropharyngeal mucosa, virus survivability in the face of the host immune response and the ability of the virus to produce infectious virions for exhalation from the respiratory tract. Reproduced with permission of the Future Medicine Group (10) (See Color Plates).

virus, suggesting that the inherent transmissibility was probably lower than that of variola major. Thus the less severe constitutional symptoms of variola minor patients perhaps enabled more opportunities for virus transmission during the infectious period resulting in similar, as apposed to dissimilar, secondary attack rates for variola major and minor. Although smallpox is typically spread by respiratory droplets over a short distance, some examples of long distance transmission of classic smallpox do exist. One such case occurred in 1978 at the University of Birmingham, UK, where a woman who was vaccinated 12 years earlier died of smallpox (5). She is identified as the last human fatality of the disease. It is widely believed that VARV traveled up through an air duct that connected a smallpox virology laboratory to her work station. Other theories suggest that this woman was exposed by using the laboratory telephone or simply from laboratory personnel. Another case occurred in a hospital at Meschede, Germany, in 1970 (7). In this case, a recent returnee from Pakistan is believed to have initiated 19 other cases of smallpox on all levels of a large general hospital despite being isolated for the 5 days of his stay. Factors that enabled VARV to travel long distances in the hospital were likely the design of the hospital that facilitated strong rising air currents when it was heated, the patient’s severe cough, and the hospital’s humidity level.

VARV as a Potential Biothreat Agent

With the global eradication of smallpox in 1979, the causative agent, VARV, no longer circulates in human populations; however, there is concern that clandestine stocks of VARV exist and could be reintroduced through bioterrorism and/or biowarfare. The reintroduction of aerosolized VARV (or perhaps MPXV) into human populations would result in high levels of mortality for several reasons. First, an efficacious anti-viral therapy is not available for the treatment of exposed individuals. Second, the routine immunization of the U.S. civil population with VACV had all but ceased in the early 1970s, resulting in the population under age 30 lacking cross-protective immunity to VARV. Third, the strength of vaccine immunity in older Americans has decreased with the passage of time, leaving these individuals with unknown protection against smallpox. Fourth, a growing segment of the American population is immunocompromised as a result of infection with human immunodeficiency virus (HIV), and the use of immunosupressive drugs for cancer treatment and the prevention of organ transplantation rejection. Fifth, individuals with skin conditions including atopic dermatitis are on the rise. A familial history of eczema is a contraindication to vaccination with the traditional smallpox vaccine to prevent eczema vaccinatum, a severe complication associated the smallpox vaccine. The attractiveness of VARV as a bioweapon, and to a certain extent MPXV, is its ability to spread from person to person. Of the National Institute of Allergy and Infectious Diseases (NIAID) Category A agents that are deemed the most serious bioterrorist threat, these orthopoxviruses are the most transmissible. In addition, there is concern that VARV or MPXV may be genetically engineered to make them more virulent and/or capable of breaking through the smallpox vaccineinduced immunity, as has been demonstrated with a recombinant ectromelia virus (ECTV) expressing interleukin-4 (9).

16.2.2 16.2.2.1

Human Monkeypox History

One could speculate that human MPXV infections have been occurring in Africa for centuries and were masked under the guise of smallpox (10). MPXV was first isolated in 1958 from the vesiculo-pustular lesions found on infected cynomolgus macaques imported to the State Serum Institute of Copenhagen, Denmark (11). During the next few years, similar outbreaks were reported in monkey colonies in the United States and in a zoo in Rotterdam, The Netherlands. In the latter case, the first

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animals affected were giant anteaters from South America, but the disease spread to various species of apes and monkeys. The viruses isolated from these animals were found to be similar and to represent a previously undescribed species. No human monkeypox was associated with these epizootics. MPXV remained primarily of academic interest throughout the 1960s, despite the observation that highly susceptible monkeys infected with MPXV have almost identical clinical manifestations as humans infected with VARV (12). The attitude of the scientific community changed upon the realization that MPXV could lethally infect humans in known smallpoxfree locales (11). Between 1970 and 1971, six cases of human MPXV infection were reported in Liberia, Sierra Leone, and Nigeria, which had been smallpox-free for at least a year; additionally, the primary human monkeypox case discovered during this same period was in a 9-month-old child in Zaire (now the Democratic Republic of the Congo [DRC]). Between 1970 and 1980, 4 of 47 cases (9%) were suspected to be from human-to-human transmission, with the remaining 43 human cases (91%) acquired from contact with animals (13, 14). The majority of infections were acquired in regions conterminous with the tropical rain forest.

16.2.2.2

Clinical Disease

The most severe human MPXV infections have been reported in the Congo basin, whereas attenuated human infections have generally occurred in West African countries. The difference is attributed to inherent differences in the virulence of the circulating strains (15, 16). In the Congo basin, human infections generally resulted from handling MPXV-infected animal tissues (17). MPXV-infected humans develop a skin rash and follow a disease course similar to that observed in smallpox victims. However, some differences exist between smallpox and human monkeypox. First, humans infected with MPXV frequently present with severely swollen lymph nodes (lymphadenopathy of the neck, inguinal, and axillary regions), which are not clinically apparent with smallpox victims. Second, a hemorrhagic form of human monkeypox has not been reported. Interestingly, humans infected with MPXV typically present with a rash similar to that observed in less severe cases of smallpox. To quantify this somewhat, approximately 58% of smallpox patients and 11% of human monkeypox (Congo Basin strain) cases had >100 pocks, respectively. The case-fatality rate for human monkeypox is ~10%, compared to 10 to 30% for variola major.

16.2.2.3

Person-to-Person Transmission of MPXV

Person-to-person transmission appears to be on the rise. Of the 338 cases documented in the intensive surveillance area within the DRC between the 1981 and 1986, 245 cases (72%) were primary or co-primary (11). Of the remaining 93 secondary cases (28%), 69 were first generation, 19 second generation, and 5 third or fourth generations. The secondary attack rate (among close susceptible contacts), which is a measure of the risk of person-to-person transmission, was 9%. Furthermore,

mathematical modeling experiments based on this data concluded that MPXV could not transmit indefinitely in the unvaccinated human population without zoonotic amplification (18). In July 1996, 42 cases, including 3 deaths (7%), occurred in a village populated with 346 inhabitants. A male, identified epidemiologically as the primary case, is believed to have directly or indirectly passed the infection through eight members of his clan and to the village inhabitants (13). These findings were interpreted as enhanced human-to-human transmission in the Congo basin as compared to previous studies (11). Moreover, in 2003, a hospital in the Republic of the Congo reported six generations of human-to-human MPXV transmission, suggesting that the transmission efficiency may also be increasing (19).

16.2.2.4 Human Monkeypox: An Emerging Infectious Disease Cases of human monkeypox are increasing in Africa, and for the first time human monkeypox was observed in the Western hemisphere during an outbreak in 2003 (13, 14, 20). This may be due to a combination of factors pertaining to the environment, human health, increased geographical range of reservoir species, or new reservoir species. Regardless of the reason(s), the increasing number of cases provides an opportunity to enhance virus transmissibility within human populations. 16.2.2.4.1

Increasing Geographic Range

The interaction of MPXV with reservoir and incidental hosts is still poorly understood, as is the potential for virus transmission to humans within and outside its geographical range. Until the U.S. outbreak in 2003, MPXV had remained fairly localized to a handful of countries in central and western Africa, with the majority of cases detected in the DRC. The U.S. outbreak added to the breadth of host species capable of supporting MPXV replication, and demonstrated the potential of the virus to expand its geographical range. No human infections were attributed to the shipment of animals that entered the United States from Africa; rather, most patients had direct contact with infected native prairie dogs that had been housed with the imported African rodents. The U.S. outbreak was caused by the less virulent West African strain, which probably made it easier to bring the outbreak under control. A more recent example (2005) of MPXV expanding its environs were 19 human monkeypox infections discovered in Sudan, a previously MPXV-free country (21). This outbreak occurred some 300 miles northeast of the edge of the tropical rainforest—the traditional home of the Congo Basin strain of MPXV. From our experience with VACV, it would not be surprising if MPXV continues to infect new species, as occurred in the United States and possibly in Sudan. 16.2.2.4.2

Increasing Incidence of Disease

Between 1981 and 1986, 404 human monkeypox cases were reported in Africa, 386 of the 404 cases (96%) were from the

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DRC. Of these 386 cases, 338 (88%) fell into the intensive surveillance area within the DRC (14). Reported cases dwindled after the 1981 to 1986 surveillance program with no reports between 1992 and 1995, possibly as a result of less surveillance. Since this respite, an upsurge was noted with 71 cases between February and August of 1996 in which six deaths (8%) occurred in a human population of 15,698. A further 511 cases were reported in 1997 (14). Reported cases continued to rise between 1998 and 2002 with 1,265 reported cases in the DRC, but only 215 specimens were collected; 88 specimens (41%) tested positive for MPXV, suggesting that human monkeypox cases may be lower (approximately 518 rather than 1,265). The reason for the upsurge in cases is not known, but many Africans may be more susceptible to severe disease due to poor nutrition or co-infections with other pathogens. Two reports have documented co-infections of MPXV and VZV, with one fatal outcome (22, 23). It is known that HIV infection increases the morbidity and mortality of humans when they are concurrently infected with Mycobacterium tuberculosis (24, 25), and a similar effect would be expected following co-infection with MPXV. Another likely contributing factor could be the cessation of smallpox vaccination by the WHO circa 1980, as vaccination for smallpox is 85% effective against severe monkeypox disease 3 to 19 years following immunization (11, 26). Alternatively, there may be more frequent contact between humans and infected animals as the ecosystem is degraded. Lastly, the broad host-range of MPXV may permit additional species to become reservoirs or incidental hosts that increase the exposure risks for humans.

16.3 Recognition of the Threat of Bioweapons and Emerging Infectious Diseases The twin threats of the intentional release of bioweapons and the continual emergence of new pathogens was well recognized prior to the attacks on the World Trade Center in New York and the Pentagon in Washington, D.C., in September 2001. The emergence of HIV in the early 1980s emphasized that pathogens respected no borders, are adaptable, and will forever be with us. In 1994, Laurie Garrett documented the stories and struggles of responding to newly emerging diseases in a post-World War II era in “The Coming Plague” (27). Between 1973 and 2003, more than 36 newly emerging infectious diseases were identified worldwide: almost one new disease a year (28). There is a consensus that emerging diseases require an integrated approach including a surveillance network that captures information locally and disseminates it globally in timely, accurate, and seamless manner; research to develop clinical and laboratory diagnostics for disease recognition; research

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to develop prophylactic, therapeutic, and supportive patient treatments; and public health outreach and education to disseminate acquired information. The reality of the bioweapons threat was revealed with the defections of two Biopreparat scientists from the former Soviet Union. Biopreparat was a clandestine Soviet organization of bioweapons research and production, with facilities sited across the Soviet Union. In 1989, Vladimir Pasechnik, a Biopreparat microbiologist defected to Great Britain, and was followed in 1992 by Kanatjan Alibekov (Ken Alibek), the first deputy chief of Biopreparat. The information provided by the defectors, in part, was made public in 1999 in Ken Alibek’s book “Biohazard” (29). The extent of the Soviet’s bioweapon program initiated a series of responses directed both at program elements that remained in the former Soviet Union, and the potential dissemination of Soviet bioweapons, technology, and/or experts to rogue nations or stateless terrorist groups. With the break-up of the Soviet Union, terrorist groups such as Al Qaeda are the greatest potential users of bioweapons. Indeed, in 1984, the followers of the Indian guru Bhagwan Shree Rajneesh contaminated salad bars at 10 restaurants in Dalles, USA, with salmonella and sickened about 750 people (30). Furthermore, in 1993, Aum Shinrikyo released aerosolized Bacillus anthracis from a high-rise building in Tokyo, Japan (to no effect) prior to a sarin gas-attack on the Tokyo subway in 1995 that resulted in the deaths of 12 commuters and serious illness for 54 others (31). The appeal of bioweapons lies in their low cost, technological simplicity, and ease of concealment. In addition, there are a large number of biological agents that could be used, their long incubation periods and delayed onset of disease could help the perpetrator escape, and their potential for secondary spread adds to a propensity to cause terror and anxiety. In FY98/99, National Institute of Allergy and Infectious Diseases (NIAID) research activities to develop countermeasures to VARV and MPXV as bioweapons began in earnest through 1-year supplements to existing grants and interagency agreements. These initial activities resulted in an extension of the coverage of the 15 million doses of Dryvax™ stock-pile by showing the vaccine could be diluted 1:5 and still generate a “take” in human volunteers (32, 33), the launch of the process of developing a safe, sterile smallpox vaccine grown in cell culture using modern technology (34); support for the development of a vaccine that can be used in populations contraindicated for vaccination with a live Dryvax or Dryvax-derived vaccine (35, 36); the launch of the development and testing of at least three antiviral drugs for use against human orthopoxvirus infections (37, 38), and increased genetic information on orthopoxviruses responsible for human infections or for modeling orthopoxviruses that infect humans (39–43). In later years, these areas of research and product development were extended and expanded upon through the conventional grant and contract process.

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16.4 Historic Prophylactic and Therapeutic Treatments for Human Poxvirus Diseases 16.4.1

Smallpox Vaccines

16.4.1.1 Traditional Vaccines: Live, Animal Passaged and Virulent First-generation vaccines evolved from locally produced products that gained regional and/or national prominence through efficacy. These vaccines were neither clonal nor highly purified and could be contaminated with microorganisms, as they were serially propagated on domesticated animals, most often calves or sheep (at least in the early years). Four major vaccines were used during the smallpox eradication program: Dryvax (United States), Lister (United Kingdom, Europe, Africa, Asia, and Oceania), Temple of Heaven (China), and EM-63 (USSR). During the intensified smallpox eradication program, these vaccines were prepared locally to a uniform potency of 1 × 108 PFU/mL that gave a presented dose of ~2.5 × 105 PFU per vaccination site when used with a bifurcated needle (44). Although vaccines, regardless of source, gave similar levels of protection from severe smallpox, they varied in the severity of post-vaccination complications. In the United States, Dryvax is the only approved smallpox vaccine (45). This vaccine was manufactured by Wyeth laboratories as a lyophilized preparation of live VACV made using the New York City Board of Health strain that had been passed 22 to 28 passages in the skin of calves. The preparation was not sterile and vaccine distribution to the public was discontinued in 1983. The expertise and manufacturing capacity to produce new batches of this type of vaccine are lacking.

16.4.1.2

Type and Frequency of Complications

Fenner and colleagues identified two major groups of VACV complications: abnormal skin eruptions (accidental infection, generalized vaccinia, eczema vaccinatum, erythema multiforme, and progressive vaccinia [vaccinia necrosum]) and disorders affecting the central nervous system (encephalopathy and encephalitis; ref. 5). In the United States, the frequency of VACV-associated complications (NYCBH strain) was thoroughly examined in 1968’s national and 10-state surveys (46, 47). The majority of vaccine-associated complications reportedly occurred after primary immunization and less frequently with re-vaccination, except in the case of progressive vaccinia. Using the 10-state survey, 1,253.8 cases per million primary immunizations were observed for all ages. More specifically, for every million vaccinations, there were 935.5 serious, but not life-threatening reactions, 52.3 life-threatening reactions, and 1.5 deaths. For a thorough description of these complications, see the review by Fulginiti et al. (48).

16.4.1.3

Contraindications to Vaccination

Five conditions were traditionally accepted as contraindicators for immunization with VACV: immune disorders, young age (less than 2 years old), eczema, pregnancy, and disorders of the central nervous system (5, 49). Although cardiac complications associated with the vaccination were not considered significant during the 1960s, several cardiac complications reported in early 2003 prompted the Centers for Disease Control and Prevention (CDC) to revise their recommendations for contraindicators of vaccination to include heart disease. Women who are breastfeeding, persons less than 18 years of age, and individuals with allergies to vaccine components have also been included as contraindicated to vaccination (50). The current number of people afflicted with the contraindicated conditions has significantly increased since the eradication program. Thus, in the event of a bioterrorist attack, or the emergence of MPXV into the human population, it is inevitable that the number of adverse events associated with a mass vaccination would be considerably more than during the eradication program. For this reason, the design and evaluation of safer vaccines became a major research thrust.

16.4.2

Vaccinia Immune Globulin (VIG)

Intramuscular administration of VIG, a product derived from the pooled plasma of vaccinated individuals, is indicated for treatment of generalized vaccinia, progressive vaccinia (vaccinia necrosum), eczema vaccinatum, and certain autoinoculations, although efficacy has not been demonstrated through controlled clinical trials. VIG was reported to halt formation of new lesions and to cause rapid clinical improvement in cases of generalized vaccinia and eczema vaccinatum (51). One large study suggested that post-exposure treatment of contacts of patients with smallpox with vaccination and VIG appeared more efficacious than vaccination alone. Smallpox developed in 5 of 326 contacts who received VIG compared to 21 of 379 controls, for a relative efficacy of 70% in preventing smallpox (52, 53). In 2005, the Food and Drug Administration (FDA) approved the manufacture of new stocks of VIG by DynPort Vaccine Company LLC.

16.4.3

Antivirals

During the smallpox eradication program, a number of compounds were shown to have efficacy against orthopoxvirus infections in tissue culture, and some were actually tested in field conditions. Thiosemicarbazone and metisazone were administered prophylactically in a series of trials in India and showed some protective effect; however, their administration was often associated with severe nausea and vomiting (5). Cytosine arabinoside and adenine arabinoside were also used to treat variola major and minor, but failed to affect the case mortality rate or the clinical progression of disease. Rifampicin showed antiviral activity against VACV in a mouse model, but was never tested

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clinically against VARV. No drugs are currently licensed for the treatment of orthopoxvirus infections.

16.5 New Risks Require New Treatment Modalities The success of the 20th century smallpox eradication program was based on the lack of an animal reservoir for VARV, and availability of cheap, stable, vaccines, a long disease incubation period allowing the tracing and post-exposure vaccination of contacts, and a relatively high level of herd immunity. In the 21st century, we are faced with the potential use of natural or recombinant VARV and MPXV as biological weapons and the emergence of MPXV as a more important zoonotic disease. Currently, the human population lacks solid herd immunity to orthopoxviruses due to the cessation of smallpox vaccination in the 1970s. Also, the traditional smallpox vaccine is no longer suitable for a growing minority of the U.S. population due to contraindicators. The threat posed by the intentional release of VARV or MPXV or an epizoonosis will require a capacity to rapidly diagnose the disease and intervene therapeutically with antivirals. Intervention is likely to take place during the diagnosis of the first wave of cases, which is at the rash stage of disease approximately 7 to 19 days p.i. Preimmunization of at-risk populations with vaccines will likely not be practical, and the therapeutic use of vaccines is ineffective 4 days p.i. with VARV (54). Instead, immunization will be used to prevent further spread of the pathogen by treating atrisk populations. Although this scenario clearly demonstrates the need for efficacious antivirals (see Figure 16.1, panel B),

presently none are licensed for use with human orthopoxvirus infections. This gap in the armamentarium was recognized by NIAID in the late 1990s.

16.6 A New Paradigm for Licensure of Human Poxvirus Vaccines and Drugs 16.6.1

Animal Efficacy Rule

Naturally occurring smallpox was eradicated in the late 1970s by a global vaccination program sponsored by the WHO. Human monkeypox, although on the rise, is still sporadic and usually occurs in the roadless tropical rain forest. In recognition of this problem, the FDA promulgated the so-called “Animal Efficacy Rule,” which acknowledges that therapeutics and prophylactics against NIAID Category A biothreat agents such as VARV and MPXV cannot be licensed under the usual regulatory standards (21 CFR 314 or 601; U.S. Code of Federal Regulations title 21, part 314, subpart I, Federal Register, 2002). The Animal Efficacy Rule permits the use of well-controlled animal efficacy data to support an application for licensure of drugs and biological products intended to treat or prevent serious or life-threatening conditions in humans resulting from exposure to biological, chemical, radiological, or nuclear substances. Product licensure requires that the Animal Efficacy Rule be utilized if human challenge or protection efficacy trials to test the product would be unethical due to the risks associated with exposure, or when clinical field trials are unfeasible (e.g., in the case of rare, naturally occurring human diseases caused by dangerous infectious agents). Although the

Table 16.1. Elements of the “Animal Efficacy Rule”. Criteria for use of animal data There is a reasonably well understood pathophysiological mechanism for the toxicity of the substance and its prevention or substantial reduction by the product. The effect is demonstrated in more than one animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represent a sufficiently well characterized animal model for predicting the response in humans.

Issues relating to smallpox ●

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The animal study endpoint is related clearly to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity.

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The data or information on the pharmacokinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allow selection of an effective does in humans Reproduced with permission from ref. 58

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Scientific knowledge is limited as the last cases of endemic smallpox occurred in 1949 in the USA, and 1977 worldwide, prior to the age of molecular biology and immunology. VARV naturally infects only humans; experimental infection of nonhuman primates is forced. Animal models using related orthopoxviruses produce disease with similarities to smallpox, but the pathogenesis varies depending on the animal species, the characteristics of the infecting virus and the route of infection. No one animal model has been established that completely mimics the human disease. There are no animal models for the major morbidities of smallpox. Orthopoxvirus doses sufficient to produce 100% morality in animal models shorten the incubation period substantially, thus making it difficult to study the effect of postexposure interventions. Interpretation of mortality studies in animals are limited by the ethical requirement to euthanize moribund animals The specific pharmacodynamic response related to antipoxviral activity cannot be measured in uninfected humans for the purpose of selecting an effective dose. Pharmacokinetics in the animal species used in orthopoxvirus infection models may not be the most relevant for dose selection in humans.

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selection of animal models is left up to the scientific judgment of the principal investigator, a typical choice would involve at least one rodent and non-human primate model. The Animal Efficacy Rule presents common and unique regulatory hurdles for licensure of vaccines and antimicrobials (55, 56). The criteria for animal data use in licensure of products under the Animal Efficacy Rule is stated in Table 16.1, and is matched to the realities of smallpox product development in animal models. The first three sections of Table 16.1 apply equally to vaccines and antivirals with the fourth section specifically addressing issues relevant with antivirals. Although the available animal models can be characterized in great detail using modern molecular and immunologic techniques, little is known about the molecular and cellular basis of VARV or MPXV pathogenesis, especially during the 10- to 12-day incubation period that was modeled on a 1950s understanding of mousepox. There is no single animal model that mimics smallpox and human monkeypox accurately. The animal models differ from human disease in the infectious dose required to initiate infection, tissues targeted for pathology, and duration of disease. In the case of antiviral development, the sole use of animal efficacy data as a means of establishing an effective human drug dose is problematic (56). Because there are no pharmacodynamic responses in animal models that can predict the human response to an anti-orthopoxvirus drug, human dose selection must be based on kinetics, which can vary considerably between humans and animals. For example, the phase I oxidative metabolism of hexadecylpropanediol-cidofovir (HDP-CDV) is much higher in cynomolgus monkeys than in mice or rabbits, yet in vitro data suggest that human metabolism will be closer to metabolism in mice and rabbits than in non-human primates (56). Although the non-human primate efficacy data is traditionally given more weight than similar data from mice, should this be the case when the pharmacokinetics is predicted to be very different from that of humans? For licensure of vaccines the key issues are selection of the correlates of immunity, relevance of various immune measurements to protective immunity, and the possible need for the candidate vaccine to protect against challenge with a live attenuated vaccine such as Dryvax (57). The licensure of vaccines and antivirals will require a profound and sustained research effort, and constant open dialog among the drug sponsor, regulatory authorities, and government agencies to reduce the Animal Efficacy Rule to practice (56).

16.6.2 Historic Animal Models for Preclinical Evaluation of Human Orthopoxvirus Vaccines and Drugs Many studies using various combinations of orthopoxviruses, hosts, and routes of inoculation have been tested. The following models that are used most frequently in preclinical testing of prophylactics and therapeutics against human orthopoxvirus infections are less than optimal as compared to others in modeling smallpox and human monkeypox.

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16.6.2.1

Vaccinia Virus in Mice

The origin of VACV remains a mystery, but recent data suggest that it is closely related to horsepox virus and may have been derived from this virus species sometime in the 19th century (58). Live VACV was used as a vaccine in the global program to eradicate smallpox. For a wide variety of mouse strains, VACV is highly infectious, but is of low virulence. Except for immunodeficient mice, it is next to impossible to observe mortality following pericutaneous or footpad inoculation of VACV even at doses as high as 107 PFU (59). Following infection, the virus replicates efficiently at the site of infection, but is rarely detected in the draining lymph node, and more rarely in the spleen or liver. Similarly intravenous and intraperitoneal doses of VACV in excess of 107 PFU are necessary to observe mortality. The non-physiological intracranial route of inoculation is the most sensitive of all routes tested and LD50 values can be achieved in the area of 10 PFU, but are dependent on the strains of VACV and the host (60, 61). The intranasal route of VACV inoculation is the most thoroughly studied of all of the infection routes, and LD50 values have been reported which range from 3.3 × 105 to greater than 6 × 108 PFU depending on the strain of VACV and strain of mouse (62–64). VACV challenge by the intracranial, intradermal, and intravenous routes has previously been used to evaluate orthopoxvirus antivirals in mice. The intradermal route of VACV challenge has been used to evaluate test compounds: interferons, polyacrylic acid, polymethacrylic acid, cytosine arabinoside, iodeoxyuridine, ethyldeoxyuridine, thiocyanatodeoxyuridine, S-adenosylhomocysteine hydrolase, ribavirin, and others (65, 66). Furthermore, a frequently employed in vivo test for antivirals is inhibition of tail lesion formation in mice inoculated with an intravenous dose of ~106 PFU of VACV. The VACV intracranial, intradermal, and intravenous mouse challenge models, however, do not resemble the human orthopoxvirus diseases that will be potential targets of an efficacious orthopoxvirus antiviral.

16.6.2.2

CPXV in Mice

CPXV was named as a result of its association with the pustular lesions on the teats of cows and the hands of milkers, although repeated studies have shown that bovine cowpox is not, and never has been, a common disease (67, 68). The natural reservoir in nature is not the cow, rather it is likely to be rodents (voles, wood mice, and rats; ref. 4). The pathogenesis of CPXV following intradermal inoculation of guinea pigs and rabbits was studied thoroughly by Downie in the 1930s. In the late 1960s, Subrahmanyan and Mims carried out less detailed studies with the Brighton strain of CPXV in various strains of mice (69, 70). Outbred mice inoculated with CPXV by intravenous, intranasal instillation and intracranial routes yielded LD50 values of approximately 1 × 107, 9.9 × 106, and 1 × 104 PFU, respectively. As with VACV, footpad inoculation of outbred mice with a high dose of CPXV (3.3 × 107 PFU) led to severe swelling of

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the footpad, but few clinical signs. Extensive replication of the virus was observed in the inoculated foot, but the infection was not fatal. Little virus replication was detected in local lymph nodes, spleen or liver (69). Similar results were obtained with lower doses (4 × 103 PFU) of the L97 strain of CPXV in BALB/ c and C57BL/6 mice (71). More recently, Bray et al. reported that 4-week-old female mice inoculated by tail scarification with 106 PFU of the Brighton strain of CPXV had a more generalized infection with 40% associated mortality (72). This more severe disease, following a peripheral route of inoculation of CPXV, may be due to the method of inoculating the virus, the mouse strain or the age of the mice. Resistance to severe CPXVinduced disease has been shown to be exquisitely sensitive to the age of the mouse because more than half of the 2-weekold outbred mice are killed by footpad inoculation of 400 PFU of the Brighton strain of CPXV, whereas 6-week-old mice are completely resistant to the lethal effects of 3.3 × 107 PFU (69). This early age dependence for severe disease prevents the use of CPXV in vaccination challenge studies, and mitigates against its use for antiviral efficacy testing, as the immune system is not fully developed at the time that the mouse is most sensitive to severe disease. Huggins and colleagues used lethal aerosol and intranasal CPXV challenge models to test a number of experimental orthopoxvirus therapeutics (73, 74). As an integral part of their study, the authors characterized CPXV pathogenesis by both the aerosol and intranasal instillation routes of infection (75). Following intranasal instillation, the pulmonary disease

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appeared more variable than for the aerosol infection, possibly due to the nonuniform distribution of the inocula. Despite this observation, it was concluded that both protocols gave similar results overall, and that either model could be used for routine in vivo testing of experimental therapeutics. With the aerosol challenge model, studies found that 4-week-old female BALB/c mice (11–13 g) exposed to 5 × 102 PFU resulted in no visible signs of disease, 5 × 104 PFU caused transient mild illness and weight loss but no deaths, and ~5 × 106 PFU resulted in uniform mortality. This high challenge dose of virus required for a lethal infection differs from that of VARV infection of humans, which is thought to be a low-dose infection (10).

16.6.3 New Models for Preclinical Evaluation of Human Orthopoxvirus Vaccines and Drugs The Animal Efficacy Rule demands a greater understanding of the animal models used to generate efficacy data for product licensure. Mousepox, rabbitpox, and the intravenous monkeypox experimental models together recapitulate most of the important features of human orthopoxvirus infections (Figure 16.3). With mousepox, a severe lethal disease is initiated with a low-dose intranasal or aerosol challenge or by natural transmission without early lung involvement, as is the case with smallpox, but death occurs prior to rash development. Similarly, the rabbitpox model is typified by a highly lethal disease induced by a low dose of virus. The disease course can be marginally longer than mousepox, and just prior

Figure 16.3. Animal models proposed for use in licensure of smallpox therapeutics under the animal efficacy rule. Reproduced with permission from SIGA Technologies (See Color Plates).

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to death an early stage of rash development can be detected by transillumination of the ear. The lethal monkeypox model in cynomologus monkeys uses an intravenous infection of a large bolus of virus to mimic a high-level secondary viremia with rash development. All of the experimental models have shortened incubation periods as compared to smallpox. The lack of prominent rash development as a clinical stage in rabbitpox and mousepox prevents therapeutic interventions at this stage, the likely post-exposure treatment stage with smallpox or human monkeypox; however, this stage is reproduced in the MPXV model where the primary viremia is bypassed by intravenous injection of virus.

16.6.3.1

Mousepox: ECTV in Mice

ECTV, the causative agent of mousepox, causes an acute exanthematous disease of mouse colonies in Europe, Japan, China, and the United States (76–78). The natural reservoir for ECTV is unknown, but one report provides evidence that wild mice may be involved. Laboratory studies have shown that ECTV, like VARV, has a very narrow host range, infecting only certain mouse species (79, 80). A number of different strains of ECTV have been isolated that have been shown to differ in their virulence for the mouse (81). The Moscow, Hampstead, and NIH79 strains are the most thoroughly studied with the Moscow strain being the most infectious and virulent for the mouse. In the late 1940s, mousepox was proposed as a model for the study of the pathogenesis of smallpox and generalized vaccinia in humans (82). This rudimentary understanding of ECTV infection of the mouse and spread to internal organs during the disease incubation period still forms the conceptual basis for the incubation period of smallpox and human monkeypox (Figure 16.4, also compare with Figure 16.2). Studies from a succession of investigators in the last five decades have resulted in a detailed description of the virologic and pathologic disease course in genetically susceptible (A, BALB/c, DBA/2, and C3H/He; death ~7 days p.i.) and resistant (C57BL/6 and AKR) inbred and out-bred mice; identification and characterization of important cell-mediated and innate responses for recovery from infection; and the discovery of rmp-1, rmp-2, rmp-3, and rmp-4 loci that govern resistance to severe mousepox (83–87). Varying mouse genotypes, virus strain and dose of virus result in distinct disease patterns for a given route of infection. Mousepox has at least four features similar to smallpox. First, a relatively small dose of virus is required to initiate disease in the upper and lower respiratory tract. Second, following a low-dose intranasal infection there is no obvious lung involvement during the course of early disease. Third, virus can be detected in respiratory gases during the pre-exanthem period (88). Fourth, both diseases present with a characteristic exanthematous rash, although in mousepox rash development is dependent on a number of parameters including mouse strain, virus strain, route of inoculation, and virus dose (89). Mousepox differs from smallpox in at least two features fol-

Figure 16.4. Pathogenesis of mousepox. Reproduced with permission from ref. (86).

lowing respiratory tract infection. First, the disease course in mousepox is shorter as compared to smallpox. Death in fatal cases of mousepox usually occur 7 to 14 days p.i., whereas deaths in ordinary smallpox occur approximately 18 to 22 days p.i. Second, the major lesions in mousepox are observed in the liver and spleen, whereas these organs are relatively uninvolved in smallpox (5, 76).

16.6.3.2

Rabbitpox: VACV in Rabbits

Two strains of RPXV have been identified. The Rockefeller strain of virus was isolated by Greene from a spontaneous outbreak of severe disease in rabbits (90). The Utrecht strain of RPXV was isolated in a similar fashion by Jansen (91). Both virus strains were associated with high levels of mortality, although experimental findings suggested the Utrecht strain was possibly more virulent. Although certain strains of VACV and RPXV cause distinctly different diseases in the domestic rabbit, the viruses are closely related as determined by biologic and genetic tests (92, 93). The genome of the

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Rockefeller strain of RPXV has been sequenced confirming RPXV as a VACV strain (94). Both VACV and RPXV require little more than one infectious particle to initiate an infection by the respiratory route; however, this is where the similarity ends. VACV failed to produce a fatal disease or even a severe infection, with doses as high as 1.3 × 104 PFU (95). Aerosol infection with RPXV, on the other hand, produced almost uniform fatalities with the lowest dose administered (15 PFU; six deaths of seven animals inoculated). The minimum infective doses of RPXV varied depending on the route of administration (intradermal, 0.1 to 1 pock-forming unit; subcutaneous, 6 to 12 pock-forming unit; intravenous, ~100 pock-forming unit; intranasal instillation, 1000 pock-forming unit; and aerosol ~1 pock-forming unit; refs. 96 and 97). Epidemiological studies suggest that RPXV transmission is mediated through aerosols. Experimental respiratory infections of rabbits with RPXV have been studied in great detail (97–100). Conversely, the resemblance of rabbitpox to smallpox is striking. Both diseases are initiated with a relatively small dose of virus (≤100 virions). There is a late onset of virus transmissibility that, for both diseases, occurs at about the beginning of the exanthem. Also, the viremia of rabbitpox resembles that reported for smallpox in its occurrence at the onset of overt disease, in the direct relationship between virus titer and severity of disease and in its absence in some fatal cases (101). Furthermore, the early deaths in rabbitpox, which differed from the late deaths by the presence of a blood coagulation defect and a progressively increasing viremia, bare an uncanny resemblance to severe purpuric or hemorrhagic forms of smallpox (102, 103). However, rabbitpox differs from smallpox in its shorter incubation period, its greater severity and its more dramatic involvement of the upper respiratory tract late in the disease.

16.6.3.3

Monkeypox: MPXV in Non-human Primates

The clinical presentation of human monkeypox is virtually indistinguishable from smallpox, except for the frequently enlarged cervical and inguinal lymph nodes in the former (5, 11, 104). Both VARV and MPXV produce systemic diseases in humans with a generalized exanthem, whereas CPXV and VACV usually produce only a localized lesion at the site of inoculation. The clinical features of natural or experimental monkeypox can vary from subclinical to fatal depending on primate species and routes of inoculation. Monkeypox in monkeys is very similar to monkeypox and smallpox in humans, justifying its choice as a preclinical model for smallpox. MPXV can be transmitted by aerosol administration or intranasal instillation, as well as by parenteral inoculation by any route (105–108). For example, intramuscular inoculation of MPXV into cynomolgus monkeys was used as a primate model for the pathogenesis of human generalized orthopoxvirus infections, smallpox and generalized vaccinia (109). This model provided a similar pattern of viremia as that seen with ECTV in mice, RPXV in rabbits and VARV in monkeys. Aerosolized administration of high doses (106 to 107)

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of VARV, MPXV, RPXV strain Utrecht, VACV or CPXV to cynomolgus monkeys resulted in a febrile reaction with variable mortalities, ulcerative bronchiolitis, bronchitis, and peribronchitis; however, as in the human disease, only the monkeys infected with VARV and MPXV developed the typical exanthem (105). More recently, aerosol and intravenous VARV infections of cynomolgus monkeys were re-examined as a model for human smallpox (110). No monkeys exposed to the highest achievable aerosol dose of 108.5 PFU developed severe disease, although all exposed animals became infected. Intravenous infection of monkeys with either 109 PFU of the Harper or India 7,124 strains of VARV produced a uniformly acute and lethal infection. A lower intravenous dose of 108 PFU resulted in less fulminant, systemic disease and 33% mortality. This lower challenge dose has been used to evaluate antivirals using death and day of death, virus load in blood, and skin lesions as endpoints. Intravenous inoculation of VARV initiates an infection of the monkey qualitatively similar to the secondary viremia of smallpox (see Figure 16.2). This instantaneous viremia eclipses the incubation and prodromal phases of the disease and seeds virus in target tissues, including the skin. Although this model is qualitatively similar to smallpox, it is quantitatively different as the amount of virus injected into blood is orders of magnitude higher than would be detected in the secondary viremia of ordinary smallpox (89% of smallpox cases) and is closer to hemorrhagic smallpox (2.4% of cases; ref. 5). The initiation of an artificially high viremia following infection likely induces pathophysiological mechanisms that are distinct and possibly more severe than those induced during secondary viremia as a consequence of a natural infection. Importantly, this model offers a robust test for antiviral efficacy because target organs are immediately seeded with virus, whereas in human smallpox this occurs much later after infection (111).

16.6.4 New Drug Applications and New Biological Licenses for Therapeutic and Prophylactic Treatments of Human Orthopoxvirus Infections NIAID research supports the development of control measures including diagnostics, therapies, and vaccines in anticipation of their need for protecting the public health. In the case of human orthopoxvirus infections, the goals have been to produce at least three antivirals and sufficient vaccine for the entire U.S. population. The following prophylactics and therapeutics have approved Investigational New Drug (IND) applications and are currently supported by NIAID for development into products.

16.6.4.1

New Vaccines

The eradication of smallpox negated the need to maintain stocks of smallpox vaccines. Aging vaccine stocks were not

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replaced and some existing stocks were destroyed as costcutting measures. With the realization of the threat of VARV as a bioweapon in the 1990s, additional stocks of vaccine needed to be manufactured. ACAM2000, a vaccine derived from Dryvax was developed for use with the majority of the U.S. population, and modified VACV Ankara (MVA), a more highly attenuated vaccine with an improved safety record, was evaluated for use with an immunodeficient portion of the U.S. population. 16.6.4.1.1

Acambis 2000 Vaccine

In partnership with CDC, NIAID supported the development and testing of a new generation of live vaccines based on the Dryvax vaccine, but to be produced using newer manufacturing approaches. Because Dryvax was produced by sequential passages in calf skin, and therefore contained a population of viruses with distinct biological properties, six virus clones were isolated from a pool of 10 vials of Dryvax. Clone 2 (named ACAM1000) was selected as the seed for the secondgeneration smallpox vaccine based on comparable behavior to Dryvax when tested in mice, rabbits, and monkeys for virulence and immunogenicity (112). In partnership with Baxter BioScience, the ACAM1000 master seed virus was used to infect Vero cells under serum-free conditions to produce a second larger master seed virus stock named ACAM2000. ACAM2000 was evaluated in three phase I clinical trials and produced major cutaneous reactions, evoked neutralizing antibody and cell-mediated responses, and had a reactogenicity profile similar to Dryvax (113). Similarly, phase II randomized, double-blinded, controlled trials found ACAM2000 to be equivalent to Dryvax in terms of cutaneous response rate, antibody responses, and safety (114). In phase III clinical trials, three cases of myo/pericarditis were observed with ACAM2000, although this was not unexpected as several cases of myo/pericarditis were identified after smallpox vaccination in three Dryax and one ACAM2000 phase II clinical trials held in 2003 (115). 16.6.4.1.2

MVA Vaccine

A significant portion of the American population is contraindicated for vaccination with Dryvax or Acambis 2000 because they are immunosuppressed through infection with HIV or as a result of immunosuppressive drugs for cancer chemotherapy and anti-organ rejection therapy. For this reason, MVA vaccine is under evaluation. MVA was developed by growing the Ankara strain of VACV for greater than 500 passages on chicken embryo fibroblasts, which dramatically reduced its virulence by restricting its ability to replicate in human cells (116). From 1968 MVA has been safely used in more than 100,000 humans without documentation of any of adverse reactions associated with other VACV vaccines. Prophylactic MVA immunization of mice protects as efficiently as Dryvax against a lethal intranasal challenge with VACV strain WR (117, 118), but fails to protect when delivered as a post-

exposure treatment (119). Similarly, cynomolgus monkeys immunized with MVA survived a lethal intravenous or respiratory challenge with MPXV, although in the former study two doses of MVA were required to match Dryvax efficacy for blocking skin lesion formation (120, 121). Importantly, there are limited data in mouse models to suggest that MVA can efficiently protect against a lethal VACV intranasal challenge under some immunosuppressive conditions (e.g., B-cell deficient and β2-microglobulin-deficient mouse strains), but not others (e.g., RAG-1 -/- mouse strain and mice with decreased CD4 or MHC class II expression and double-knockout mice deficient in MHC class I- and II-restricted T-cell activities; refs. 119 and 122). The safety, immunogenicity, and efficacy of the MVA vaccine (strain TBC, Therion Biologics Corporation) has been demonstrated against a Dryvax challenge in vaccinia-naïve and vaccine-immune volunteers (123). IMVAMUNE, an MVA vaccine (strain BN, Bavarian Nordic GmbH) has been tested for safety and immunogenicity in human volunteers (124), and is scheduled for phase II clinical testing in the NIAID Vaccine and Treatment Evaluation Network. Because the MVA virus does not replicate efficiently in human cells, one to two vaccine doses containing ~100 times more MVA virus than Dryvax may be required to induce equivalent immune responses and protection, making this a potentially expensive vaccine in the absence of adjuvants.

16.6.4.2

New Antivirals

An early objective of NIAID was the licensure of at least three antivirals against human orthopoxvirus infections. This goal was supported independently by reports generated by the Institute of Medicine and the National Academy of Sciences in the United States (125, 126). The second report discussed a large number of potential targets for antiviral development. To date, two candidate antivirals have received IND status. 16.6.4.2.1

CMX001

As of 2006, Chimerix and SIGA Inc have received IND status for their compounds CMX001 and ST-246, respectively. CMX001 is a HDP-CDV salt synthesized by covalently coupling CDV to an alkoxyalkanol to form a prodrug (127). The conjugate was designed to act as a natural lipid and be absorbed intact from the small intestine. Whereas the parental compound, CDV, lacks oral bioavailability and shows nephrotoxicity, 88% of HDP-CDV is bioavailable and is distributed to tissues via plasma and/or lymph without significant concentration in the kidney, and would be predicted to lack nephrotoxicity. Only after cellular uptake is CDV released from the conjugate, phosphorylated by host kinases, and available to competitively inhibit the virus-encoded host polymerase (128, 129). Exhaustive preclinical antiviral efficacy studies have been carried out in mice with ectromelia, vaccinia and CPXVs and in rabbits with RPXV. Phase I clinical trials are ongoing. Most importantly, CDV and HDP-CDV have broad spectrum antiviral activity against viruses that encode DNA

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polymerases including all orthopoxviruses known to infect humans, adenoviruses, and herpes viruses (human cytomegalovirus, human herpes simplex virus 1 and 2, 6 and 8, varicella zoster virus, and Epstein Barr virus; ref. 130). 16.6.4.2.2

ST-246

ST-256 (4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3dioxo-4,6-etheno-cyclo-prop[f]isoindol-2(1H)-yl)-benzamide is active against multiple species of orthopoxviruses, including two strains of VARV (131). Resistance mapping studies indicates that ST-246 targets the VACV F13L ortholog family. The F13L ORF encodes a major envelope protein, p37, which is required for production of extracellular, but not intracellular virus. Thus, ST-246 is unique in that it does not affect the actual production of infectious virus, only its efficient release from cells. Preclinical efficacy studies have shown ST-246 to be highly effective when administered shortly after CPXV, VACV, and ECTV intranasal infections of mice. Importantly, ST-246 was highly effective at treating CPXV and ECTV infections as late as 72 hrs p.i. (132). Considering that death of untreated controls occurs in 7 to 10 days p.i., this is quite an impressive feat. Phase I clinical trials are ongoing.

16.6.5 Financing Development of Products for Human Orthopoxvirus Infections That Have No Commercial Market The intensity of the federal government’s response to emerging infectious diseases and bioweapons gained momentum throughout the 1990s with the majority of the funding distributed through the CDC, Defense Advanced Research Projects Agency, FDA, and National Institutes of Health (NIH). The NIH’s mission is to support relevant basic research, develop medical interventions, and provide research resources; NIAID is the lead institute in implementing this program. NIAIDsponsored product development research is supported at all three phases of the process: discovery, preclinical evaluation, and clinical evaluation in human trials. Because the federal government is the only market for narrow spectrum prophylactics and therapeutics against biothreat agents, companies have few incentives to invest in these products when faced with limited returns on investments, regulatory hurdles, and extensive clinical trial requirements. In recognition of this reality, staged federal funding programs are available via NIAID throughout the product development cycle. Conventional principal investigator initiated grant proposals, such as R01s, are supplemented with product targeted STTR and SBIR grants to fund concept development through preclinical testing. These funding mechanisms are supplemented with various NIAID contract resources that permit products to be tested in vitro in cell based assays and in vivo in animal models under the NIAID Antiviral Testing Program, and in humans via the Collaborative Antiviral Study Group. Both CMX001 and ST-246 were developed using some or all

157

of these resources, and each received a contract in the range of $10 to 20 million to fund product development from the preclinical testing stage to IND submissions. Previously, there was no mechanism to support the movement of potential products such as CMX001 and ST-246 from pilot-level to fullscale production and subsequent procurement through Project BioShield. The Project BioShield Act became law in 2004, and provides $5.6 billion in appropriations over 10 years to authorize procurement of countermeasures for biological, chemical, radiological, and nuclear attacks for the strategic national stockpile. Although Project BioShield provides for payment of deliverables, it left the financial burden of funding the “production line” on the company, a risk that few companies were willing to undertake. In December 2006, passage of the Pandemic and All-Hazards Preparedness Act (Bioshield 2) mitigated against this disproportionate weighting of the risk of development of a non-commercial product on the company. The act establishes the Biomedical Advanced Research and Development Authority (BARDA) to issue contracts and grants for advanced research and development. Product development for medical countermeasures eligible for funding under BARDA is defined broadly, and includes clinical testing, design, and development of animal models for testing, scaling-up manufacture to commercial scale and activities to improve technologies for administering the product. Under BARDA, the costs associated with pilot-level to full-scale production can be contracted with milestone payments, which provide more equitable sharing of risk between government and companies. Procurement of the product is carried out under Project BioShield.

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Chapter 27 Disease Surveillance in Georgia: Benefits of International Cooperation Lela Bakanidze, Paata Imnadz, Shota Tsanava, and Nikoloz Tsertsvadze

27.1

Introduction

Effective communicable disease control relies on high- quality disease surveillance, which is the systematic and regular collection of information on the occurrence, distribution, and trends of an event on an ongoing basis with sufficient accuracy and completeness to provide the basis for action. Such a system therefore provides information for planning, implementation, monitoring, and evaluation of public health programs. It includes case detection and registration, case confirmation, data reporting, data analysis, outbreak investigation, response and preparedness activities, feedback, and communication. Health authorities must also provide appropriate supervision, training, and resources for the surveillance system to operate properly. The Georgian National Health Policy, adopted in 1999, declares the reduction of communicable and socially dangerous diseases to be a major priority for maintaining and improving the health of the Georgian population over the next decade.

27.2

Surveillance System in Georgia

Ideally, a surveillance system is sensitive enough to correctly identify all cases of a particular disease occurring in the community, especially in the case of dangerous pathogens, when even a single case must be detected. All clinically diagnosed or laboratory confirmed cases of communicable diseases that come to health facilities for treatment or consultation, regardless of whether they are reported urgently or once a month, must be registered. The usefulness of public health surveillance data depends on its uniformity, simplicity, and timeliness. State and local public From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

health officials use the information about occurrence of diseases to accurately monitor trends, plan and make decisions, and evaluate the effectiveness of interventions. Established surveillance systems should be regularly reviewed based on the explicit criteria of usefulness, cost, and quality, and existing systems should be modified as a result of such reviews. Attributes of quality include: (1) sensitivity; (2) specificity; (3) representativeness; (4) timeliness; (5) simplicity; (6) flexibility; and (7) acceptability. The sensitivity of a surveillance system is its ability to detect health events (completeness of reporting). Its specificity is inversely proportional to the number of false-positive reports. Representativeness can be measured by comparing surveillance data covering part of the population to either nationwide data, where available, or to random samplesurvey data. Simplicity in a system means it is easy to understand and implement, and is usually expected to be relatively cheap and flexible. A flexible system can easily be adapted by adding new disease entities, conditions, and/or by extending surveillance to additional population groups. Acceptability depends on the perceived public health importance of the event under surveillance, recognition of individual contributions, and the time required for reports. Internationally regulated dangerous infections, such as plague, cholera, yellow fever, poliomyelitis, viral hemorrhagic fevers, tularemia, anthrax, rabies, SARS, smallpox, tick-borne encephalitis (TBE), and influenza caused by a new virus subtype, must be reported to officials immediately. Urgent notification must also be done for groups of cases of any infectious disease, excluding acute respiratory infections and influenza.

27.3 The National Center for Disease Control and Medical Statistics (NCDC) of Georgia The NCDC was founded in 1996 to succeed the Georgian Station for Plague Control. An integral part of the Georgian Public Health system, the NCDC reports to the Ministry 253

254

of Labor, Health and Social Affairs of Georgia. The main responsibilities of NCDC include conducting surveillance on communicable and non- communicable diseases; controlling important diseases; carrying out preventive measurements; promoting healthy lifestyles; and gathering and processing medical statistical data. In addition, NCDC houses the Georgian National collection of especially dangerous pathogens. The NCDC network comprises 11 regional public health centers (CPH) and 66 district (rayon) CPH. Medical facilities and physicians are also part of the network. Surveillance on communicable and non-communicable diseases, including especially dangerous pathogens, continues to be one of the main responsibilities of the NCDC. All institutions and providers delivering healthcare services to the population regardless of their ownership, including laboratories and private care providers, must notify the local public health service whenever they diagnose, suspect, or even receive positive laboratory results that might indicate a disease of interest. The NCDC determines and annually updates the list of notifiable and reportable diseases based on the current epidemiological situation. Following the collapse of Soviet Union, the Republic of Georgia found itself in a very difficult economic situation. The healthcare system seemed nearly non-existent, and only international healthcare agencies were operating with the support of international organizations by conducting surveillance on communicable and non-communicable diseases. With the help of these agencies, NCDC succeeded in control and prevention of diseases important to the public health through health promotion programs, gathering and processing medical statistical data, running immunization programs, and so forth. During the 1990s, there were numerous achievements, like situation analysis and public health strategy design and piloting. Communicable disease surveillance guidelines for CPHs and healthcare providers, job aids for district-level CPHs and facility workers, etc., were established; human resource capacity was strengthened; district-level training and continuous supervision and support were carried out; and nationwide policy (endorsed by MoLHSA Decrees) was developed and implemented. Standard case definitions were determined for AFP/polio, measles, diphtheria, mumps, rubella/CRS, pertussis, tetanus, acute viral hepatitis, rabies, shigellosis, salmonellosis, cholera, bacterial meningitis, and influenza H5N1. Case definitions for anthrax, plague, tularemia, brucellosis, TBE, hemorrhagic fevers (HFRS, CCHF), and other diseases are forthcoming. The NCDC has research potential as well. About 60% of the staff members are specialists with university educations, and 32% of them are doctors of science or are candidates for scientific doctorates. The list of implemented and ongoing projects jointly funded by the United States and other international agencies demonstrates the diversity of their expertise: ●

Internaional Training and Research in Emerging Infectious Diseases (1997–2002), with Fogarty International Center, National Institutes of Health

L. Bakanidze et al. ●

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●

●

●

●

●

●

●

●

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Establishing Epidemiological Network on the Territory of Georgia (1997), with the “Open Society Georgia” Foundation Improvement of Epidemiological Network in Georgia (1998), with the “Open Society Georgia” Foundation Reproductive Health Survey (1999–2000), with United Nations Population Fund (UNFPA), United Nations Children’s Fund (UNICEF), United States Agency for International Development (USAID), UN High Commissioner for Refugees (UNHCR), American International Health Alliance (AIHA), and the Centers for Disease Control and Prevention (CDC) Nutritional Status of Children Under Five Years of Age in Six Regions of Georgia (2000–2001), with the USAID/ Save the Children-US, Georgia Field Office Provision of Epidemiological Survey Services on Baku– Tbilisi–Ceyhan Pipeline Route (2003), with and the British Petroleum Company, International Training and Research in Emerging Infectious Diseases (ITREID), the Fogarty International Center and NCDC Reproductive Health Survey 1999–2000, conducted jointly by the NCDC (Tbilisi, Georgia) and CDC (Atlanta, GA), funding provided by UNFPA, UNICEF, USAID, UNHCR, and AIHA; Nutritional Status of Children Under Five Years of Age in Six Regions of Georgia: 2000–2001; conducted jointly by the NCDC (Tbilisi, Georgia), with technical assistance provided by Irwin Shorr (private consultant) and the CDC (Atlanta, GA), and managed by the Georgia Field Office of “Save the Children”-USA; funding provided by USAID Enhanced Epidemiologic and Laboratory Diagnostic Capacity for the Control of Botulinum Intoxication in Georgia. Partner: U.S. Department of Health and Human Services (DHHS)/Biotechology Engagement Program (BTEP); Collaborators: CDC (Atlanta, GA) Prevention of Amebiasis and Creation of Diagnostic TestSystems for E. histolytica Strains Isolated in Georgia. Partner: U.S. DHHS/BTEP; Collaborators: University of Virginia Health System, Charlottesville, VA; Molecular Epidemiology and Antibiotic-Resistance of Bacterial Infections in Georgia. Partner: U.S. DHHS/BTEP. Collaborators: University of Maryland School of Medicine, Department of Epidemiology and Preventive Medicine, Baltimore, MD Clinical and Molecular Epidemiology of Drug-Resistant Tuberculosis in the Republic of Georgia and the Caucasus. Partner: U.S. DHHS/BTEP; Collaborators: Emory University, Atlanta, GA Epidemiology, Molecular Characteristics and Clinical Course of HCV Infection in Georgia. Partner: U.S. DHHS/BTEP; Collaborators: Johns Hopkins University, Baltimore, MD Ecology, Genetic Clustering, and Virulence of Yersinia pestis Strains Isolated from Natural Foci of Plague in Georgia. Funding Agency: DTRA, etc.

The U.S. Defense Threat Reduction Agency (DTRA) has begun implementation of a project aiming to improve the

27. Disease Surveillance in Georgia

surveillance system through standardized and repeatable disease monitoring systems, mobile epidemiological response teams, and secure transportation of infectious agents; systems of communications and information technology, including electronic communicable disease reporting system; biosafety and physical security of central reference laboratories, safe transportation of pathogens, and to provide help in promulgating new national rules and regulations, and their relations to BWPPP. All these elements are supplied with verifiable training in their field. A central Epidemiological Monitoring Station (EMS) was arranged at NCDC. It is equipped with modern sophisticated equipment, like light cyclers, RT- PCR, and so forth. EMS carries out surveillance on especially dangerous infections. Here, the first avian flu case in Georgia was identified. The Republic of Georgia is benefiting greatly from these projects. Biosecurity and biosafety at biological facilities will increase, and the disease surveillance infrastructure and

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capabilities with state-of-the-art technology will be improved, leading to detection and response to outbreaks or epidemics in a timely manner and improvement of early warning systems and data collection necessary for action linked to control measures. This sustainable disease surveillance system will continue to benefit the Republic of Georgia through reduced risk of disease proliferation risk. The laboratory capacity of NCDC reference laboratories has been improved, promoting opportunities to carry out various collaborative research endeavors.

References 1. Abt. Associates, Inc (2005) Surveillance and Control of Communicable Diseases: Guidelines for Public Health Service in Georgia. 2. Thacker SB, Parrish RG, Trowbridge FL (1988) A method for evaluating systems of epidemiological surveillance. World Health Stat Q 41:11-18.

Chapter 4 Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology H. L. Stevenson, N. Ismail, and D. H. Walker

4.1

Introduction

The etiologic agents of human tick-borne ehrlichioses include two genera, Ehrlichia and Anaplasma, which are members of the family Anaplasmataceae in the order Rickettsiales and are small α-proteobacteria. Human ehrlichioses, including that caused by Ehrlichia chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis (HME), vary greatly in disease severity and clinical presentation, ranging from a mild flu-like illness to severe septic shock-like syndrome. These agents, as well as the geographical distribution and vertebrate and invertebrate hosts, are listed in Table 4.1. The presence of the different arthropod vectors of the pathogens, for example, the ticks Amblyomma americanum and Rhipicephalus sanguineus, for E. chaffeensis and Ehrlichia canis, respectively, strongly correlates with reported human disease cases worldwide (1). The incidence of ehrlichial species throughout the world has been increasing with recent identification of cases being reported in Mexico (1, 2), Brazil (3), Spain (4), Africa (5, 6), the Netherlands (7), Russia (8–10), China (11), Japan (12), and Thailand (13–15). For many of these countries, including Mexico, Brazil, and Spain, Ehrlichia spp. had not been previously identified, and several other reports disclosed the presence of species that were previously thought to be endemic only in other regions, such as the detection of Ehrlichia ewingii in Cameroon, Africa. Additionally, an astute physician in Cape Girardeau, MO, completed a study in an outpatient primary care clinic and found that 29 of 102 patients had confirmed or suspected HME, an incidence two orders of magnitude higher than expected (16). The increased incidence and prevalence of ehrlichial species worldwide is thought to be multi-factorial and involves changes in host-vector ecology, better detection techniques, and increased awareness of the pathogens (17–19). It is a very exciting time in the field of ehrlichiosis, with many recent discoveries providing answers to previously controversial From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

or unknown questions. Ehrlichia are obligately intracellular bacteria that lack genes for lipopolysaccharide (LPS) and peptidoglycan (20), and reside within the early endosomes of monocytic cells inhibiting phagolysosome fusion (21). Ehrlichiae possess other unique features, with many of them being limited to the genus, including the expression of several outer membrane glycoproteins (22), as well as the ability to repress genes that are critical for induction of host innate immune responses (21, 23). Cholesterol taken up from the host is a requirement for ehrlichial survival and, along with outer membrane glycoproteins, may provide cell membrane stability in the absence of peptidoglycan and LPS (22, 24). Both Anaplasma and Ehrlichia have been found to contain ankyrin domains, and recently one of these proteins, gp200, was demonstrated to be translocated to the nucleus, where it is suspected to modulate host cell gene expression (25, 26). This chapter focuses on E. chaffeensis and HME; however, the other agents of human ehrlichioses are also addressed, mainly to compare similarities or differences among members of the family Anaplasmataceae. It mainly focuses on recent advances in the field and concentrates on potential targets for future investigations.

4.2 Genomic Studies and Potential Virulence Factors Until this year, only four Anaplasmataceae genomes had been published (two Wolbachia spp., Anaplasma marginale, and Ehrlichia ruminantium), none of which have been reported to cause human disease. Four additional genomes of medically important members of the family have now been sequenced including A. phagocytophilum, E. chaffeensis, Neorickettsia sennetsu (27), and E. canis (25), thereby increasing our understanding of the characteristics and physiology of these unique microorganisms. All species in the order Rickettsiales have relatively small genomes (0.8–1.5 Mb) that have arisen through reductive evolution as they developed dependence on 37

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Table 4.1. Agents of human ehrlichiosis in the family Anaplasmataceae. Organism Ehrlichia chaffeensis Ehrlichia ewingii Ehrlichia muris Ehrlichia canis Neorickettsia sennetsua Anaplasma phagocytophilumb

a b

Disease

Geographical Distribution

Vertebrate Host

Vector

HME Ehrlichiosis ewingii HME Asymptomatic Sennetsu fever HGA

North America, China (PCR) North America Russia Presumably worldwide Japan and Malaysia North America, Europe

Humans, deer, dogs, raccoons, canids Humans, deer, dogs Mice Dogs Humans Humans, deer, rodents, cats, sheep, cattle, horses, llamas, bison, others?

Ticks Ticks Ticks Ticks Trematodes Ticks

Ticks are the arthropod vector for all listed microorganisms except N. sennestu, for which trematodes are suspected to be the vector and reservoir Neutrophils are the main target cell for A. phagocytophilum; all other species infect host mononuclear phagocytes

the host cell for necessary functions (27). In addition, they all have an unresolved evolutionary relationship with the progenitor of the mitochondria, and ultrastructural studies of the E. canis-E. chaffeensis-E. muris genogroup have observed a very close apposition of mitochondria and endoplasmic reticulum with the ehrlichial vacuole membrane (28). However, whether these organelles have a functional role in the life cycle of the bacterium is unknown. Hotopp et al. reported that E. chaffeensis, A. phagocytophilum, N. sennetsu, and W. pipientis have the ability to synthesize all nucleotides, and E. chaffeensis, A. phagocytophilum, and N. sennetsu are able to synthesize most vitamins and cofactors (27). These characteristics are thought to provide benefit not only for the bacterium itself by decreasing competition for these molecules, but possibly also for the host cell or vector as well. These bacteria all have also genes for type IV secretion systems, which are structures known to use a complex of transmembrane proteins and a pilus to mediate the translocation of macromolecules across the cell envelopes of both Gram-negative and Gram-positive bacteria (27, 29). Other virulence genes, such as those that encode twocomponent regulatory systems, have also been described and studied. Cheng et al. discovered that the genomes of E. chaffeensis and A. phagocytophilum contain three potential pairs of two-component regulatory systems, including three sensor kinases and three response regulators (30). The twocomponent systems are composed of a pair of sensor histidine kinases and a response regulator, usually a transcription factor, which is activated in response to environmental changes detected by the sensor. These systems up- or downregulate genes necessary for survival in diverse environments and have been proposed to be employed when bacteria, such as Ehrlichia, transition between their vertebrate and invertebrate hosts (27). Activation of the sensor component involves autophosphorylation by histidine kinases, and inhibition of this kinase by the drug closantel abrogates E. chaffeensis survival within in the human THP-1 macrophage cell line (30). Additionally, this two-component system appears to be required for inhibition of phagolysosome fusion as treatment with closantel results in fusion of these cytoplasmic vacuoles with subsequent killing of the bacteria (31). Furthermore, a large protein

superfamily found only in the Ehrlichia spp. and Anaplasma spp., namely Omp-1/MSP2/p44, is thought to allow persistence of the organisms within their vertebrate and arthropod reservoirs by providing the necessary machinery for surface protein variation, facilitating the survival of the bacteria in their tick vectors and vertebrate hosts (27). The E. canis genome sequence also confirmed the presence of type IV secretion systems, and the proposed structure is shown in Figure 4.1 (29, 32). Mavromatis et al. specifically described two of these systems, namely VirB and VirD, which are thought to play a role in pathogen–host interactions and are possibly associated with protein secretion and inhibition of bacterial inclusion trafficking to lysosomes. This hypothesis conflicts with the observation made by Kumagai et al. that inhibition of two-component regulatory systems was enough to allow fusion of the phagosome with the lysosome (31); however, the study by Mavromatis et al. (25) examined these systems in E. chaffeensis, and not E. canis. Nevertheless, it is possible that the two-component system controls type IV secretion, as this has been described in other pathogenic bacteria including Agrobacterium tumefaciens and Escherichia coli (24, 31, 33). All of the Rickettsiales have genes for type IV secretion co-transcribed with enzymes, such as superoxide dismutase, that catabolize reactive oxygen species, but whether these are secreted by type IV secretion complexes needs to be investigated. In addition, E. chaffeensis has retained the capability for synthesizing most of its own amino acids including arginine, similar to E. ruminantium, thus allowing for better resistance of the bacterium to intracellular nitric oxide, the synthesis of which depletes host cell arginine (27). Interestingly, VirB, which is common among all of the Rickettsiales, has been associated with toxin secretion. This possibility warrants further investigation, particularly the determination of its role in initiating septic shock-like syndrome in diseases such as HME, ehrlichiosis ewingii, and human granulocytic anaplasmosis (HGA). Further comparison of ehrlichial genomes will provide insight and facilitate investigations of bacterial virulence factors, disease pathogenesis, and mechanisms of immune modulation, and will provide targets for vaccines or new antimicrobial therapies (27).

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TYPE IV SECRETION SYSTEM

B5

B5 B1

outer membrane

B2 B7

B7

inner membrane B9

B9

B2

B3

B4

B5

B8 B11

D4

ATP

B1

B10

B6

B4 B3

ATP

B6

ATP

B7

B8

B9

B10

B11

D4

03080 2/22/02

Figure 4.1. Type IV secretion system of Ehrlichia canis (www.genome.ad.jp/keg; ref. 32). The type IV secretion systems are multi-subunit cell envelope spanning structures composed of a secretion channel and often a pilus. In Gram-negative bacteria, the type IV secretion apparatus spans both membranes, the periplasm, and cell wall (29) (See Color Plates).

4.3 Ehrlichial Monocyte Entry, Developmental Stages, Differential Outer Membrane Protein Expression, and Manipulation of Host Defenses After ehrlichiae enter their vertebrate host and evade initial host immune responses, they must quickly be engulfed by host cells where they can replicate within early endosomes by binary fission, resulting in the characteristic morulae that can be observed by Diff-Quik staining (Figure 4.2). For E. chaffeensis and A. phagocytophilum, this process involves caveolae and glycosylphosphatidylinositol-anchored proteins, which mediate an endocytic pathway that bypasses phagolysosomal pathways (24). In addition, in human THP-1 macrophages, E. chaffeensis downregulates genes such as SNAP 23 (synaptosomal-associated protein, 23 kDa), Rab5A (member of RAS oncogene family), and STX16 (syntaxin 16), which are involved in membrane trafficking (21). The presence of two different morphologic forms of the bacterium has long been noticed during ultrastructural analysis of

ehrlichial morulae; however, the biological significance of the dense-cored cell (DC) or reticulate cell (RC) was only recently elucidated (Figure 4.3). Zhang et al. have determined that the DC of E. chaffeensis is the infectious form that enters host monocytes, a hypothesis that had been previously proposed without experimental support (34). DC forms predominate during the first hour post-infection, correlating with the time during which intermediate forms, presumably DC transforming into RC, are also observed. By 24 hours post-infection, single RC are observed within each morula, with some of them undergoing binary fission. At 48 hours post-infection, morulae contain several RC forms, which appear to be a result of extensive binary fission during the previous 24 hours. DC forms again predominated at 72 hours, correlating with the time when DC are released to begin a new cycle. DC forms are never observed undergoing binary fission; to the contrary, the RC forms are never found adhering to the host cell membrane or in a vacuole at 0 hours post-infection. Furthermore, DC and RC preferentially express gp120 and p28-19, respectively, supporting the role of gp120 as an adhesin required for attachment of the bacterium to the host cell membrane (35). In addition, host cell receptors such as E- and

40

Figure 4.2. Diff-Quik stain of an Ehrlichia muris-infected DH82 monocyte. The cytoplasm contains numerous morulae, and each usually harbors between 1 and 40 organisms (69) (See Color Plates).

Figure 4.3. Illustration of the developmental cycle of E. chaffeensis. A dense-cored (DC) cell attaches to the host cell, and it enters into the host through phagocytosis. In the phagosome, the DC transforms into the intermediate phase (IM)-1 and subsequently RC. RC multiplies by binary fission for 48 h and transforms into IM-2 cell and eventually matures into DC at 72 h, which is released by exocytosis or lysis of the host cell. Figure from: Zhang et al. 2007. (34)

L-selectin have been implicated in the binding of E. chaffeensis to human THP-1 monocytes (36). Ehrlichiae contain a multigene family of 28 kDa outer membrane proteins (i.e., p28), which is conserved among members of the genus. Additionally, certain immunodominant glycoproteins including gp 120 of E. chaffeensis and gp140 of E. canis migrated as larger-than-predicted molecular masses provoking

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further investigation to determine if post-translational modifications of these bacterial proteins occur. O-linked glycosylation was reported and is proposed to play a role in eliciting T cellindependent immune responses (22). These pathogens have also been found to differentially express macrophage- and tick cell-specific p28 outer membrane proteins, with p28-19 and -20 predominating within DH82 macrophages and p28-14 within ISE6 embryonic tick cells (37). The observation that p28-19 is preferentially expressed in host monocytes is consistent with the data presented by Zhang et al., who also reported a predominance of p28-19 in ehrlichial morulae later in the course of cell culture infection (i.e., ³ 6 days post-inoculation; ref. 36). Further supporting the preferential expression of p28-19 (OMP-19) in host cell macrophages, Nandi and Winslow reported that prior vaccination with OMP-19 protected C57BL/6 mice from fatal Ixodes ovatus Ehrlichia (IOE) infection, with the mice producing high titers of anti-OMP antibodies (38). These observations support the long evolutionary relationship between the bacterium and its vector and its niche within the host monocytes. How differential expression of these proteins provides an advantage to the organism within these extremely different environments is not known. Thus, in vivo investigations of ehrlichial infection in animal models and natural tick vectors will be necessary to determine the biological significance of these in vitro observations. To this end, a recent study by Ganta et al. determined that C57BL/6 mice have different responses to infection depending on the source of the inoculum (39). ISE6 tick cell-derived Ehrlichia inoculated into mice resulted in a more persistent infection, which includes relapses of increasing bacterial load. The ability of ehrlichiae not only to enter a host macrophage, inhibit phagolysosome fusion and replicate, but also to suppress or induce genes to facilitate these and other processes have also been reported (21, 23). Lin and Rikihisa found that E. chaffeensis infection downregulated surface expression of Toll-like receptors TLR-2 and TLR-4 and CD14, and inhibited the activation of several transcription factors that are involved in the induction of proinflammatory innate immune responses (21, 23). The mechanism by which downregulation of TLR-2 and TLR-4 benefits survival within the macrophage is not well understood, as pathogen-associated molecular patterns (PAMPs) have not been identified in ehrlichiae, and as mentioned previously, the traditional ligands for these receptors, peptidoglycan and LPS, that activate TLR-2 and TLR4, respectively, are not present in the bacterium (25). Some of the genes encoding traditional PAMPs may have been lost during reductive evolution of these bacteria allowing them to persist within their tick vectors, as ticks are known to have strong innate defenses toward these structures (24). Within the mammalian host, activation of other innate cells including natural killer T (NKT) cells has been observed in infections with pathogens that do not activate TLRs (40). NKT cell interaction with antigen-presenting cells, such as dendritic cells, may overcome the requirement for pattern recognition receptor activation in induction of the adaptive immune response. Recently, these non-traditional lymphocytes were found to be

4. Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology

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directly activated by E. muris and are critical for controlling the bacterial burden in a mouse model of mild HME (41). Microarray analysis of THP-1 cells infected with E. chaffeensis revealed repression of Th1 cytokines such as IL-12, IL-15, and IL-18, which are important inducers of adaptive Th1-mediated immune responses, as well as induction of a number of genes that inhibited apoptosis (21). All of the repressed genes are unique to ehrlichial infection of macrophages and have not been identified in other bacterial infections. These data need to be interpreted with caution as other factors involved in host defenses, including those that induce macrophage activation and target cell apoptosis, were not present in the in vitro system studied. For example, the same organism, E. chaffeensis, causes a variety of manifestations in humans, most likely due to differences in host factors, and this diversity has not been replicated in vitro. Neverthless, the in vitro studies have provided insight into the ability of ehrlichial organisms to survive inside one of the main cells that represents host defense against bacterial invasion, the mononuclear phagocyte.

have been observed to undergo apoptosis without evidence of ehrlichial infection (42, 43). These characteristics, along with the clinical presentation resembling toxic shock-like syndrome, support the hypothesis that dysregulation of the host immune response leads to tissue damage and eventually multi-system organ failure. Recently, Dierberg and Dumler reported a significantly greater amount of hemophagocytosis (macrophage activation) and an increased number of CD8+ cells in the lymph nodes of patients that died of HME (44). An investigation of liver tissues from autopsy cases in immunocompetent patients with HME showed that lymphohistiocytic foci and marked monocytic infiltration are common features, and Ehrlichia-infected cells are rarely identified (45). Unfortunately, most pathological evidence of HME is derived from autopsy cases; therefore, little is known about the organ pathology that occurs in acute disease that is followed by complete recovery.

4.4 Clinical Manifestations and Pathology of HME

The difficulty in obtaining clinical specimens to study the pathogenesis of and host defenses against HME has led to development of several useful animal models. E. chaffeensis infection does not cause disease that mimics that seen in humans, even in immunocompromised mice, and is completely cleared from wild-type animals within two weeks (46). Animal models using other ehrlichial species closely related to E. chaffeensis, including Ehrlichia muris and IOE, induce pathology that more closely mimics HME. Intraperitoneal (i.p.) inoculation of E. muris results in persistent asymptomatic infection in C57BL/6 mice, and lesions are observed in the liver, lungs, and spleen (18). Lymphohistiocytic infiltrates are present in both the liver and lung, and by day 20 poorly formed granulomas are being developed. Welldefined granulomas are present on day 30 post-infection, at which time ehrlichial organisms are no longer detected by immunohistochemistry. Organisms are detectible by real-time polymerase chain reaction (PCR) up to 150 days post-infection, which corresponds with very high serum antibody titers by indirect immunofluorescence assays. In Russia, E. muris infection of Ixodes persulcatus ticks has also been associated with the occurrence of a human disease similar to HME in the same region. Serum antibodies from patients presenting with fevers of unknown origin and symptoms similar to HME were found to react with antigens that are shared with E. chaffeensis. Ticks in the area were analyzed by PCR for the presence of ehrlichial DNA, and at this point only E. muris has been identified (47–49). IOE, a bacterium even more closely related to E. chaffeensis than E. muris, was isolated from ticks in Japan (50). The cell wall components demonstrated by immuno-ultrastructural studies indicated a close antigenic relationship between the ehrlichiae (51). i.p. IOE inoculation resulted in a progressive, fatal disease that closely mimics severe HME with mice developing toxic shock-like syndrome and multisystem

The clinical presentation of HME may be nonspecific Table 4.2, and the disease is difficult to diagnose. A characteristic sign, such as a rash, is observed in relatively few patients, and history of a tick bite may be lacking. Interstitial pneumonia, hepatic dysfunction, aseptic meningitis, and hemorrhages have been described. The severity of the disease is greater in elderly and immunocompromised patients; however, even in immunocompetent patients HME can be fatal. Despite the susceptibility of E. chaffeensis to doxycycline, the case fatality rate is still approximately 3%. Overwhelming ehrlichial infections are observed mainly in immunocompromised patients, and fatal ehrlichiosis in immunocompetent patients occurs with a low bacterial load. In addition, hepatocytes Table 4.2. Symptoms, clinical characteristics, and laboratory findings occurring during human monocytotropic ehrlichiosis (HME). Symptoms/Clinical characteristics (n = 237)a Fever Headache Myalgia Nausea Vomiting Rash Cough Pharyngitis Diarrhea Lymphadenopathy Abdominal pain Confusion a

Selected laboratory findings (n = 33)b 97% 81% 68% 48% 37% 36% 26% 26% 25% 25% 22% 20%

Elevated AST Elevated ALT Thrombocytopenia Leukopenia Anemia

90% 84% 73% 72% 55%

Adapted from Fishbein et al. 1994 Adapted from Eng et al. 1990, AST = aspartate aminotransferase, ALT = alanine aminotransferase b

4.5

Current Status of Animal Models

42

failure including hepatic injury characterized by elevated liver enzymes (AST>ALT) and a marked decrease in the frequency of CD4+ lymphocytes in the spleen, which may correlate with the lymphopenia observed in severe cases of HME (52, 53). Apoptosis of hepatocytes and sinusoidal lining cells occurs as early as day 5. By day 9 post-infection, a focal hepatic necrosis is observed, mainly in the midzone of the hepatic lobules. The lung pathology is also similar to that in HME with the interstitium focally thickened and an infiltration of monocytes present on day 9 post-infection. Ehrlichial morulae are not present within the cytoplasm of apoptotic cells as determined by double staining of the TUNEL preparations with anti-Ehrlichia antibodies. IOE has also been used to study secondary challenge infections in C57BL/6 mice (54). In this study, mice were given low-dose IOE and survived the initial infection; however, when given an additional inoculation with the same dose four weeks later, mice succumbed to the disease. The fatal outcome was CD8+ T cell-dependent and associated with high levels of serum TNF-α and CCL2. These observations further support the hypothesis that pathogenic CD8+ T cells play a role in fatal ehrlichiosis. The development of the E. muris and IOE models is a significant advance in the field, enabling the study of differences between mild and severe disease; however, the antigenic and genetic differences in the ehrlichiae cannot be ignored when

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interpreting the results. Recently, our laboratory reported that IOE when inoculated intradermally also resulted in mild disease with characteristics similar to E. muris that all mice inoculated intradermally survived an infectious dose that would be fatal had mice been inoculated intraperitoneally (53). This route of inoculation also better mimics natural ehrlichial transmission because the intradermal (i.d.) location is the inoculation site for ehrlichiae by feeding ticks. Furthermore, i.d. inoculation of IOE resulted in a lower systemic bacterial burden and much less inflammation and apoptosis in the liver when compared to i.p. inoculation of mice with the same dose of organisms. The hepatic lesions observed in these three mouse models (Figure 4.4) reflect the difference in the clinical severity. Mild forms of the disease initiated by either i.p. inoculation with E. muris or i.d. inoculation with IOE, resulted in a more localized, contained inflammatory response with low levels of serum TNF-α and IL-10, and relatively little apoptosis. To the contrary, i.p. inoculation with IOE produced dispersed hepatic inflammation and extensive apoptosis of both hepatocytes and mononuclear cells, which correlates with an uncontrolled inflammatory response leading to high levels of serum TNF-α and IL-10 (53, 55). At this time, IOE has not been associated with human disease; however, its ability to infect both invertebrate and vertebrates hosts in Japan and its striking virulence in mice, warrants further investigation of potential human involvement.

Figure 4.4 Comparison of liver pathology in three mouse models of human ehrlichiosis. On day 7 post-infection, E. muris inoculated intraperitoneally (A) and IOE inoculated intradermally (B) both result in mild disease characterized by perivascular lymphohistiocytic infiltration. In contrast, IOE inoculated intraperitoneally results in severe apoptosis (C) and multi-focal hepatic necrosis (D) (See Color Plates).

4. Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology

4.6 Characteristics of the Protective and Detrimental Immune Responses to Ehrlichiae Both cell-mediated and humoral immunity are required for complete protection against Ehrlichia. Cell-mediated immunity, orchestrated mainly by CD4+ Th1 cells, is required for protection against severe disease (46, 56). Interestingly, although Ehrlichia are obligately intracellular organisms, anti-Ehrlichia antibodies provide passive protection against i.p. inoculation of IOE (52, 56–60). Mouse models of HME also allow the determination of the factors that lead to the development of severe ehrlichiosis, which include high concentrations of TNF-α and IL-10 in the serum, a high frequency of TNF-α-producing CD8+ T cells in the spleen, low IL-12 levels in the spleen, and a 40-fold decrease in the number of IFN-γ-producing CD4+ T cells (52). The decrease in IL-12, one of the main Th1-promoting cytokines, may be due to multiple factors including intracellular repression of IL-12 by the organism itself (36) or apoptosis of host macrophages, one of the main antigen-presenting cells involved in early IL-12 production. In several different mouse strains, induction of Th1 immunity has been essential to host survival, and when CD4+ T cell help is compromised, the immune response becomes dysregulated and destructive (52). Overall, the pathogenesis of ehrlichial disease appears to be multifactorial as in other septic conditions, which explains the inability to control disease severity by inhibiting one component such as TNF-α (55). Whether features of the defective immune response that have been characterized in mice, such as high levels of serum TNF-α and IL-10, occur in humans needs to be determined by performing more clinical studies, particularly in regions where potential vectors of HME are prevalent.

4.7 Tick Vectors, Ecology, and Ehrlichial Transmission As previously mentioned, the distribution of the arthropod vectors and vertebrate hosts correlates with the presence of the diseases that they transmit (11, 61–63). A prototypical example of this tight vector-host relationship is that of HME cases and the distribution of its primary vector (Amblyomma americanum) in the United States. This relationship also correlates with the regions in which the reservoir host is found; however, this association is not as striking for HME because the main reservoir, the white-tailed deer (Odocoileus virginianus) is extremely widely distributed in North America. The transmission cycle for E. chaffeensis (Figure 4.5) is representative of all of the ticktransmitted human ehrlichioses. In addition, E. chaffeensis has been identified in several other mammals including canids, goats, raccoons, and opossums (19). Other vectors of E. chaffeensis include other Metastriata ticks in the Ixodidae family such as Ixodes pacificus (64), Ixodes ricinus (65), Haemaphysalis yeni (66), Amblyomma testudinarium (66), possibly Amblyomma maculatum (67), and Dermacentor variabilis (68). In addition,

43

Wen et al. (11) reported the presence of E. chaffeensis DNA by PCR in several tick species including A. testudinarium, H. yeni, I. ovatus, I. persulcatus, Boophilus microplus, and Dermacentor silvarum, in both northern and southern China; previously this bacterium was thought to exist only in North America and possibly Thailand (13). If E. chaffeensis is successfully transmitted by these various metastriata tick vectors, the distribution of HME may be more widespread than predicted. However, many of the previously mentioned reports used only serology and/or PCR for detection of either anti-Ehrlichia antibodies or ehrlichial DNA, which may limit interpretation of the results due to crossreactivity or genomic similarities among ehrlichial species, respectively. Other more conclusive studies that include isolation of the bacteria and further genetic characterization are necessary. The occurrence of HME-like illness described in patients with febrile illness in Russia, along with the presence of I. persulcatus ticks throughout the country (9), makes this region ideal for studying of not only vector–host ecology, but also of its relationship with the incidence of human disease. Ehrlichiae have not been isolated from I. persulcatus; even though genetically they appear to be the mildly virulent E. muris, the agent deserves further study including isolation and experimental infection of mice to confirm its pathogenicity. The Anan and IOE (formerly H565 strain) strains differed by only three nucleotides of 1,449 base pairs in the 16S rRNA gene; however, when inoculated intraperitoneally into mice, these strains caused 0 and 100% fatalities, respectively (50). The main reservoir for E. muris in Japan is wild mice (Apodemus spp.), which makes this vector–host relationship feasible for manipulation in the laboratory (12). The range of I. persulcatus ticks extends contiguously from Eastern Europe to Korea and Japan, an area comprising nearly one-fifth of the global population (48). Further studies in these regions are necessary to determine if this species is a competent vector of a HME. I. ovatus ticks, the species from which IOE was isolated, are found throughout Japan which also provides an ideal environment for future ecological studies. Although significant advances have been made in our understanding of the host immune responses to ehrlichiosis, our knowledge could be further expanded if the effect of natural tick transmission and salivary components could be determined. Experimental transmission studies utilizing these organisms and their corresponding vectors have not been reported.

4.8

Conclusions

Ehrlichiae have evolved many mechanisms to survive within their arthropod and mammalian hosts, including immune evasion strategies such as the absence of identified PAMPs (e.g., LPS and peptidoglycan), the ability to vary surface antigens by differential expression of bacterial glycoproteins, and the inhibition of phagolysosome fusion involving two-component regulatory systems. In addition, ehrlichiae are able to synthesize several nucleotides, vitamins and cofactors to aid in their

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Figure 4.5. A life cycle of E. chaffeensis. Noninfected larvae obtaining blood from a bacteremic vertebrate reservoir host (e.g., white-tailed deer [shaded]) become infected and maintain ehrlichiae to the nymphal stage. Infected nymphs may transmit E. chaffeensis to susceptible reservoir hosts (unshaded) or to humans during acquisition of blood. Infected adult ticks, having acquired ehrlichiae either by transstadial transmission from infected nymphal stage or during blood meal acquisition as noninfected nymphs on infected deer, may also pass E. chaffeensis to humans or other susceptible reservoirs. Transovarial transmission has not been demonstrated, and eggs and unfed larvae are presumably not infected (19).

intracellular survival, and have genes that encode enzymes to counteract host defenses. Continued comparison of the genomes of these pathogens and further characterization of encoded proteins will provide valuable insight into the pathogenesis initiated by these unique bacteria. The availability of the different animal models will facilitate our understanding of the host response to these agents. The range of clinical manifestations seen even in immunocompetent hosts proves that host factor differences cannot be ignored when studying these diseases. Furthermore, the ability of organisms that lack PAMPs (such as LPS and peptidoglycan) to cause toxic shock-like syndrome will provide valuable information about novel mechanisms of inducing immune-mediated pathology. Human studies investigating these immune phenomena are much needed and will help to confirm many of the intriguing mechanisms elucidated using animal models. Finally, the emergence of these infectious diseases and increasing reports of human cases worldwide necessitates

further studies of potential vector–host relationships. As awareness of these diseases and surveillance increases, climate change and ecological disturbances continue, and effective methods to detect these pathogens are more readily available, the prevalence of human ehrlichioses throughout the world will continue to challenge current concepts.

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4. Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology 4. Aguirre E, Sainz A, Dunner S, Amusategui I, Lopez L, RodriguezFranco F, Luaces I, Cortes O, Tesouro MA (2004) First isolation and molecular characterization of Ehrlichia canis in Spain. Vet Parasitol 125:365–372. 5. Loftis AD, Reeves WK, Szumlas DE, Abbassy MM, Helmy IM, Moriarity JR, Dasch GA (2006) Rickettsial agents in Egyptian ticks collected from domestic animals. Exp Appl Acarol 40:67–81. 6. Ndip LM, Ndip RN, Esemu SN, DIckmu VL, Fokam EB, Walker DH, McBride JW (2005) Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii. Vet Micrbiol 111:59–66. 7. Wielinga PR, Gaasenbeek C, Fonville M, de Boer A, de Vries A, Dimmers W, Op Akkerhuis GJ, Schouls LM, Borgsteede F, van der Giessen JW (2006) Longitudinal analysis of tick densities and Borrelia, Anaplasma and Ehrlichia infection of Ixodes ricinus ticks in different habitat areas in the Netherlands. Appl Environ Microbiol 72:7594–7601. 8. Alekseev AN, Dubinina HV, Pol I, Van DeSchouls LM (2001) Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol 39:2237–2242. 9. Rar VA, Fomenko NV, Dobrotvorsky AK, Livanova NN, Rudakova SA, Fedorov EG, Astanin VB, Morozova OV (2005) Tickborne pathogen detection, Western Siberia, Russia. Emerg Infect Dis 11:1708–1715. 10. Shpynov SN, Rudakov NV, Iastrebov VK, Leonova GN, Khazova TG, Egorova NV, Borisova ON, Preider VP, Bezrukov GV, Fedorov EG, Fedianin AP, Sherstneva MB, Turyshev AG, Gavrilov AP, Tankibaev MA, Fournier PE, Raoult D (2004) [New evidence for the detection of ehrlichia and anaplasma in ixodes ticks in Russia and Kazakhstan]. Med Parazitol (Mosk) 2:10–14. 11. Wen B, Cao W, Pan H (2003) Ehrlichiae and ehrlichial diseases in China. Ann NY Acad Sci 990:45–53. 12. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, Hiramitsu Y, Tajima T (2006) Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl Environ Microbiol 72:1102–1109.. 13. Heppner DG, Wongsrichanalai C, Walsh DS, McDaniel P, Eamsila C, Hanson B, Paxton H (1997) Human ehrlichiosis in Thailand. Lancet 350:785–786. 14. Parola P, Cornet JP, Sanogo YO, Miller RS, Thien HV, Gonzalez JP, Raoult D, Telford III Sr, Wongsrichanalai C (2003) Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol 41:1600–1608. 15. Suksawat J, Xuejie Y, Hancock SI, Hegarty BC, Nilkumhang P, Breitschwerdt EB (2001) Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand. J Vet Intern Med 15:453–462. 16. Olano JP, Masters E, Hogrefe W, Walker DH (2003) Human monocytotropic ehrlichiosis, Missouri. Emerg Infect Dis 9:1579–1586. 17. Kovats RS, Campbell-Lendrum DH, McMichael AJ, Woodward A, Cox JS (2001) Early effects of climate change: do they include changes in vector-borne disease? Philos Trans R Soc Lond B Biol Sci 356:1057–1068. 18. Olano JP, Hogrefe W, Seaton B, Walker DH (2003) Clinical manifestations, epidemiology, and laboratory diagnosis of human monocytotropic ehrlichiosis in a commercial laboratory setting. Clin Diagn Lab Immunol 10:891–896. 19. Paddock CD, Childs JE (2003) Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin Microbiol Rev 16:37–64.

45 20. Lin M, Rikihisa Y (2003) Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect Immun 71:5324–5331. 21. Zhang JZ, Sinha M, Luxon BA, Yu XJ (2004) Survival strategy of obligately intracellular Ehrlichia chaffeensis: novel modulation of immune response and host cell cycles. Infect Immun 72:498–507. 22. McBride JW, Yu X-J, Walker DH (2000) Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis. Infect Immun 68:13–18. 23. Lin M, Rikihisa Y (2004) Ehrlichia chaffeensis downregulates surface toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF-κB, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol 6:175–186. 24. Rikihisa Y (2006) Ehrlichia subversion of host innate responses. Curr Opin Microbiol 9: 95–101. 25. Mavromatis K, Doyle CK, Lykidis A, Ivanova N, Francino MP, Chain P, Shin M, Malfatti S, Larimer F, Copeland A, Detter JC, Land M, Richardson PM, Yu XJ, Walker DH, McBride JW, Kyrpides NC (2006) The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies. J Bacteriol 188:4015–4023. 26. Nethery K, Doyle C, Elsom B, Herzog N, Popov V, McBride J (2005) Ankyrin repeat containing immunoreactive 200 kDa glycoprotein (gp200) orthologs of Ehrlichia chaffeensis and E. canis are translocated to the nuclei of infected monocytes, abstr. O-60. Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logrono, Spain, June 18–21. 27. Hotopp JC, Lin M, Madupu R, Crabtree J, Angiuoli SV, Eisen J, Seshadri R, Ren Q, Wu M, Utterback TR, Smith S, Lewis M, Khouri H, Zhang C, Niu H, Lin Q, Ohashi N, Zhi N, Nelson W, Brinkac LM, Dodson RJ, Rosovitz MJ, Sundaram J, Daugherty SC, Davidsen T, Durkin AS, Gwinn M, Haft DH, Selengut JD, Sullivan SA, Zafar N, Zhou L, Benahmed F, Forberger H, Halpin R, Mulligan S, Robinson J, White O, Rikihisa Y, Tettelin H (2006) Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet 2:e21. 28. Popov VL, Han VC, Chen SM, Dumler JS, Feng HM, Andreadis TG, Tesh RB, Walker DH (1998) Ultrastructural differentiation of the genogroups in the genus Ehrlichia. J Med Microbiol 47:235–251. 29. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E (2005) Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol 59:451–485. 30. Cheng Z, Kumagai Y, Lin M, Zhang C, Rikihisa Y (2006) Intraleukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel. Cell Microbiol 8:1241–1252. 31. Kumagai Y, Cheng Z, Lin M, Rikihisa Y (2006) Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion. Infect Immun 74:5014–5022. 32. Kanehisa M, Goto S, Hattori M, oki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357. 33. Zahrl D, Wagner M, Bischof K, Koraimann G (2006) Expression and assembly of a functional type IV secretion system elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. J Bacteriol 188:6611–6621. 34. Zhang JZ, Popov VL, Gao S, Walker DH, Yu XJ (2007) The developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell Microbiol 9:610–618.

46 35. Popov VL, Yu X, Walker DH (2000) The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry. Microb Pathog 28:71–80. 36. Zhang J-Z, McBride JW, Yu X-J (2003) L-selectin and E-selectin expressed on monocytes mediating Ehrlichia chaffeensis attachment onto host cells. FEMS Microbiol Let 227:303–309. 37. Singu V, Liu H, Cheng C, Ganta RR (2005) Ehrlichia chaffeensis expresses macrophage- and tick cell-specific 28-kilodalton outer membrane proteins. Infect Immun 73:79–87. 38. Nandi B, Winslow G (2006) Identification of T cell epitopes Ehrlichia outer membrane proteins that elicit protective immunity, abstr. 124. 20th Meeting of The American Society for Rickettsiology in conjunction with the 5th International Conference on Bartonella as Emerging Pathogens, Asolimar, CA, September 2–7. 39. Ganta RR, Cheng C, Miller EC, McGuire BL, Peddireddi L, Sirigireddy KR, Chapes SK (2006) Differential clearance and immune responses to tick cell vs. macrophage culture-derived Ehrlichia chaffeensis in mice. Infect Immun 75:135–145. 40. Munz C, Steinman RM, Fujii S (2005) Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. J Exp Med 202:203–207. 41. Mattner J, Debord KL, Ismail N, Goff RD, Cantu C III, Zhou D, Saint-Mezard P, Wang V, Gao Y, Yin N, Hoebe K, Schneewind O, Walker D, Beutler B, Teyton L, Savage PB, Bendelac A (2005) Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434:525–529. 42. Dumler JS, Brouqui P, Aronson J, Taylor JP, Walker DH (1991) Identification of Ehrlichia in human tissue. N Engl J Med 325:1109–1110. 43. Walker DH, Dumler JS (1997) Human monocytic and granulocytic ehrlichioses. Discovery and diagnosis of emerging tickborne infections and the critical role of the pathologist. Arch Pathol Lab Med 121:785–791. 44. Dierberg KL, Dumler JS (2006) Lymph node hemophagocytosis in rickettsial diseases: a pathogenetic role for CD8 T lymphocytes in human monocytic ehrlichiosis (HME)? BMC Infect Dis 6:121. 45. Sehdev AE, Dumler JS (2003) Hepatic pathology in human monocytic ehrlichiosis. Ehrlichia chaffeensis infection. Am J Clin Pathol 119:859–865. 46. Ganta RR, Cheng C, Wilkerson MJ, Chapes SK (2004) Delayed clearance of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice. Infect Immun 72:159–167. 47. Feng HM, Walker DH (2004) Mechanisms of immunity to Ehrlichia muris: a model of monocytotropic ehrlichiosis. Infect Immun 72:966–971. 48. Ravyn MD, Korenberg EI, Oeding JA, Kovalevskii YV, Johnson RC (1999) Monocytic Ehrlichia in Ixodes persulcatus ticks from Perm, Russia. Lancet 353:722–723. 49. Vorobyeva NN, Korenberg EI, Grigoryan YV (2002) Diagnostics of tick-borne diseases in the endemic region of Russia. Wien Klin Wochenschr 114:610–612. 50. Shibata S, Kawahara M, Rikihisa Y, Fujita H, Watanabe Y, Suto C, Ito T (2000) New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan. J Clin Microbiol 38:1331–1338. 51. Sotomayor E, Popov V, Feng H-M, Walker DH, Olano JP (2001) Animal model of fatal human monocytotropic ehrlichiosis. Am J Pathol 158:757–769. 52. Ismail N, Soong L, McBride JW, Valbuena G, Olano JP, Feng HM, Walker DH (2004) Overproduction of TNF-α by CD8+ type 1

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Chapter 34 Biological Basis and Clinical Significance of HIV Resistance to Antiviral Drugs Mark A. Wainberg and Susan Schader

34.1

Introduction

HIV-1 drug resistance has emerged as a major factor that limits the effectiveness of antiviral drugs in treatment regimens. Many studies have shown that the development and transmission of drug-resistant (DR) HIV-1 is largely a consequence of incompletely suppressive antiretroviral regimens; HIV-1 drug resistance can significantly diminish the effectiveness and duration of benefit associated with combination therapy for the treatment of HIV/AIDS (1–6). Resistance-conferring mutations in both the HIV-1 reverse transcriptase (RT) and protease (PR) genes may precede the initiation of therapy due to both spontaneous mutagenesis and the spread of resistant viruses by sexual and other means. However, it is also generally believed that multiple drug mutations to any single or combination of antiretrovirals (ARVs) are required in order to produce clinical resistance to most ARVs and that these are in fact selected following residual viral replication in the presence of incompletely suppressive drug regimens (7–9). In the case of the PR inhibitors (PIs; refs. 10–12), and most nucleoside analog RT inhibitors (NRTIs), the development of progressive high-level phenotypic drug resistance follows the accumulation of primary resistance-conferring mutations in each of the HIV-1 PR and RT genes (13–15). Non-nucleoside RT inhibitors (NNRTIs) have low genetic barriers for the development of drug resistance and, frequently, a single primary drug resistance mutation to any one NNRTI may be sufficient to confer high-level phenotypic drug resistance to this entire class of ARVs (16, 17). Furthermore, differences have also been reported in regard to the development and evolution of ARV drug resistance between subtype B HIV-1 and several group M non-B subtypes. Non-B subtypes, e.g, subtype C HIV-1 variants, are known to possess naturally occurring polymorphisms at several From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

RT and PR codons that are implicated in drug resistance (18, 19). In some studies, the presence of these polymorphisms did not significantly reduce susceptibility to ARVs in phenotypic resistance assays or limit the effectiveness of an initial ARV therapy (ART) regimen for a period of up to 18 months (18–20). However, it has also been suggested that polymorphisms at resistance positions may sometimes facilitate selection of novel pathways leading to drug resistance, especially with incompletely suppressive ARV regimens (18). This, in turn, may have important clinical implications with respect to choice of effective ART. This warrants increased genotypic surveillance on a worldwide basis, as the prevalence of non-B HIV-1 infection is increasing rapidly. As illustrated in Figures 34.1-34.3, it has been possible to select numerous drug resistance mutations for all licensed ARVs and investigational agents such as the HIV-1 entry inhibitors that are currently undergoing final stages of clinical testing (5, 6, 21). In view of the hypervariability of HIV-1 and limitation of existing ARV combinations to completely suppress viral replication, it is essential that new anti-HIV drug discovery initiatives focus on the identification of new therapeutic targets and the development of ARV agents with more robust genetic barriers and a broader spectrum of activity against DR HIV-1 variants.

34.2 Generation of HIV-1 Drug Resistance Resistance mutations to ARVs may arise spontaneously as a result of the error-prone replication of HIV-1 and, in addition, are selected both in vitro and in vivo by pharmacological pressure (22–24). The high rate of spontaneous mutation in HIV-1 has been largely attributed to the absence of a 3’->5’exonuclease proof-reading mechanism. Sequence analyses of HIV-1 DNA have detected several types of mutations including base substitutions, additions and deletions (22). The frequency of spontaneous mutation for HIV-1 varies considerably as a result 309

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of differences among viral strains studied in vitro (25). Overall mutation rates for wild-type (WT) laboratory strains of HIV-1 have been reported to range from 97 X 10–4 to 200 X10–4 per nucleotide for the HXB2 clonal variant of HIV-1 to as high as 800 X 10–4 per nucleotide for the HIV-1 NY5 strain (22, 25). In addition to the low fidelity of DNA synthesis by HIV1 RT, other interdependent factors that affect rates of HIV mutagenesis include RT processivity, viral replication capac-

ity, viral pool size, and availability of target cells for infection (26–29). It follows that an alteration in any or combination of these factors might influence the development of HIV drug resistance. There is also data showing that thymidine analog mutations (TAMs) in RT can significantly increase the likelihood of further mutant HIV-1 distributions and evolution of drug resistance; furthermore, this can happen in the presence or absence of concomitant NRTIs (30, 31).

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VML Primary mutation Secondary mutation Significance unknown

Figure 34.3. The letter designation at the top of a box refers to the amino acid that is present in the wild-type sequence of PR. The letters at the bottom refer to substitutions associated with drug resistance. Sometimes, several different amino acid changes at the same codon can conferresistance, drug as for example both I and L in the case of position 46 (See Color Plates).

34.3

Inhibitors of RT

The RT enzyme is encoded by the HIV pol gene and is responsible for the transcription of double-stranded proviral DNA from viral genomic RNA. Two categories of drugs have been developed to block RT; these are NRTIs that act to arrest DNA chain elongation by acting as competitive inhibitors of RT and NNRTIs that act as non-competitive antagonists of enzyme activity by binding to the catalytic site of RT. NRTIs are administered to patients as precursor compounds that are phosphorylated to their active triphosphate form by cellular enzymes. These compounds lack a 3’ hydroxyl group necessary for elongation of viral DNA. These analogs can compete effectively with normal deoxynucleotide triphosphate (dNTP) substrates for binding to RT and incorporation into viral DNA (32, 33). NNRTI antiviral activity is incompletely understood but is known to involve the binding of these non-competitive inhibitors to a hydrophobic pocket close to the catalytic site of RT (34, 35). NNRTI inhibition reduces the catalytic rate of polymerization without affecting nucleotide binding or nucleotideinduced conformational change (36). NNRTIs are particularly active at template positions at which the RT enzyme naturally pauses. NNRTIs do not seem to influence the competition between dideoxynucleotide triphosphates (ddNTPs) and the

naturally occurring dNTPs for insertion into the growing proviral DNA chain (37). Both types of RT inhibitors have been shown to successfully diminish plasma viral burden in HIV-1 infected subjects. However, monotherapy with all drugs has led to drug resistance. Patients who receive combinations of three or more drugs are less likely to develop resistance, since these “cocktails” can suppress viral replication with much greater efficiency than single drugs or two drugs in combination. Although mutagenesis is less likely to happen in this circumstance, it can still occur, and the emergence of DR breakthrough viruses has been demonstrated in patients receiving highly active ART (HAART; refs. 38 and 39). Furthermore, the persistence of reservoirs of latently infected cells represents another major impediment to currently applied anti-HIV chemotherapy (40). Replication of HIV might resume once therapy is stopped or interrupted and, therefore, eradication of a latent reservoir of 105 cells might take as long as 60 years, a goal that is not practical with currently available drugs and technology (40, 41). Resistance to 3TC ((-)-2’, 3’-dideoxy-3’-thiacytidine, lamivudine) develops quickly whereas resistance to other NRTIs commonly appears only after about six months of therapy. Phenotypic resistance is detected by comparing the IC50 (or drug concentration capable of blocking viral replication by 50%) of pretreatment viral isolates with those obtained after

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therapy. Thus, higher IC50 values obtained after several months of treatment reflect a loss in viral susceptibility to ARV agents. Selective polymerase chain reaction (PCR) analysis of the RT genome confirms that the number of mutations associated with drug resistance increases concomitantly with increases in IC50 values. Mutations associated with drug resistance have been reported in response to the use of any single NRTI or NNRTI (42). However, not all drugs elicit the same mutagenic response; sensitivity and resistance patterns must be considered on an individual drug basis. For example, patients on 3TC monotherapy may develop high-level, i.e. 1000-fold resistance within weeks, whereas six months or more are often required in order for sensitivity to ZDV to drop by 50-100-fold. In contrast, HIV may appear to remain reasonably sensitive, even after prolonged monotherapy, to four of the other commonly used nucleoside analogs: ddI (didanosine), ddC (zalcitabine), d4T (stavudine) and ABC (abacavir). In the case of ZDV, increases in IC50 below threefold are regarded as non-significant, while 10-50 fold increases usually represent partial resistance, and increases above 50-fold denote high-level resistance. Patient resistance to nucleoside analogs can often develop independently of the dose of drug that is administered. Tissue culture data have shown that HIV-1 resistance can be easily demonstrated against each of NRTIs, NNRTIs and PIs, by gradually increasing the concentration of compound in the tissue culture medium (43, 44). Cell lines are especially useful in this regard, since HIV replication occurs very efficiently in such hosts. Tissue culture selection provides an effective pre-clinical means of studying HIV mutagenesis, especially since the same resistance-conferring mutations that arise in cell culture also appear clinically. Owing to the high turnover and mutation rate of HIV-1, the retroviral quasispecies will also include defective virions and singly mutated DR variants that are present prior to commencement of therapy. Multiply mutated variants appear later, because it requires time to accumulate multiple mutations within a single viral genome, and are not commonly found in the retroviral pool of untreated patients. An exception to this involves cases of new infection with DR viruses transmitted from extensively treated individuals. Patients with advanced infection have a higher viral load and a broader range of quasispecies than newly-infected individuals. Such patients are often immunosuppressed and may also have diminished ability to immunologically control viral replication, possibly leading to more rapid development of drug resistance. Site-directed mutagenesis has shown that a variety of RT mutations encode HIV resistance to both NRTIs and NNRTIs. Crystallographic and biochemical data have demonstrated that mutations conferring resistance to NNRTIs are found in the peptide residues that make contact with these compounds within their binding pocket (34, 35). Resistance-encoding mutations to NRTIs are found in different regions of the RT enzyme, probably due to the complexity of nucleoside incorporation, which involves

M. A. Wainberg and S. Schader

several distinct steps. These mutations can decrease RT susceptibility to nucleoside analogs. A summary of primary RT mutations has been published elsewhere (42). It has also been shown that a family of insertion and deletion mutations between codons 67 and 70 can cause resistance to a variety of NRTIs including ZDV, 3TC, ddI, ddC, and d4T. Usually, these insertion mutations confer multidrug resistance (MDR) when present in a ZDV-resistant background. Another less frequently observed resistance mutation, K65R, has been shown to be associated with prior treatment with ABC-containing regimens and results in reduced antiviral susceptibility to both ABC and the acyclic RT nucleotide analog tenofovir (TDF). Hence, resistance to these ARV agents can develop via genetic pathways involving either the TAMs or K65R as hallmark drug resistance mutations (45). In recent years, the proportion of genotyped clinical samples containing K65R has increased from less than 1 to almost 4%, reflecting the increased use of TDF in treatment regimens. Diminished sensitivity to NNRTIs appears quickly both in culture selection protocols and in patients (34, 37). NNRTIs share a common binding site, and mutations that encode NNRTI resistance are located within the binding pocket that makes drug contact (34, 35, 37, 44, 46–49). This explains the finding that extensive cross-resistance is observed among all currently approved NNRTIs (49, 50). A substitution at codon 181 (tyrosine to cysteine; Y181C) is a common mutation that encodes cross-resistance among many NNRTIs (46, 51, 52). Replacement of Y181 by a serine or histidine also conferred HIV resistance to NNRTIs (53). A mutation at amino acid 236 (proline to leucine; P236L), conferring resistance to a particular class of NNRTIs that include delavirdine, can also diminish resistance to nevirapine and other NNRTIs, particularly if a Y181C mutation is also present in the same virus (54). Other important substitutions are Y188C and Y188H that can also confer NNRTI resistance. Another drug resistance mutation, namely K103N (lysine to asparagine), is commonly observed and is responsible for reduced susceptibility to all approved NNRTIs (46, 51, 52). Substitution of K103N results in alteration of interactions between NNRTIs and RT. The K103N mutation shows synergy with Y181C in regard to resistance to NNRTIs, unlike antagonistic interactions involving Y181C and P236L (55). Resistance to NNRTIs is also observed in cell-free enzyme assays (51, 53, 56–58). Both Y181I and Y188L mediate decreased sensitivity to NNRTIs without affecting either substrate recognition or catalytic efficiency, supporting the idea that resistance to NNRTIs is attributable to diminished ability of these drugs to be bound by RT.

34.4

PR Inhibitors

DR viruses have been observed in the case of all PR inhibitors (PIs) developed to date (11, 12, 59). In addition, some strains of HIV have displayed cross-resistance to a variety of

34. HIV Resistance to Antiviral Drugs

PIs after either clinical use or in vitro drug exposure (11, 12, 59). In general, the patterns of mutations observed with PIs are more complex than those observed with RT antagonists. First, a greater number of mutations within the PR gene are involved. This involves greater variability, as well, in temporal patterns of appearance of different mutations and the manner in which different combinations of mutations can give rise to phenotypic resistance. These data suggest that the PR enzyme can adapt more easily than RT to pressures exerted by antiviral drugs. At least 40 mutations in PR have been identified as responsible for resistance to PIs (11, 12, 59). Certain of the mutations within the PR gene are more important than others and can confer resistance, virtually on their own, to at least certain PIs (11, 12, 59). One mutation, in particular, D30N, is probably unique to nelfinavir, a potent HIV PR inhibitor. However, a variety of other mutations may confer cross-resistance among multiple drugs within the PI family. In addition, wide arrays of secondary mutations have been observed, that, when combined with primary mutations, can cause increased levels of resistance to occur. On the other hand, the presence of certain of these secondary mutations on their own may not lead to drug resistance, and, in this context, some of these amino acid changes should be considered to represent naturally-occurring polymorphisms. In addition, it should be noted that resistance to PIs can also result from mutations within the substrates of the PR enzyme, i.e. the gag and gag-pol precursor proteins of HIV. A variety of studies have now shown that mutations at cleavage sites within these substrates can be responsible for drug resistance, both in tissue culture as well as in treated patients. However, the full clinical significance of cleavage site mutations in regard to PR resistance is not yet understood.

34.5 ARV Drug Resistance in Non-B Subtypes of HIV-1 Group M Genotypic divergence of pol gene sequences between different HIV-1 subtypes is only beginning to be investigated, although the RT and PR enzymes are the main targets of anti-retroviral therapy (60–64). Group O and HIV-2 viruses carry natural polymorphisms Y181C and Y181I that confer intrinsic resistance to NNRTIs (65–67). Subtype F isolates, showing 11% nucleotide sequence variation from subtype B and group M viruses, have also been reported to have reduced sensitivity to some NNRTIs while retaining susceptibility to others such as nevirapine and delavirdine, NRTIs and PIs (68, 69). In contrast, the drug sensitivity of subtype C isolates from treatment-naive patients in Zimbabwe was reported to be similar to that of subtype B isolates (69, 70). Recent studies conducted with Ethiopian subtype C clinical isolates showed natural resistance to NNRTIs in one case and resistance to ZDV in another, due to natural polymorphisms at positions G190A and K70R, respectively (71). Another study reported no differences in drug susceptibility among subtypes A, B,

313

C, and E; subtype D viruses showed reduced susceptibility due to rapid growth kinetics (72). High prevalence (i.e. 94%) of a valine polymorphism (GTG) at position 106 in RT from subtype C HIV-1 clinical isolates has also been reported (73). In tissue culture experiments, selection of subtype C with efavirenz (EFV) was associated with development of high-level (i.e. 100-1000 fold) phenotypic resistance to all NNRTIs. This was a consequence of a V106M mutation that arose in place of the V106A substitution that is more commonly seen with subtype B viruses (73). This V106M mutation conferred broad cross-resistance to all currently approved NNRTIs and was selected on the basis of differential codon usage at position 106 in RT, due to redundancy in the genetic code. Genotypic diversity and drug resistance may be particularly relevant in establishing treatment strategies against African and Asian strains. First, since many antiviral drugs have been designed based on sequences of subtype B RT and PR enzymes, and drug resistance profiles, if not responses, may be different for non-B viral strains. Second, drug resistance may develop more rapidly in resource-poor countries if only sub-optimal therapeutic regimens are available. Global phenotypic and genotypic screening of non-B subtypes is warranted so as not to jeopardize the outcome of recently introduced ARV treatments (74).

34.6 Transmission of HIV Drug-Resistance As stated, HAART, including drugs that inhibit the RT and PR enzymes of HIV-1, has resulted in declining morbidity and mortality (75). The failure to completely suppress viral replication allows for the development of genotypic changes in HIV-1 that confer resistance to each of the three major classes of ARV drugs (76–78). Cumulative data indicate that single DR variants can be transmitted to approximately 10 to 15% of newly infected persons in western countries in which ARVs have been available for many years, with transmission of dual and triple-class MDR observed in 3–5% of cases (79–82). There is concern that the transmission of MDR viruses in primary HIV-1 infection (PHI) may limit future therapeutic options. Treatment failure has been observed in several individuals harboring MDR infections (82–84). Some reports have shown an impaired fitness of transmitted MDR variants compared with WT infections acquired in PHI (85), and the mutations that were transmitted in such patients persisted in the absence of treatment (85). This persistence differs from the rapid outgrowth of WT viruses in established infections upon treatment interruption, due to the selective growth advantage and fitness of WT variants (85–87). Taken together, these findings suggest that archival WT viruses may not exist in MDR infections transmitted during PHI. Several reports have also documented cases of intersubtype superinfection (A/E and B) in recently infected (RI) intravenous drug users (IDUs; refs. 88 and 89). Other

314

studies have failed to confirm superinfection following intravenous drug exposure, suggesting that superinfection is a relatively rare event (90, 91). Several subsequent reports demonstrated superinfection in subtype B infections. In one case, a WT superinfection arose following a primary MDR infection (92, 93). It is important to assess the virological consequences of transmission of DR variants in primary infection, as well as the time to disappearance of resistant virus in those patients not initially treated. Genotypic analysis indicates that a single dominant HIV-1 species can persist for more than 2 years in circulating plasma and peripheral blood mononuclear cells, regardless of route of transmission. Resistant and MDR infections can persist for 2 to 7 years following PHI. Superinfection with a second MDR strain in a patient originally infected with a MDR strain from an identified source partner has also been described (85). Despite a rapid decline in plasma viremia suggestive of an effective immune response, this patient was susceptible to a second infection which occurred concomitant with a dramatic rise in viral load. Five other subtype B superinfections have been described, as well as three intersubtype A/E and B superinfections (85, 88, 89, 92–95). Six of the seven superinfections described have occurred in the first year following initial infection. Many have attributed superinfection to co-infection during primary infection. Two longitudinal studies involving IDU populations (n = 37 in both studies) indicated that superinfection is a rare phenomenon that was not observed during 1 to 12 years of follow-up spanning 215 and 1072 total years of exposure (92, 93). However, it is not known whether any patients were recruited within the first year of HIV-1 exposure in these studies. In the case of the MDR infections cited previously, identification of the source partner of infection argues against co-infection (95). Findings of HIV-1 superinfection are a matter of concern insofar as such results challenge the assumption that immune responses can protect against re-infection. Of course, the impaired viral fitness of the initial MDR infection described above may be a factor in permitting superinfection. The initial MDR strain showed a 13-fold impaired replicative capacity from a WT variant strain from the isolated source partner following a treatment interruption. Fitness considerations may also have been important in a WT superinfection of an initial MDR infection and cases of subtype B superinfection following A/E infections that elicited low-level viremia (88, 89). In newly infected individuals, multi-mutated viruses conferring MDR may represent a new determinant of virological outcome. Persistence of MDR in the absence of treatment raises serious issues regarding HIV-1 management. For RI MDR patients, drug resistance analysis and viral fitness may provide useful information in regard to ultimate therapeutic strategies.

M. A. Wainberg and S. Schader

It is interesting to note that the presence of the M184V mutation in RT, associated with high-level resistance to 3TC, seems to have been associated with the persistence of low viral load. In two PHI cases, rebounds to a high level of plasma viremia occurred only at times when the M184V mutation in RT could no longer be detected. A third PHI patient maintained low plasma viremia over 5 years, and his virus also contained the M184V mutation throughout this time. In an additional individual, high viral loads were present at times after primary infection in spite of the M184V mutation, but virus could only be isolated from this individual in coculture experiments after loss of the M184V mutation (85). These data are consistent with previous findings on loss of fitness conferred by the M184V mutation in RT, alongside multiple other pleiotropic effects, including diminished processivity, diminished rates of nucleotide excision, and diminished rates of initiation of reverse transcription (96–98). Other studies suggest that despite reduced ARV susceptibility, MDR infections may be of some immunological and virological benefit due to the impaired replicative capacity of MDR variants (86, 99–101). Moreover, in all cases, RT assays and competitive fitness assays showed MDR viruses to have compromised replicative capacity. The absence of genotypic changes in these viruses over time further supports the concept of expansion of predominant MDR quasispecies during primary infection. Recombination events can also occur in this period. It is also important to point out that the replication fitness of a given virus vs its transmission fitness may represent two very different concepts. ART, by reducing HIV-1 replication, has been shown not only to impact significantly on morbidity and mortality but also to reduce the spread of HIV-1 (102, 103). Treatment effectiveness is hampered by the development of DR strains, leading inexorably to virological failure (76). The transmissibility of DR strains is not fully understood and may differ from that of WT strains for at least two reasons: first, the relative fitness of DR strains compared with WT in the absence of therapy and second, the degree to which partially active therapies can reduce viral load in persons harbouring resistant viruses (80, 104). As a consequence of widespread use of ART in North America, the transmission of DR strains in RI individuals has increased from 3.8% in 1996 to 14% in 2000. Such an increase of primary DR is of public health concern since a clear association between DR and early treatment failure has been reported (82). However, several groups in Europe and Australia have reported a recent stable or decreasing trend in DR transmission for RT and/or PIs (105, 106), and have attributed this decline to the widespread use of suppressive triple ARV regimens since 1996. This presupposes that transmission of DR variants may have earlier been more common due to the widespread use of suboptimal biotherapy or even monotherapy regimes prior to 1996 and the likelihood that these suboptimal regimens may have selected for drug resistance mutations with very high frequency (106).

34. HIV Resistance to Antiviral Drugs

34.7

Conclusion

The accumulation of specific resistance-conferring mutations is associated with the development of phenotypic resistance to anti-HIV drugs which can significantly diminish the effectiveness and longevity of ART. Cross-resistance among drugs of the same class also occurs frequently and is most problematic with NNRTIs due to their lower genetic barrier for rapid selection of drug resistance compared to other classes of ARVs. There is now also data indicating that cross-resistance amongst the NRTIs may in fact be more widespread than was initially thought (107). Furthermore, the emergence of new drug resistance mutations is helping to establish new mutant distributions with additional pathways for developing crossresistance to ARVs (108). These new patterns of cross-resistance together with increasing transmission of MDR HIV-1 variants are problematic and seriously limit the number of effective treatment options that are now available for longterm management of HIV-infection. Additional strategies, in addition to new drug discovery programs, are urgently required to help curb the development of DR HIV-1. One possible approach that merits further consideration is based on the maintenance of specific fitnessattenuating drug mutations in therapeutic regimens for HIV1 infection (108, 109). The M184V substitution in RT has been extensively studied in this regard because of its ability to impair viral replication capacity while limiting the development of subsequent drug resistance mutations in HIV-1 RT, e.g. TAMs and the Q151M multi-drug complex resistance mutation associated with use of AZT and d4T (110, 111). Of course, restricted evolution of drug resistance in these circumstances may also result from other alterations of RT function by M184V (96). One recent study has shown that viruses containing the M184V mutation may be transmitted less frequently than viruses containing other mutations associated with drug resistance (98), perhaps because M184V compromises viral replicative capacity. Further work on these and other topics is needed to improve our understanding of HIV drug resistance in the context of clinical relevance, successful antiviral chemotherapy, and likelihood of transmission of resistant strains (112).

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55.

of HIV-1, even in patients on effective combination therapy. Nat Med 5:512–517. Wong JK, Hezareh M, Gunthard HF, et al (1997) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278:1291–1295. Schinazi RLB, Mellors J (1997) Mutations in retroviral genes associated in drug resistance. Intl Antiviral News 5:129–142. Laboratories AVMFH. NAIDS, National Institute for Allergy and Infectious Disease. Japour AJ, Mayers DL, Johnson VA, et al (1993) Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Group, Study the AIDS Clinical Trials Group Virology Committee Resistance Working Group. Antimicrob Agents Chemother 37:1095–1101. Winston A, Mandalia S, Pillay D, Gazzard B, Pozniak A (2002) The prevalence and determinants of the K65R mutation in HIV-1 reverse transcriptase in tenofovir-naive patients. AIDS 16:2087–2089. Richman D, Shih CK, Lowy I, et al (1991) Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc Natl Acad Sci USA 88:11,241–11,245. Vandamme AM, Debyser Z, Pauwels R, et al (1994) Characterization of HIV-1 strains isolated from patients treated with TIBO R82913. AIDS Res Hum Retroviruses 10:39–46. Chong KT, Pagano PJ, Hinshaw RR (1994) Bisheteroarylpiperazine reverse transcriptase inhibitor in combination with 3¢-azido-3¢-deoxythymidine or 2¢,3¢-dideoxycytidine synergistically inhibits human immunodeficiency virus type 1 replication in vitro. Antimicrob Agents Chemother 38:288–293. Esnouf R, Ren J, Ross C, Jones Y, Stammers D, Stuart D (1995) Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors. Nat Struct Biol 2:303–308. Fletcher RS, Arion D, Borkow G, Wainberg MA, Dmitrienko GI, Parniak MA (1995) Synergistic inhibition of HIV-1 reverse transcriptase DNA polymerase activity and virus replication in vitro by combinations of carboxanilide nonnucleoside compounds. Biochemistry 34:10,106–10,112. Byrnes VW, Sardana VV, Schleif WA, et al (1993) Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors. Antimicrob Agents Chemother 37:1576–1579. Balzarini J, Karlsson A, Perez-Perez MJ,Camarasa MJ, Tarpley WG, De Clercq E (1993) Treatment of human immunodeficiency virus type 1 (HIV-1)-infected cells with combinations of HIV-1-specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy. J Virol 67:5353–5359. Sardana VV, Emini EA, Gotlib L, et al (1992) Functional analysis of HIV-1 reverse transcriptase amino acids involved in resistance to multiple nonnucleoside inhibitors. J Biol Chem 267:17,526–17,530. Dueweke TJ, Pushkarskaya T, Poppe SM, et al (1993) A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors. Proc Natl Acad Sci USA 90:4713–4717. Nunberg JH, Schleif WA, Boots EJ, et al (1991) Viral resistance to human immunodeficiency virus type 1-specific pyridinone reverse transcriptase inhibitors. J Virol 65:4887–4892.

34. HIV Resistance to Antiviral Drugs 56. Jonckheere H, Taymans JM, Balzarini J, et al (1994) Resistance of HIV-1 reverse transcriptase against [2¢,5¢-bis-O-(tert-butyldimethylsilyl)-3¢-spiro-5²-(4²-amino-1²,2²- oxathiole-2²,2²-dioxide)] (TSAO) derivatives is determined by the mutation Glu138-->Lys on the p51 subunit. J Biol Chem 269:25,255–25,258. 57. Loya S, Bakhanashvili M, Tal R, Hughes SH, Boyer PL, Hizi A (1994) Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. AIDS Res Hum Retroviruses 10:939–946. 58. Boyer PL, Currens MJ, McMahon JB, Boyd MR, Hughes SH (1993) Analysis of nonnucleoside drug-resistant variants of human immunodeficiency virus type 1 reverse transcriptase. J Virol 67:2412–2420. 59. Murphy RL (1999). New antiretroviral drugs part I: PIs. AIDS Clin Care 11:35–37. 60. Vanden Haesevelde M, Decourt JL, De Leys RJ, et al (1994) Genomic cloning and complete sequence analysis of a highly divergent African human immunodeficiency virus isolate. J Virol 68:1586–1596. 61. Cornelissen M, van denBurg R, Zorgdrager F, Lukashov V, Goudsmit J (1997) pol gene diversity of five human immunodeficiency virus type 1 subtypes: evidence for naturally occurring mutations that contribute to drug resistance, limited recombination patterns, and common ancestry for subtypes B and D. J Virol 71:6348–6358. 62. Gao Q, Gu Z, Salomon H, Nagai K, Parniak MA, Wainberg MA (1994) Generation of multiple drug resistance by sequential in vitro passage of the human immunodeficiency virus type 1. Arch Virol 136:111–122. 63. Shafer RW, Winters MA, Palmer S, Merigan TC (1998) Multiple concurrent reverse transcriptase and protease mutations and multidrug resistance of HIV-1 isolates from heavily treated patients. Ann Intern Med 128:906–911. 64. Becker-Pergola G, Kataaha P, Johnston-Dow L, Fung S, Jackson JB, Eshleman SH (2000) Analysis of HIV type 1 protease and reverse transcriptase in antiretroviral drug-naive Ugandan adults. AIDS Res Hum Retroviruses 16:807–813. 65. Descamps D, Collin G, Loussert-Ajaka I, Saragosti S, Simon F, Brun-Vezinet F (1995) HIV-1 group O sensitivity to antiretroviral drugs. AIDS 9:977–978. 66. Descamps D, Collin G, Letourneur F, et al (1997) Susceptibility of human immunodeficiency virus type 1 group O isolates to antiretroviral agents: in vitro phenotypic and genotypic analyses. J Virol 71:8893–8898. 67. Tantillo C, Ding J, Jacobo-Molina A, et al (1994) Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J Mol Biol 243:369–387. 68. Apetrei C, Descamps D, Collin G, et al (1998) Human immunodeficiency virus type 1 subtype F reverse transcriptase sequence and drug susceptibility. J Virol 72:3534–3538. 69. Shafer RW, Eisen JA, Merigan TC, Katzenstein DA (1997) Sequence and drug susceptibility of subtype C reverse transcriptase from human immunodeficiency virus type 1 seroconverters in Zimbabwe. J Virol 71:5441–5448. 70. Birk M, Sonnerborg A (1998) Variations in HIV-1 pol gene associated with reduced sensitivity to antiretroviral drugs in treatment-naive patients. AIDS 12:2369–2375.

317 71. Loemba H, Brenner B, Parniak MA, et al (2002) Genetic divergence of human immunodeficiency virus type 1 Ethiopian clade C reverse transcriptase (RT) and rapid development of resistance against nonnucleoside inhibitors of RT. Antimicrob Agents Chemother 46:2087–2094. 72. Palmer S, Alaeus A, Albert J, Cox S (1998) Drug susceptibility of subtypes A, B,C,D, and E human immunodeficiency virus type 1 primary isolates. AIDS Res Hum Retroviruses 14:157–162. 73. Brenner B, Turner D, Oliveira M, et al (2003) A V106M mutation in HIV-1 clade C viruses exposed to efavirenz confers crossresistance to non-nucleoside reverse transcriptase inhibitors. AIDS 17:F1–F5. 74. Petrella M, Brenner B, Loemba H, Wainberg MA (2001) HIV drug resistance and implications for the introduction of antiretroviral therapy in resource-poor countries. Drug Resist Updat 4:339–346. 75. Palella FJ Jr, Delaney KM, Moorman AC, et al (1998) Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 338:853–860. 76. Wainberg MA, Friedland G (1998) Public health implications of antiretroviral therapy and HIV drug resistance. JAMA 279:1977–1983. 77. Hirsch MS, Brun-Vezinet F, Clotet B, et al (2003) Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: 2003 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis 37:113–128. 78. D’ Aquila RT, Schapiro JM, Brun-Vezinet F, et al (2003) Drug resistance mutations in HIV-1. Top HIV Med 11:92–96. 79. Salomon H, Wainberg MA, Brenner B, et al (2000) Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study. AIDS 14:F17–F23. 80. Yerly S, Kaiser L, Race E, Bru JP, Clavel F, Perrin L (1999) Transmission of antiretroviral-drug-resistant HIV-1 variants. Lancet 354:729–733. 81. Boden D, Hurley A, Zhang L, et al (1999) HIV-1 drug resistance in newly infected individuals. JAMA 282:1135–1141. 82. Little SJ, Holte S, Routy JP, et al (2002) Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med 347:385–394. 83. Hecht FM, Grant RM, Petropoulos CJ, et al (1998) Sexual transmission of an HIV-1 variant resistant to multiple reverse-transcriptase and protease inhibitors. N Engl J Med 339:307–311. 84. Gandhi RT, Wurcel A, Rosenberg ES, et al (2003) Progressive reversion of human immunodeficiency virus type 1 resistance mutations in vivo after transmission of a multiply drug-resistant virus. Clin Infect Dis 37:1693–1698. 85. Brenner BG, Routy JP, Petrella M, et al (2002) Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection. J Virol 76:1753–1761. 86. Verhofstede C, Wanzeele FV, Van Der Gucht B, De Cabooter N, Plum J (1999) Interruption of reverse transcriptase inhibitors or a switch from reverse transcriptase to protease inhibitors resulted in a fast reappearance of virus strains with a reverse transcriptase inhibitor-sensitive genotype. AIDS 13:2541–2546. 87. Devereux HL, Youle M, Johnson MA, Loveday C (1999) Rapid decline in detectability of HIV-1 drug resistance mutations after stopping therapy. AIDS 13:F123–F127.

318 88. Jost S, Bernard M-C, Kaiser L, et al (2002) A Patient with HIV-1 superinfection. N Engl J Med 347:731–736. 89. Ramos A, Hu DJ, Nguyen L, et al (2002) Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users. J Virol 76:7444–7452. 90. Gonzales MJ, Delwart E, Rhee SY, et al (2003) Lack of detectable human immunodeficiency virus type 1 superinfection during 1072 person-years of observation. J Infect Dis 188:397–405. 91. Tsui R, Herring BL, Barbour JD, et al (2004) Human immunodeficiency virus type 1 superinfection was not detected following 215 years of injection drug user exposure. J Virol 78:94–103. 92. Altfeld M, Allen TM, Yu XG, et al (2002) HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus. Nature 420:434–439. 93. Koelsch KK, Smith DM, Little SJ, et al (2003) Clade B HIV1 superinfection with wild-type virus after primary infection with drug-resistant clade B virus. AIDS 17:F11–F16. 94. Smith DM, Wong JK, Hightower GK, et al (2004) Incidence of HIV superinfection following primary infection. JAMA 292:1177–1178. 95. Allen TM, Altfeld M (2003) HIV-1 superinfection. J Allergy Clin Immunol 112:829. 96. Petrella M, Wainberg MA (2002) Might the M184V substitution in HIV-1 RT confer clinical benefit. AIDS Rev 4:224–232. 97. Turner D, Brenner B, Routy JP, et al (2004) Diminished representation of HIV-1 variants containing select drug resistanceconferring mutations in primary HIV-1 infection. J Acquir Immune Defic Syndr 37:1627–1631. 98. Wainberg MA, Hsu M, Gu Z, Borkow G, Parniak MA (1996) Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase. AIDS 10:S3–S10. 99. Baxter JD, Mayers DL, Wentworth DN, et al (2000) A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS. AIDS 14:F83–F93. 100. Colgrove RC, Pitt J, Chung PH, Welles SL, Japour AJ (1998) Selective vertical transmission of HIV-1 antiretroviral resistance mutations. AIDS 12:2281–2288.

M. A. Wainberg and S. Schader 101. Dickover RE, Garratty EM, Plaeger S, Bryson YJ (2001) Perinatal transmissionmajor, of minor, and multiple maternal human immunodeficiency virus type 1 variants in utero and intrapartum. J Virol 75:2194–2203. 102. Quinn TC, Wawer MJ, Sewankambo N, et al (2000) Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med 342:921–929. 103. Yerly S, Vora S, Rizzardi P, et al (2001) Acute HIV infection: impact on the spread of HIV and transmission of drug resistance. AIDS 15:2287–2292. 104. Phillips A (2001) Will the drugs still work? Transmission of resistant HIV. Nat Med 7:993–994. 105. Chaix ML, Descamps D, Harzic M, et al (2003) Stable prevalence of genotypic drug resistance mutations but increase in non-B virus among patients with primary HIV-1 infection in France. AIDS 17:2635–2643. 106. Ammaranond P, Cunningham P, Oelrichs R, et al (2003) No increase in protease resistance and a decrease in reverse transcriptase resistance mutations in primary HIV-1 infection: 1992–2001.AIDS 17:264–267. 107. Kuritzkes D (2002) Drug resistance. Navigating resistance pathways. AIDS Read 12:395–400, 407. 108. Nijhuis M, Deeks S, Boucher C (2001) Implications of antiretroviral resistance on viral fitness. Curr Opin Infect Dis 14:23–28. 109. Brenner BG, Turner D, Wainberg MA (2002) HIV-1 drug resistance: can we overcome. Expert Opin Biol Ther 2:751–761. 110. Ait-Khaled M, Rakik A, Griffin P, et al (2002) Mutations in HIV-1 reverse transcriptase during therapy with abacavir, lamivudine and zidovudine in HIV-1-infected adults with no prior antiretroviral therapy. Antivir Ther 7:43–51. 111. Ait-Khaled M, Stone C, Amphlett G, et al (2002) M184V is associated with a low incidence of thymidine analogue mutations and low phenotypic resistance to zidovudine and stavudine. AIDS 16:1686–1689. 112. Daar ES, Richman DD (2005) Confronting the emergence of drug-resistant HIV type 1: impact of antiretroviral therapy on individual and population resistance. AIDS Res Hum Retroviruses 21:343–357.

Chapter 36 Epidemiological Surveillance of HIV and AIDS in Lithuania Saulius Caplinskas

36.1

Introduction

Epidemiological surveillance of HIV and AIDS is regulated by Law on Prevention and Control of Human Communicable Diseases of the Republic of Lithuania and its modifications (1). The Law provides for basics on management of human communicable diseases prevention and control, controversy elimination and harm restitution, responsibility of violation of judicial acts on issues of communicable disease prevention and control; rights and duties of natural person’s and body’s in the field of communicable disease prevention and control; specialities of budgeting of communicable diseases prevention and control, and compensation of their costs. In September 1999, the government of the Republic of Lithuania established the state registries of communicable diseases and their agents (1, 2) and the functions of chief manager were delegated to the Center of Communicable Diseases Prevention and Control. All cases of communicable diseases are reported to the State Registries of communicable diseases and communicable diseases agents. Personal healthcare institutions report to the territorial public health institutions on all individual cases of HIV and AIDS, and summarized data from the public health institutions are submitted to the registry. The Government of the Republic of Lithuania, the Ministry of Health and subordinated institutions are in charge of the management of communicable disease prevention and control. District governors are in charge of the management of communicable diseases prevention and control on district level according to legal regulations, and the mayors on the municipal level according to their competence. Governmental supervision of implementation of the communicable diseases prevention and control is delegated to the Ministry of Health, and institutions subordinated to the Ministry of Health, the Chief Epidemiologist of the Republic of Lithuania, district doctors and district From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

chief epidemiologists; municipal doctors are in charge of program implementation. The functions of government services, chief epidemiologists, district and municipal doctors in the field of prevention and control of communicable diseases management and supervision of the program implementation are regulated by present law and other legal regulations (Figure 36.1). The Lithuanian AIDS Center is in charge of the epidemiological surveillance of STI, HIV, AIDS on national level, evaluation of epidemiological situation, prognosis, conclusions, proposals, methodical government of personal and territorial public health case institutions on issues of epidemiological STI, HIV, and AIDS surveillance.

36.2

HIV Epidemiological Situation

Although Lithuania, with a mainly Catholic population of around 3.4 million (3), still appears to be a low prevalence country for HIV, it is situated in a sub-region bordered by neighbors worse affected than most European countries. Notably, one close neighbor, Estonia, had a similarly low prevalence state of HIV for many years, until the turn of the millennium, when suddenly it found itself to have the second highest prevalence of HIV infection in Europe due to an outbreak that spread rapidly in their intravenous drug users (IDUs) (3). Similarly worryingly high rates are found in Latvia, Belarus, Ukraine, Kaliningrad, and the rest of the Russian Federation.

36.3

General Overview

The first HIV-positive person in Lithuania was reported in 1988. From January, 1988 to January 1, 2006 a cumulative total of 1100 (29.41 per 100,000 population; refs. 4 and 5) HIV infections were registered in Lithuania, of these 96 have developed AIDS and 99 have died. However, the actual number of HIV-infected people may be higher than this. For example, international agencies estimated that there were 3,300 people 327

328

S. Caplinskas

Ministry of Health Governmental Public Health Care Service under the Ministry of Health

District administraion

Health Information Centre

EuroHIV

Statistical data

Centre of Communicable Diseases Prevention and Control Analysis and prognosis of epidemiological situation

Centre of Extremal Health Situation

Lithuanian AIDS Centre (Dept. .of Epidemiological Surveillance)

Laboratory of the Lithuanian

Test result Territorial Public Health Care Institutions

Personal Health Care Institutions Lithuanian AIDS Centre

Figure 36.1. Data flows.

Figure 36.2. HIV/AIDS registered per year cases in Lithuania (January 1, 2006) (See Color Plates).

living with HIV in Lithuania at the end of 2005 (Figure 36.2; ref. 6). HIV cases were identified in the majority of the Lithuanian districts. The highest prevalence is reported in Klaipeda (on the Baltic Sea): 28.9% of all HIV cases in the country (Figure 36.3). By the end 2005 the highest HIV prevalence in 100,000 population was reported in Klaipeda: 154.28/10000, the

capital Vilnius ranked only sixth by HIV prevalence with 24.93 cases per 100,000. There were 49 HIV-positive foreigners registered, including 14 Russian citizens, 16 Latvians, four from Belarus, two from Estonia and one HIV case from France, Denmark, Spain, Poland, Ukraine, Uzbekistan, Thailand, Vietnam; and five unknown cases (7).

36. Epidemiological Surveillance of HIV and AIDS in Lithuania 35

HIV prevalence

329

HIV incidence

30

26,13

22,78

25

29,4

19,6

20 15

11,44

9,72

10 5

1,45

0

0,33

1996

3,82 2,33 1,47 0,87 1997

1998

7,63

5,72

1999

2000

3,93

3,18

2,07

1,86

1,87

3,51

2001

2002

2003

2004

2005

Figure 36.3. HIV prevalence and incidence per 100,000 population (1996–2004) in Lithuania (See Color Plates).

450 400 350 300 250

299

200

26

34

2 12 10 80

4 5 15 4 55

31

37

20 04

15 12 2 3 45

20 02

19

20 01

6 3 4 4 7 12 22 29 29 19

99 20 00

98

97

19

96

92 19 93

9

3 17 1 8 5 5 15 2417 23

1 11 7 12

1 10

11

2

19

19

1 4

19

1

19 95

1 7

19 94

1

19 91

19 89

19 88

0 1

19 90

50

11 4 10 49

20 03

100

20 05

150

HIV outbreak in Alytus Corrective Institution Lithuanian AIDS center(outpatien depatment) detoxication, rehabilitation and methadone needle exchange cabinet (include AIDS centre needle exchange) Pre-Trial Wards health care institucion

Figure 36.4. HIV infection cases by place of diagnosis. Source: Lithuanian AIDS Center, 2006 (See Color Plates).

seksual

Intravenous drug use

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

1991

1990

1989

1988

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

unknow

Figure 36.5. HIV mode of transmission by year in Lithuania (%) (See Color Plates).

The HIV epidemic in Lithuania appears to have passed through two phases. The first phase runs from the mid-1980s to the year 2002 (7). This was a quiet phase with only a few endemic infections, mainly due to contacts abroad (8). The second phase started when the official statistics jumped from 328 registered HIV cases at the end of 2001 to 845 HIV cases at the end of 2003. This was due to an outbreak of HIV infection

transmitted through the use of contaminated needles used in the Alytus Correctional Facility (CF; Figure 36.4). Until 1997, these infections were transmitted either heterosexually or homosexually: in 1989–1993 the virus spread among men having sex with men (MSM); in 1993–1996 heterosexual HIV transmission prevailed, especially in seafarers who have been infected in African countries. Starting from 1997, HIV

330

S. Caplinskas 100%

50%

heteroseksual

homoseksual

Intravenous drug use

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

1991

1990

1989

1988

0%

unknow

Figure 36.6. Tendencies HIV of transmission mode in Lithuania (See Color Plates).

has been mainly spread through blood in the IDU population. The annual share of HIV infection acquired through injection equipment reached 70–77%, except of 2002—the year of an HIV outbreak in Alytus CF. In 2002, the mode of HIV transmission through drug injection bounced up to 95% (9). Correspondingly, the proportion of new diagnosed heterosexual case has steadily increased from 9.1% in 2001 to 17.7% in 2004 and to 16.6% in 2005 (Figure 36.5). The number and proportion of new cases reported among MSM remains low (2004: four cases; 2005: three cases) and stable (2.5–2.9%; Figure 36.6). Until 2006, in Lithuania IDUs clearly dominate: 866 (78.7%) people were infected via illicit drug injection, 114 (10.4%) heterosexually, 70 (6.4%) homosexually; the transmission route remains unknown in 50 cases (10). HIV has been mostly reported in the 25–29 age group (25.8%) and 30–34 age group (18.4%), while 76.8% of the total cases were identified in the 20–39 age group, which is continually replenished with newly infected IDUs (Table 36.1). The youngest patient in Lithuania was 15 years old and the oldest was 68 years old at the time of the HIV diagnosis. Average age according to the mode of transmission also differs; 37 years of age for those who have been infected via sexual intercourse and 30 years of age for

Table 36.1. HIV infection cases by age in Lithuania, December 31, 2005 Age

Males

Females

IDU

Total

15–19

41

9

42

50

20–24

159

31

166

190

25–29

254

30

240

284

30–39

327

44

307

371

40–49

128

12

88

140

50–59

32

3

10

35

60 and older

9

3

—

12

Unknown

16

2

13

18

infection through contaminated drug injecting equipment (Table 36.1). The annual rate of female HIV cases has continuously increased, thus leading to a decrease in the male/female ratio: in 2002 it was 12/1; in 2003, 7/1; in 2004, 5/1; and in 2005, 3/1 (Figure 36.7) (11). The majority of the reported females are at their reproductive age, at average 31 years. The majority of them were infected through intravenous drug use (67.9%). The number of female HIV cases occurring heterosexually has annually increased. This fact is seriously increasing the threat of further HIV spread in general population. So far, there are no cases reported of mother-to-child HIV transmission, nor HIV infection in children younger than 15 years of age. Until 2006, a total of 11 pregnant women with HIV infection have been reported. Based on 100,000 population, the prevalence of HIV infection has been on a steady increase. Thus, HIV prevalence in 1996 was 1.45 for 100,000 of population (Table 36.2) and 29.4 in 2005 (which is the lowest in the Baltic Sea region). The HIV incidence has also gradually increased. In 1996, the HIV incidence for 100,000 population was 0.33, and in 2005 it was 3.51 (Table 36.3) (ref. 10, 12). A primary study of the HIV-1 molecular typification in Lithuania proved as predominating the subtype B virus. Thus, HIV-1 subtype B, which is the most prevalent in Western Europe, was also the most common subtype in all three Baltic countries (Lithuania, Latvia, and Estonia) and Russia, and was linked to homosexual transmission. However, after the HIV outbreak in the Alytus CF in 2002, the HIV-1 subtype A has been prevailing (13).

36.4

AIDS Cases

The first case of AIDS diagnosed in Lithuania was a man in 1988. In 1999, 11 years later, the first woman in Lithuania was diagnosed with AIDS. The overall AIDS incidence rate

36. Epidemiological Surveillance of HIV and AIDS in Lithuania

331

450

moteris

400

vyrai

350 300 250 200 150 100 50 0 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Figure 36.7. Dynamics of HIV spread by gender (See Color Plates).

Table 36.2. HIV prevalence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

Estonia

0

Latvia

0

Lithuania

0

0

0.06

0.19

0.51

0.51

0.58

0

0.04

0.04

0

0.22

0.11

0.04

0

0

0

0.03

0.03

0.21

0.03

Belarus

0

0

0.2

0.11

0.12

0.14

Poland

0.03

0.02

0.08

0.16

1.36

0

0

0.02

0.03

0.18

Russian Federation

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

0.33

0.8

0.74

0.55

0.19

0.27

0.28

1.29

0.62

0.7

0.85

27.99

107.07

66.07

63.3

56.7

46.02

1.01

6.61

9.94

19.25

33.54

22.66

17.5

14.1

0.13

0.11

0.24

0.3

12.93

0.32

0.84

1.4

1.78

1.76

1.95

10.87

3.18

3.93

0.12

0.2

0.1

0.05

3.51

0.08

9.9

6.35

5.4

4.02

5.17

5.7

9.05

7.08

7.91

7.76

2.12

1.46

1.25

1

0.07

0.06

0.06

0.07

1.1

1.4

1.43

1.5

1.65

1.36

1.63

1.46

1.49

1.58

1.72

1.7

0.11

0.14

1.03

2.96

2.76

13.64

40.66

61.01

35.15

27.64

23.62

—

Table 36.3. HIV incidence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

Estonia

0

0

0

0.06

0.19

0.51

0.51

0.58

0.33

0.8

0.74

0.55

0.62

0.7

0.85

27.99

107.07

66.07

63.3

56.7

46.02

Latvia

0

0

0.04

0.04

0

0.22

0.11

0.04

0.19

0.27

0.28

1.29

1.01

6.61

9.94

19.25

33.54

22.66

17.5

14.1

12.93

Lithuania

0

0

0

0.03

0.03

0.21

0.03

0.13

0.11

0.24

0.3

0.32

0.84

1.4

1.78

1.76

1.95

10.87

3.18

3.93

3.51

Belarus

0

0

0.2

0.11

0.12

0.14

0.12

0.2

0.1

0.05

0.08

9.9

6.35

5.4

4.02

5.17

5.7

9.05

7.08

7.91

7.76

Poland

0.03

0.02

0.08

0.16

1.36

2.12

1.46

1.25

1

1.1

1.4

1.43

1.5

1.65

1.36

1.63

1.46

1.49

1.58

1.72

1.7

0

0

0.02

0.03

0.18

0.07

0.06

0.06

0.07

0.11

0.14

1.03

2.96

2.76

13.64

40.66

61.01

35.15

27.64

23.62

—

2001

2002

2003

2004

2005

Russian Federation

Table 36.4. AIDS incidence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

Estonia

0

0

0

0

0

Latvia

0

0

0

0

0

Lithuania

0

0

0

0.03

0

0.03

0.03

Belarus

0

0

0

0

0

0

0.04

Poland

0

0.002

0.01

0.01

0.07

0.06

0.12

Russian Federation

0

0

0.002

0.003

0.02

0.03

0.02

1995

1996

0

0

0.13

0.07

0.07

0.04

0.04

0.12

0.03 00.04 0.09 0.02

0.07

0.2

0.48

0.21

0.28

0.14

0.22

0.15

0.37

0.74

1.92

2.22

0.08

0.12

0.2

0.16

0.49

0.74

0.95

1.66

2.3

2.44

3.33

3.11

0

0.05

0.03

0.13

0.08

0.24

0.16

0.19

0.27

0.26

0.26

0.61

0.29

0.02

0.02

0.03

0

0.02

0.04

0.05

0

0.06

0.21

0.37

0.85

1.38

0.18

0.26

0.3

0.29

0.32

0.33

0.35

0.31

0.34

0.32

0.33

0.44

0.49

0.01

0.02

0.03

0.03

0.05

0.04

0.03

0.01

-

-

-

-

-

has been increasing slowly during the past 12 years (from 0.03 in 1990 to 0.29 in 2005), but still remains quite low in comparison to the rates in the neighboring countries (Table 36.4).

1997

1998

1999

2000

From the introduction of the HIV registry, AIDS as the advanced HIV stage was diagnosed in 96 persons (Table 36.5). There were 41 deaths of AIDS.

332

S. Caplinskas

Table 36.5. AIDS cases in Lithuania, December 31, 2005 AIDS cases Number of AIDS diagnosed Number of deaths

Males

Females

IDU

Total

84

12

17

96

9

12

54

99

From AIDS

40

1

4

41

Others reasons

47

11

50

58

Table 36.6. HIV/TB co-infection in Lithuania, December 31, 2005 1992 1993 1994 1999 2000 2001 2002 2003 2004 2005 Total HIV/TB

1

1

1

2

3

3

2

5

8

7

33

Of all registered AIDS cases, 56% are MSM, 24% AIDS cases were caused by heterosexual contacts, and only 12% might be attributed to IDU; in 8% of the AIDS cases, the transmission was unknown. In 3.8% of the cases, AIDS was diagnosed at the time of HIV/AIDS diagnosis (14).

36.5

HIV/TB Co-infection in Lithuania

The first case of active TB in person with HIV was identified in 1992. Up to 2006, the number of HIV/TB co-infection reached 33 (Table 36.6). This means that about 3% of all HIV cases are also TB-infected. The prevalence of active tuberculosis in people with HIV is 10 times higher than in the general population (4, 5).

36.6

HIV Outbreak in Alytus CF

The regulations in the Lithuanian prison system on HIV testing in the inmates provides for testing: · · · ·

in 3 months upon entry to the pre-trial house or CF 3 months prior to release from CF once a year in convicts according to epidemiological or medical indications (Figure 36.8)

Until 2002, there were no cases of HIV in prison inmates reported. With the continuous increase in the number of HIV cases in Lithuania, chances of incarcerating people with HIV also increased. In some cases prison inmates upon entry into the penitentiary system have already been included into outpatient care system of the Lithuanian AIDS Center. However, about 30 to 40% of HIV cases used to be identified only on entry to pre-trial wards. The first person with HIV was imprisoned in 1992. The mode of transmission in this case was sexual intercourse. The second person with HIV has entered the prison in 1996. The spread of HIV infection among IDUs had

Figure 36.8. HIV/AIDS in Lithuanian prison settings (See Color Plates).

a significant impact on the increase of HIV cases in penitentiaries. By the end 2001, 118 cases of HIV infection were newly identified in or already served their sentence in prison; by the end of 2002, there were 8 cases more (Figure 36.9) (15).

36.6.1 State Mental Health Center, Lithuanian AIDS Center, and Prison Department Data, 2005 In May 2002, the diagnosis of HIV infection in a person recently released from prison, necessitated a follow up study of the possible contacts of the former inmate. This study led to the discovery of the HIV outbreak in the Alytus CF (Figure 36.9) (16).

36.6.2

Prison Department Data, 2005

From May to June, 2005, 2,000 prisoners were tested for HIV infection in the Alytus CF and 207 new HIV cases were identified. During July-August of the same year, 1,813 tests were done repeatedly and 77 positive persons were identified. 17 of them were new prisoners, 60 become positive, with existing high probability to be infected after first wave of testing performed. In September-October, 1,481 HIV tests were done and only 15 HIV-positive persons were identified. The majority of the inmates with HIV infection admitted that a possible mode of their infection could be intravenous drug use at their home and needle/syringe sharing. Only 13 of those tested were revealed to have a CD4 cell count less than 500 cell/mm3. Serological markers of HCV infection were found in 252 (98%) of 257 HIV-infected persons, 22 (7%) have had HBV surface antigen. Due to the active national HIV epidemiological surveillance system, the HIV outbreak in the Alytus CF was practically identified at the very first stage of its development. The latter was confirmed by laboratory testing. The rapid spread of HIV in the Alytus CF was successfully confined due to all preventive measures taken, from administrative-organizational to technical-educational. The measures taken to fight HIV in prisons are

333

180

375

160

145,1

140

77,4

77,5

135,2 13,3%

95,3

100 80

126,3

117,2

120 83,3

156,8

14,4% 212

11,3%

20% 18% 18,1% 16% 14%

15,6%

12%

229

10% 8%

8,8%

60

6,6%

7,5%

128

6%

40

4%

20 0 1997

1998

22

32

33

1999

2000

2001

2% 0% 2002

2003

2004

2005

2006

Percent of drug users in prison settings

Dinamisc of drug and toxic substances addiction (100 000 population)

36. Epidemiological Surveillance of HIV and AIDS in Lithuania

Prevalence of HIV infection in penitentiaries (abs. Nu)

Chronology of HIV and measures taken in prisons of Lithuania

2003 May - HIV/AIDS Couseling Center established in Central Prison Hospital 2003 − Local Zone cancelled 2002 − HIV outbreak in Alytus 1998 - opening of

1997 − several persons with the Local Zone HIV positive. Problem of their accomodation during their term of imprisonment 1992 − the first HIV infected person

Figure 36.9. Drug and toxic substances addiction, percentage of drug users in prison settings and prevalence of HIV infection in penitentiaries (See Color Plates).

effective at the moment. But total prevention of drug use in penal establishments is hard to achieve; therefore, the World Health Organization (WHO) has recommended that the prison system should follow strict procedures to stop the transmission of blood-borne infections, including improving the accessibility of disinfectants; establishing of drug-free zones; and providing addiction treatment. Screening in other penitentiaries in the same year did not reveal any new HIV infection cases. Although HIV testing in other correctional facilities did not reveal any new HIV infection cases among the inmates, the HIV and drug use prevention was reinforced in all Lithuanian penitentiary institutions. (Figure 36.10)

Thanks to the joint multi-sectorial actions, the outbreak in Alytus prison was stopped: in 2003 there were 15 new cases reported, two cases in 2004, and four cases in 2005 (Figure 36.11) (16).

36.7 HIV Transmission Through Sexual Contacts According to WHO estimations (2003), there might have been 7,000 to 11,000 IDUs, 17,000 to 44,000 MSM, 5,000 to 8,000 sex workers (SWs), and 11,400 (327 per 1,000,000) prisoners in Lithuania.

334

S. Caplinskas 500

Total HIV cases in Lithuania

400

HIV cases discovered in prisons

300 200 100 0 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Figure 36.10. Registered HIV cases in prisons and total HIV number registered in Lithuania (See Color Plates).

207

new HIV cases in Alytus prison

77 15 2002 May-June

2002 July-August 2002 September-October

15 2003

2 2004

4 2005

Figure 36.11. HIV outbreak in Alytus prison (See Color Plates).

36.8

Homosexual Transmission

The first HIV-positive cases in Lithuania were diagnosed in homosexual men’s community in 1989. It was estimated that the virus entered this community in 1980-1983. There were seven HIV-positive persons registered in 1990. These cases were disclosed mostly by partner notification. In 1989–1993, the virus spread among MSM. Until 1997, the HIV transmission has been mostly through sexual contacts: 26 HIV cases by heterosexual transmission and 20 cases by homosexual transmission (Figure 36.6). Since 1996, the number and proportion of new cases reported among MSM remained low (2004, four cases; 2005, three cases) and stable (2.5–2.9%) (17). One major way of transmitting HIV is through sexual intercourse. In this regard, the Lithuanian national program laid plans for prevention activities with the most at-risk populations (MARPs): SWs, their clients, IDUs, MSM, seafarers, long-distance truck drivers, soldiers, and police officers. It has also planned for improving the availability of good-quality condoms to these high-risk groups, and youth and general public education. It was estimated by national experts during a WHO and The Joint United Nations Programme on HIV/AIDS (UNAIDS) workshop in 2003, that there might have been from 17,000 to 44,000 MSM in the country. This is still a marginalized group

that had remained relatively underground and far from being reached by any prevention activities. It is no surprise that again very few non-government organizations (NGOs) have shown any interest in working with this MARP. Also the mass media generally give a very negative picture relevant to MSM issues. In order to provide some prevention services, the LAC established a “Target Group” Health Consulting Room to provide services for MSM, and other at-risk groups. This provides health advice and counselling to MSM and even clinical services, including testing. All the attendees were tested for Hepatitis B, syphilis, gonorrhoea, HIV, hepatitis C, and Chlamydia infection. One case of each gonorrhoea and chlamydia infection, three cases of Candida infection, and one of hepatitis C were identified in 50 consultations with a dermato-venerologist. This outlet also runs a popular website where there is some discussion of safer sex, sexually-transmitted infections (STI), and prevention. The services were expanded to include anonymous STI clinic services with anonymous testing and treatment accessible to all patients (MSM, SW, and others). In 2005, in total 4,369 visits were registered (the majority [2686] attended a dermato-venerologist office while 1,683 attended a gynaecologist office). In a survey of 90 MSM in 2003, 61 declared that they have used a condom with their last partner (68%), while an internet questionnaire resulted in 117 out of 213 saying that they have used a condom with the last sex partner (55%).

36. Epidemiological Surveillance of HIV and AIDS in Lithuania

Sentinel surveillance performed by the Lithuanian AIDS Center in MSM proved the following statistics: in 2003, of 242 MSM, two were HIV-positive (0.8%); in 2004, 79; and in 2005, also 79 MSM were tested for HIV and no cases of infection were found.

36.9

Heterosexual Transmission

The first HIV cases of heterosexual HIV transmission were identified in 1988. In 1993-1996, heterosexual HIV transmission prevailed, especially in seafarers who were infected in African countries. Until 1997, the HIV transmission through sexual contacts prevailed, with 26 cases of heterosexual HIV transmission reported (Figure 36.12) (18). Since then, the proportion of newly diagnosed heterosexual cases has steadily increased from 9.1% in 2001 to 17.7% in 2004 and 16.6% in 2005. It has been estimated by national experts during a WHO and UNAIDS workshop in 2003, that there might have been about 5,000 to 8,000 SWs (both male and female) in the country. In 2005, about 120 street SWs regularly visited the AIDS Center site of low threshold services. This facility offered testing to SWs and 111 voluntarily underwent HIV, syphilis, HBV, and HCV tests. No cases of HIV were found at that time, but 15 cases of gonorrhoea, five of chlamydeous infection, five of hepatitis C (all were IDUs), one case of hepatitis B, and one case of syphilis were identified. Some SWs also agreed to answer a questionnaire on their behavior. Sixty-five percent claimed that they have used a condom with their most recent client in 2003, while in 2004, 70% said that have used a condom with their most recent client. This model should be expanded to all the major cities, by supporting the establishment of new NGOs with similar scope of activities.

36.10

SWs

Sentinel surveillance of SWs in the Women Health Site at the Lithuanian AIDS Center provided the following statistics: in 2003 a total of 89 SW were tested; in 2004, 86; in 2005, 111; and no cases of HIV have been identified (Figure 36.13).

unknown 5%

IDU 12%

homo 38%

hetero 8% homo 5%

hetero 50%

Year 1988-1996 (n = 52)

IDU 82% Year 1996-2005 (n = 1048)

Figure 36.12. Distribution of newly diagnosed HIV cases by transmission group (See Color Plates).

335

96% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

75% 68%

0,4% 1999

2000

2002

2004

Figure 36.13. Low threshold center for street prostitutes in LAC. STI among focus group of Vilnius street prostitutes (n = 56). The Lithuanian AIDS Center data, 2005 (See Color Plates).

Percentage of IDUs prostitutes out in Vilnius streets: in 1998, 23%; in 2001, 65%; and in 2004, 78%. Prostitution trends have changed from prostitution ® drug use into drug use ® prostitution. So far, 19 HIV-positive cases were identified in SWs (the rate of heterosexually infected women has also increased significantly).

36.11

IDU

The first HIV-positive IDU in Lithuania were diagnosed in 1994. Two years later, four HIV-positive IDUs were reported in the Klaipeda harbor. Starting from 1997, HIV has been mainly spread through blood in the IDU population. In 1997, IDUs accounted for 70% of all HIV-positive registered cases (19). The Drug Control Department is working with the European Monitoring Center for Drugs and Drug Addiction to improve the current estimates of the size of the drug problem in Lithuania. Their main priorities are to prevent addiction, especially among children and young adults, as well as to: institute policies to decrease supply; provide healthcare; rehabilitation and social rehabilitation; and establish a strong IT database. Recent surveys have demonstrated that 80% of the 5,371 registered drug users use opiates, with most of these drug users using poppy straw, and 402 are on methadone. The estimated real number of IDUs is more than 7,000 IDU. However, additional funding is still needed to carry out a more scientific study of the true extent of the problem. A main area of concern is promoting safer drug injection behavior. The principles include: ensuring that there is a good distribution of harm-reduction activities, especially easily accessible clean needles and syringes and/or bleach for cleaning injection equipment; peer counselling; programs that teach persons from the IDU scene to counsel other drug users in their own environment and in their own language; drug substitution/methadone programs should be used to getting IDUs away from risky use of illegal substances and try to channel them into a controlled, curative program (rehabilitation). All these measures must be provided in a suitably supportive social and legal environment.

336

S. Caplinskas

The first needle and syringe exchange programs were initiated in 1997 in the capital city of Vilnius. In 1998, the LAC had opened a “low threshold” consulting office aiming at promoting counselling and similar services among IDUs, and reducing HIV transmission and other STIs among IDUs and their sexual partners. During 1998-2005, a total of 2,408 IDU clients of Low Threshold Site at the Lithuanian AIDS Center for drug users were surveyed, and 90 HIV cases identified (3.74%). The majority of site visitors are IDUs with HIV infection; therefore, data on HIV prevalence in this subgroup might be higher compared to the whole IDUs population in Vilnius. In 2004, this “low threshold” services unit registered 397 new visitors; of those, 325 (81.9%) were men, and 72 (18.1%) were women. The majority of clients (375 or 94.5%) are injecting drug users and family members or sexual partners of these individuals. The number of visits is increasing every year with 10,635 for syringe exchange registered in 2004 (compared to 8,271 visits in 2003). 27,428 syringes and 28,709 injection needles were provided and 33,570 syringes and 35,437 needles were collected in 2004. In 2005, 134 new clients and 5,848 visits were registered. 12,808 syringes and 15,275 needles were exchanged while 14,512 syringes and 17,204 needles were collected in all, and 1,550 condoms distributed. In 2005, the unit tested 345 visitors (134 were new clients) and found three HIV-positive individuals (2.2%). When tested for the hepatitis C virus, 94 were found to be positive out of 229 clients (94 out of 134 new, 70.1%). Sentinel surveillance of IDUs performed on outreach basis (mobile needle exchange site “Blue bus” of Vilnius Center for Treatment of Addictive Disorders) in Vilnius in 2005 resulted in iidentification of 22 (3%) HIV-positive cases (n = 681;Table 36.7). Comparison of risky behavior and knowledge survey in IDUs (%) The Lithuanian AIDS Center data, 2005.

36.12

trend toward “feminization” of the HIV epidemic in Lithuania would eventually have an influence on HIV mother-to child transmission rates. So far in Lithuania there have been 11 HIV-positive women observed during their pregnancy (have taken ARV) and all of the infants tested negatively.

36.13

Mother-to-Child Transmission

In Lithuania, there is an HIV test available to pregnant women, and antiretroviral therapy is available for free to pregnant women, women in child-birth and newborns. This

Conclusions

1. From the data available, it appears that Lithuania might be one of the countries with a significantly localized HIV epidemic, particularly confined among IDUs which is also affecting MSM and “bridging groups,” e.g., SWs and their clients, sex partners of IDUs, and prisoners, while the general population is likely to have had very little exposure to HIV so far. 2. By risk groups: in Lithuania, the HIV epidemic has developed similarly to other countries of Eastern and Central Europe: the first cases were diagnosed in the MSM community, then in IDUs, and, recently, infection has been more rapidly spreading among heterosexuals, though the prevailing transmission mode still is injection drug use in men. 3. HIV infection cases are more concentrated in the seaports and the capital Vilnius. 4. The low TB/HIV co-infection rate and the lack of MTC cases in Lithuania may have the characteristics of an early HIV epidemic. 5. The HIV epidemic peaked in Lithuania in 2002 and, until recently, there was a downward trend in incidence rates. However, in the last few years, the incidence rate has once again increased, fuelled by infections through the use of contaminated injecting equipment and in prisons, leading to the likelihood of an outbreak affecting wider population. 6. Treatment of drug addiction, harm reduction, re-integration of ex-prisoners into society is a main factor in HIV prevention on this stage of the HIV epidemic. There are signs of spill-over and spread of HIV from IDUs to other population groups. This indicates the possibility for further spread of HIV infection from high risk groups by bridging populations (IDUs sexual partners) into the general population.

Table 36.7. Comparison of risky behavior and knowledge survey in IDUs (%) Mean duration of drug use, in years Drugs injected exceptionally with new needle/syringe Knowledge on HIV risk because of injection equipment sharing Never have got drug treatment Use of other drugs while in MT Stable sexual partner in last 12 months Awareness of HIV test result Unsafe sex with commercial partner during recent intercourse Abstinence of commercial sex in last 12 months Underwent HIV test some when The Lithuanian AIDS Center data, 2005.

2000

2004

4.0 60 98.6 28.9 85.2 45.5 94.4 86.4 76.1 69

6.4 77.7 100 56.8 95.6 64.7 87.5 70 98.1 94.1

36. Epidemiological Surveillance of HIV and AIDS in Lithuania

7. Though the male IDUs with HIV still prevail, feminization of the HIV epidemic is being presently observed in Lithuania. 8. Counselling and care of HIV-infected pregnant women should be expended. 9. The number of TB/HIV co-infection cases has increased in Lithuania, though it is still not a serious problem among patients living with HIV/AIDS. However, even wider and deeper cooperation between HIV and TB control and prevention programs must be stimulated, HIV/TB counselling, surveillance, and treatment should be expanded accordingly. 10. Second generation epidemiological surveillance should be intensified in the target groups and country regions.

References 1. Resolution of the Government of the Republic of Lithuania (1999) No. 1046, On establishment of the state registry of communicable diseases agents and approval of regulations. Official Gazette No. 81, Publ. No. 2401. 2. Resolution of the Government of the Republic of Lithuania (1999) No. 1047, On establishment of the state regsitry on communicable diseases and approval of regulations. Offical Gazette No 81, Publ. No. 2402. 3. HIV/AIDS epidemic in the Baltic countries in 1987-2005: comparative study / S. Caplinskas, A. Ferdats, I. Januskevica, T. Pertel // 7th Nordic-Baltic congress on infectious diseases “Current challenges and new opportunities”, Riga, September 18-20, 2006: poster. – P. 49. ”, , 22-26 2006: abstract. – 2006, . 10, 2, c. 51. 4. Statistical Department of Lithuania Website. Available at: www. std.lt. 5. Health Information Center of Lithuania Website. Available at: www.lsic.lt. 6. Ministry of Health, Lithuanian Health Information Center (2006) Health and health care institutions in 2005. Vilnius ISSN 392- 8155. 7. Caplinskas S (2004) Epidemiology of HIV/AIDS in Lithuania in 1988–2001: review of present situation and prognosis of HIV transmission trends. Medicina 40 tomas, Nr. 2. 8. Likatavicius G, Caplinskas S, Rakickiene J (2004) HIV/AIDS epidemiology in Lithuania. Acta medica Lithuanica. Supplement 6. 9. Trends by transmission category in HIV/AIDS in Lithuania (1988-2005) / Oksana Strujeva, Saulius Caplinskas // 7 th NordicBaltic congress on infectious diseases “Current challenges

337 and new opportunities”, Riga, September 18-20, 2006: poster. – P. 32-33. 10. Epidemiology of the human immuno deficiency virus (HIV) in Lithuania: 19 year surveillance result / Oksana Strujeva, Saulius Caplinskas // 4th IAS conference on HIV pathogenesis, treatment and prevention, Sydney, July 22-25, 2007: abstract. – Hannover, 2007. – P. 80. 11. Characteristics of HIV transmission in women in Lithuania / Oksana Strujeva, Vilma Uzdaviniene, Saulius Caplinskas // 7 th NordicBaltic congress on infectious diseases “Current challenges and new opportunities”, Riga, September 18-20, 2006: poster. – P. 33. 12. The epidemiology of HIV infection in Lithuania, 1988-2005 / S. Caplinskas, O. Strujeva, V. Uzdaviniene. – Diagr. – Lent. – Bibliogr.: 19 pavad. - Tas pats rus.: p. 19-25 // EpiNorth. – 2007, vol. 8, no. 2, p. 19-26. 13. Molecular epidemiology of HIV-1 in Eurasia / E. V. Karamov, Ivanovsky institute of virology, Moscow; V. V. Lukashov, Academic medical center, university of Amsterdam; V. F. Eremin, Research institute for epidemioplogy and microbiology, Minsk; S. Caplinskas, Lithuanian AIDS center; I.G. Sidorovich, Institute of immunology, Moscow // Status of HIV vaccine research: an exploratory workshop on perspectives and potential for vaccine development, St. Petersburg, June 1-2, 2007: abstract. – St. Petersburg, 2007, p. 14-15. 14. HIV/AIDS ir Lithuania / Saulius Caplinskas. – Diagr. - Lent. – Bibliogr.: 8 pavad. // HIV/AIDS in Russia and Eurasia / ed.by Judyth L. Twigg. – {New York, 2006]. – P.171-186. 15. Caplinskas S, Likatavicius G (2002) Recept sharp rise in registered HIV infections in Lithuania. Eurosurveillance Weekly 6:27/06/2002. 16. HIV outbreak in prison follow-up / Saulius Caplinskas, Oksana Strujeva, Irma Caplinskiene // 10th European AIDS conference / EACS, Dublin, Ireland, Nov 17-20, 2005: abstract. – [Dublin, 2005]. - P.152-153. 17. HIV transmission trends in Lithuania / S.Caplinskas, I.Caplinskiene, O.Strujeva // 6-th Nordic-Baltic Congress on Infectious Diseases In Cooperation with the Task Force on Communicable Disease Control in the Baltic Sea Region “Current Strategies for Prevention and Treatment of Infectious Diseases”, Palanga, Jun 3-6, 2004: poster. – [Vilnius, 2004]. – P.43. 18. HIV/AIDS epidemiology in Lithuania / G. Likatavi ius, V. Uzdaviniene, V. Lipnickiene, J. Rakickiene, S. Caplinskas // 14th international conference on the reduction of drug related harm “Strengthening partnerships for a safer future”, Chiangmai, Thailand, April 6—10, 2003: abstract. – [Chiangmai, 2003]. – P. 174. 19. Links between HIV and injection drug use (IDU) in Lithuania / Irma Caplinskiene // 10th European AIDS conference / EACS, Dublin, Ireland, Nov 17-20, 2005: abstract. – [Dublin, 2005]. - P.131.

Chapter 31 HIV Latency and Reactivation: The Early Years Guido Poli

31.1 HIV as a Retrovirus: A New Pathogenic Entity HIV is a short, 9.2-Kb retrovirus encoding a handful of structural (gag, pol, env) and non-structural genes. These are divided into “regulatory” (tat, rev) and “accessory” (nef, vif, vpu, vpr) genes to underscore the fact that the virus cannot efficiently replicate in vitro in the absence of the former, but can handle quite well the deletion of the latter (1). The discovery of HIV (2, 3) was shortly followed by the identification of its primary receptor, the CD4 molecule displayed on the very subset of T lymphocytes selectively and progressively depleted during AIDS (4). In addition, CD4 is expressed also on circulating monocytes and tissue macrophages later shown to be infected in vivo and infectable in vitro (5, 6). It took another 12 years, however, to completely unlock the secrets of how HIV infects T cells and macrophages with the discovery of the second receptor, or co-receptor, mandatory for the virus to interact with in order to enter these cells (7). This second receptor is a chemokine receptor, either CCR5 (8) or CXCR4 (7). The virus external glycoprotein gp120 Env must bind firstly to CD4 to undergo a conformational change enabling it to recognize the chemokine receptor. This sequential interaction frees the second Env glycoprotein, gp41, that, springing like a jackknife, inserts itself into the target cell membrane and promotes its fusion with the viral particle (virion) membrane. Once inside the cell, with the virion membrane and proteins dispersed in the plasma membrane, the virus is uncoated and initiates its most characteristic biochemical process: the transcription of its single-stranded RNA genome into an equivalent cDNA form utilizing a unique enzyme known as reverse transcriptase (RT). This process begins in the cytoplasm but is completed into the nucleus as a consequence of the interaction between the so-called pre-integration complex and specific From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

determinants of the nuclear membrane. Linear DNA genomes of the virus can integrate in hot spots of the host DNA (9) by effect of the viral enzyme integrase, while surplus circular DNA remains as a scar of recent infection. Because RT does not possess a proof reading capacity, this process spontaneously introduces random mutations into the viral genome, thus contributing to one of the greatest problems in the clinical management of HIV infection: its extraordinary molecular variability (10). Once integrated as a “provirus,” the virus DNA lives as long as the cell that has just been infected and no actual drug regimen is capable of affecting this condition (although integrase inhibitors are on the verge of being introduced into the clinical arena). From this moment on, the virus behaves as a multi-gene complex subjected to the rules of activation and inhibition of transcriptional and post-transcriptional processes typical of the infected cell, as described later in greater detail. Novel HIV RNA is synthesized by the host RNA polymerase II complex and is subjected to splicing into fully spliced (2Kb) and partially spliced (4.5-Kb) mRNA coding for Tat, Rev, and Nef and for Env, respectively. The Tat protein reenters the nucleus and binds to its target RNA sequence known as TAR, thus potentiating the process of viral transcription. Similarly, Rev binds to dispersed RRE sequences promoting the export of the partially spliced (4.5-Kb) and unspliced (9.2-Kb) RNA from the nucleus to the cytoplasm where they can be translated into novel viral proteins (10). A p55 Gag precursor polyprotein needs to be cleaved by the viral protease to generate novel infectious particles; protease inhibitors have represented indeed the breakthrough in the mid-1990s that, in together with different kinds of RT inhibitors, have generated the combination protocols known as highly active antiretroviral (HAART) therapy, a very important landmark in the partial conquer of HIV disease (Figure 31.1A). Assembly of new proteins and genomic HIV RNA at the inner side of the plasma membrane of the infected cell is the prerequisite for the production of a new virion progeny after sealing of the particles by the p6 Gag protein (10). 279

280

G. Poli

Figure 31.1. In vivo and in vitro HIV infection. (A) Typical course of natural HIV-1 infection in the absence of antiretroviral therapy. The three phases (acute infection, clinical latency, and AIDS) are associated with variations in the levels of virus replication that is partially (in some case effectively) controlled during the intermediate phase. Modified from ref. 62. (B; left panel) Schematic profile of in vitro cytopathic infection of CD4+ cells. Peak virus replication coincides with the peak of virus-induced cytopathic effect (variable according to the type of cell, type and amount of infectious virus and stimulatory conditions). After the peak, virus replication tends to fade away while cell proliferation regains pre-infection levels. These “surviving” cells frequently carry integrated proviruses in their genome; virus replication, unless stimulated by specific factors, is frequently latent or at low levels. (B; right panel) Limiting dilution cloning of cells surviving acute HIV infection has resulted in clonal cell lines such as U1 and ACH-2. Modified from ref. 63.

31.2 Surrogate Model Systems for Studying HIV Infection In Vitro All features briefly described previously have been learned on the basis of previous knowledge of the retroviral life cycle and, to some extent, from the study of infected cells of HIV+ individuals. However, the relatively easy possibility to infect a variety of human cell types, including both primary cells (such as mitogen-stimulated leukocytes or T-cell blasts and, later, monocyte-derived macrophages [MDMs]) and several cell

lines of T lymphocytic and myelomonocytic origin, has been of substantial help to grow significant amounts of HIV for sequencing and drug testing. In addition, it was noted earlier that not all the cells in culture died as a consequence of HIV infection (Figure 31.1B). Surviving cells, regaining viability and proliferative capacity, were frequently infected although not always productively. This immediately suggested that they could be used as surrogate models for studying the molecular mechanisms underlying viral latency as opposed to unchecked replication and to fish out relevant factors capable of converting a latently infected cell culture into a viral factory.

31. HIV Latency and Reactivation

Classical cell activators already known as inducers of viral replication, including mitogens such as phytohemagglutinin (PHA), phorbol esters (PMA), and demethylating agents, indicated that indeed HIV could be reactivated from bulk infected cultures entered into a quiescent phase and (Figure 31.2). In addition, the immortal nature of the cell lines used allowed a much more detailed analysis of virus–host cell interaction upon limiting dilution cell cloning. Clonal cell lines were generated and scrutinized for their pattern of either latent or

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active viral replication. Both “truly latent” (i.e., methylated), “restricted” (i.e., inducible by multiple factors), and “permissive” chronically infected cell lines were generated from lymphocytic and myelomonocytic tumor cells (11). In our Laboratory of Immunoregulation at the National Institute of Allergy and Infectious Diseases (NIAID), both directed by A.S. Fauci, under the guidance of Thomas M. Folks and in close collaboration with the Laboratory of Molecular Microbiology headed by Malcolm A. Martin, also at NIAID, two

Figure 31.2. U1 and ACH-2 cell lines as surrogate models for studying modulation of virus expression from integrated proviruses. (A) Conversion from latent to active virus production occurs after 24 to 72 hours in these cell lines as a function of the stimulus applied. Virus production does not cause a cytopathic effect unlike what observed in acutely infected cells. (B) Northern blot analysis and indirect immunofluorescence for expression of HIV proteins in the U1 cell line stimulated with PMA. The kinetics of HIV RNAs accumulation after cell stimulation resemble those observed during acute infection (starting with 2 Kb spliced messages to full length 9.2 Kb RNA). Spontaneous expression of HIV proteins occurs in a 1 to 2% of unstimulated cells, whereas cell stimulation results in a potent induction of expression in most cells at a given time point, as here observed after 24 hours of stimulation. Modified from ref. 64.

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cell lines became focus of general interest: the U1 cell line, derived from acutely infected U937 promonocytic cells, and the ACH-2 cell line, originated from similarly infected CEMderived A301 T lymphocytic cell line (12, 13). They were infected with the same laboratory-adapted viral strain known as HIV-1LAI/IIIB, using CXCR4 for infection (X4 virus) and represented an easy-to-handle model of relative (“restricted”) viral latency. U1 possessed two integrated, defective proviruses, while ACH-2 had only one that, provirus per cell, could generate an infectious progeny of passageable virus. Later, U1 was shown to be defective in the Tat/TAR axis (14, 15), contrary to early claims, while the ACH-2 provirus was shown to be defective but self-repairing during viral reactivation (16). Nonetheless, the exploitation of these two models, and of similar others generated independently (like the HL60-derived OM10.1 cell line), has provided a significant contribution to dissect out factors and modeling mechanisms regulating HIV replication and its latent phase.

31.3 Cytokines as Physiological Factors Controlling HIV Latency and Replication After the demonstration that integrated HIV provirus could be reactivated by either its own regulatory protein Tat (either endogenously synthesized or exogenously loaded as a protein) or cell-activating factors such as mitogens (PHA), cytokine-reach supernatants derived from either activated T cells or MDM (17) or PMA (known activators of protein kinase C, PKC, dependent pathways; Figure 31.2), the search for physiological modulators of virus replication begun. PMA and related stimuli were shown to induce the activation of a dormant cytoplasmic transcription factor originally thought to be restricted in its action to the regulation of Ig light chain synthesis: nuclear factor κB (NF-κB). Almost simultaneously a potent cytokine, tumor necrosis factor (TNF)α (cachectin, and its related molecule TNF-β/lymphotoxin) was shown to activate transcription of proviruses integrated in either T cells or macrophages by the same NF-κB-dependent pathway (18–20). Thus, the first molecular pathway through which an extracellular stimulus (such as a cytokine) could directly influence the expression of an integrated provirus through activation of a cellular transcription factor was demonstrated. Endogenously released TNF-α could activate HIV expression in U1 cells in an autocrine fashion, i.e. interacting with cell surface receptors, after PMA stimulation of either U1 or ACH-2 cells (21), as later demonstrated also in primary PBMC stimulated with IL-2 and in MDM as discussed further. Was the TNF–NF-κB axis the only regulatory pathway capable of influencing virus replication or was it simply the first example described out of several? Interleukin (IL)1 (either –α or –β, another fundamental pro-inflammatory cytokine) was shown to act like TNFs in terms of activation of NF-κB-dependent transcription in at least some macro-

G. Poli

phage infection models (19, 22). Another late inflammatory molecule, IL-6, crucial for inducing the synthesis of liver proteins collectively responsive for inducing the acute phase response, induced HIV expression in the monocytic U1 cell line and in primary MDM. However, after several attempts of demonstrating activation of NF-κB, we realized that IL-6 must have exploited a different modality of upregulating virus production. At the same time, we noticed that cells stimulated with IL-6 and TNF together showed a much greater induction of virus expression than cells stimulated with each cytokine separately (and even outscoring a simple additive effect of the two stimuli). Synergy was thus demonstrated between TNF and IL-6 (but also between IL-1 and IL-6, as later demonstrated; ref. 23). At the molecular level, still in the pre-polymerase chain reaction (PCR) era, we also noticed that IL-6 stimulation alone failed to induce detectable levels of HIV RNA (as assessed by Northern blotting) and yet it induced levels of virion production only two- to threefold lower than those induced in the same cells by TNF or PMA. Thus, we proposed that IL-6 stimulation alone was acting predominantly at one or more post-transcriptional levels (i.e., increasing the efficiency of translation of low levels viral RNAs) although it also synergized at the transcriptional level in the presence of TNF (23). Only in recent years it has been defined (24, 25) that IL-6 mostly, if not exclusively, activates HIV expression in U1 cells via activation of another family of transcription factors: Fos and Jun, forming AP-1 complexes capable of binding to target sequences present both in the HIV LTR and in an intragenic enhancer present in the gag gene (26, 27). AP-1 binding results in transcriptional activation of HIV expression, although, even by quantitative PCR, IL-6 stimulation of viral RNA synthesis is usually 10fold lower than that induced by TNF-α. After the demonstration that multiple cytokines could upregulate HIV expression in monocytic cells by activating different synergistic pathways, the next question was whether HIV-suppressive cytokines existed. It was already known that class I interferons (IFNs), IFN-α/-β, suppressed acute HIV infection of T cells and macrophages and that they inhibited virion production from chronically infected cell lines by acting on the very late step of budding and release of new progeny virions (28). But did cytokines distinct from IFNs capable of interfering with HIV replication exist? In 1986, Walker and Levy demonstrated the existence of “non-cytolytic soluble suppressor factor(s)” secreted by CD8+ T lymphocytes that inhibited virus replication during experiments of HIV isolation from PBMC of infected individuals (29). Depletion of CD8+ cells (including NK cells, later shown to be also effective in terms of suppression of virus replication) became a common procedure to increase the frequency of virus isolation, while the search for the inhibitory soluble factor(s) (also known as “CAF” for CD8-antiviral factor) remained elusive for at least 10 years, when HIV-inhibitory chemokines (RANTES, MIP1α, MIP-1β; ref. 30) and IL-16 (31, 32) were claimed to mediate such a biological activity.

31. HIV Latency and Reactivation

Our laboratory at the NIH investigated the hypothesis that anti-inflammatory cytokines could counteract the HIVinductive effects of pro-inflammatory cytokines also in terms of viral replication. The hypothesis turned to be correct, at least in part. Transforming growth factor (TGF)-β and, later, IL-10 (likely the most potent known anti-inflammatory cytokines) did inhibit and even suppressed HIV replication in U1 cells and MDM. TGF-β inhibited virus expression induced by stimulation of U1 cells with either PMA, IL-1, or IL6 (but not with TNF; ref. 33). If U1 cells were stimulated with PMA in the presence of anti-TNF-α neutralizing Ab (which reduced the levels of virus expression approximately of 50% by interfering with the endogenous TNF-α dependent loop of HIV expression) and TGF-β virus expression was virtually abolished. Finally, TGF-β inhibited HIV transcription as demonstrated by both Northern blotting and runon experiments (33). Thus, the existence of HIV-suppressive cytokines was indeed demonstrated; later on, retinoic acid, a well known differentiating agent, was shown to exert inhibitory effects on virus expression from monocytic cells similar to those of TGF-β (34). However, in primary MDM, TGF-β (as well as retinoic acid) demonstrated a bipolar behavior: it inhibited acute viral replication when given after infection, but, quite surprisingly, it exerted upregulatory effects when cells were prestimulated with TGF-β (34). Independent investigators had earlier described inductive effects of TGF-β on acute infection of primary MDM or activated PBMC (35– 37). Similarly, IL-10 could inhibit HIV replication in MDM (38); we confirmed this observation but observed that this occurred at fully biologically active concentrations capable of inhibiting the secretion of endogenous pro-inflammatory cytokines such as (TNF-α, and IL-6; (39). In contrast, when MDM were incubated with lower concentrations of IL-10 (not effective in suppressing cytokine secretion) enhancement rather than inhibition of virus replication was observed; in addition, IL-10 potentiated the HIV-inductive effects of these cytokines in U1 cells (although it did not activate virus expression per se; ref. 40). It was shortly demonstrated that IL-10 enhanced endogenous TNF-α dependent HIV expression in U1 cells (41, 42). IFN-γ also showed opposite effects on HIV replication, depending on the experimental conditions. Alone, it stimulated virus expression in U1 cells, but it inhibited virus production in combination with PMA and shut-off TNF-mediated HIV expression in these cells (43). However, an ultrastructural analysis showed unequivocally that IFN-γ was a per se inducer of virus production in U1 cells, but that it, in the presence of PMA, shifted the major site of virion assembly and release from the plasma membrane to intracytoplasmic vacuoles. IFN-γ actually potently synergized with TNF in terms of virus transcription, but it simultaneously cooperated in promoting cytokine-mediated cell death thus resulting in an abortive activation of virus production (43, 44). If U1 cells were co-stimulated with anti-Fas Ab and IFN-γ, no induction of virus was observed (since Fas engagement does not lead to NF-κB activation as

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in the case of TNF receptors) but the cells were synergistically killed by the two stimuli (44). Thus, bimodal cytokines, acting as either inducers or inhibitors of virus expression as a function of the experimental conditions were described. It was later realized that several cytokines and chemokines do exert opposite effects on viral entry (via direct engagement of chemokine co-receptors, as in the case of CCR5-binding chemokines, or through downregulation of CD4 and chemokine receptors, as shown for TNF and IFN-γ) and post-integration events in the HIV life cycle. In this regard, also chemokines such as IL-8, MCP-1, and CCR5-binding chemokines were shown to upregulate virus expression as in infected cells recently reviewed (45).

31.4 Cytokine-mediated Modulation of HIV Replication: From Cell Lines to Primary Cells Infected In Vitro or In Vivo The demonstration of a complex network of cytokines and related factors showing regulatory activities on virus expression in different cell lines made urgent understanding whether these phenomena were restricted to a bizarre set of randomly selected tumor cell lines or were they demonstrable in primary cells, including cells isolated from infected individuals. As already mentioned, there were already good evidence that both endogenous and exogenous cytokine stimulation modulated virus replication in primary MDM. The demonstration of their relevance for infected CD4+ T lymphocytes initially difficult because the standard protocol through which these primary cells were infected (i.e., via pre-activation with PHA and then incubation with IL-2) did not show evidence of cytokine-driven virus replication. Addition of specific anticytokine Ab did not result in meaningful and reproducible effects and the direct measurement of cytokine production after removal of PHA and change of its conditioned medium was essentially negative. However, we observed that stimulation of PBMC with IL-2 in the absence of preactivation with PHA resulted in a productive infection so-called T-cell- and macrophage-tropic viruses (46). Indeed, before the discovery of CXCR4 and CCR5 as entry co-receptors there were several, partially overlapping classification of primary isolates and laboratory-adapted viral strains, including their tropism for these two main target cells, coinciding quite well with that based on the capacity to infect and induce cytopathic effects in a reference cell line: the HTLV-1 transformed MT-2 T-cell line. Viruses scoring positive in the infection of this cell line were defined as syncytia-inducing (SI) while those testing negative were simply defined as non-SI (NSI) viruses (47, 48). It was later shown that MT-2 cells, like most cell lines, are exclusively positive for CXCR4 expression and, therefore, permit infection and replication of only those viruses displaying a gp120 Env utilizing this chemokine receptor; conversely, NSI viruses engage selectively CCR5.

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Figure 31.3. Multiple effects of IL-2 on HIV replication. (A) PBMC or lymph node mononuclear cells (LNMC) obtained from HIV-infected individuals were stimulated in vitro with either IL-2 or IL-12. In the presence of CD8+ cells, only IL-12 induced virus replication. Removal of CD8+ cells unleashed the upregulatory effects of IL-2 outscoring those of IL-12; the lower panels indicate RT activity, a much less sensitive assay than the p24 Gag assay indicated in the top panels. Modified from ref. 53. (B) A schematic model of multiple IL-2 mediated effects on HIV infected cells. HIV- Suppressive effects mediated by CD8+ cells are dominant over the inductive ones and are hypothetically proposed to contribute to the net gain in CD4+ T cells observed in individuals receiving intermittent IL-2 therapy (references in the text).

31. HIV Latency and Reactivation

IL-2 stimulated PBMC secreted several cytokines already characterized as inducers of virus expression in cell lines (TNF-α, IL-1β, IFN-γ, and IL-6) and their peak level of production coincided or preceded that of virus replication. More importantly, their neutralization by Ab or IL-1 receptor antagonist resulted in a significant inhibition of virus replication. This was an important demonstration of two concepts: first, cytokines do act in an autocrine/paracrine fashion in terms of regulation of HIV replication in primary cells, as independently reported (49); second, IL-2 is not a direct inducer of virus replication but it is the induction of this pro-inflammatory cytokine cascade responsible for the virus-inductive effects triggered by this cytokine. This observation had relevance for the initial studies just published on the potential clinical use of IL-2 in HIV-infected individuals, showing expansion of CD4+ T lymphocytes without alterations of steady state viremia levels (but inducing only spikes of transient virus replication) (50, 51). After several successful phase II trials, intermittent IL-2 therapy is currently under phase III evaluation in thousands of patients worldwide (52). Even more convincingly, follow-up studies conducted with either peripheral or lymph node cells of infected individuals demonstrated that among other effects IL-2 potently activated CD8+ cells to exert their “CAF” activity on infected CD4+ cells in that removal of CD8+ cells unleashed the upregulatory effect of IL-2 on virus replication (53), as independently observed (Figure 31.3; ref. 54). After the demonstration of the existence of latent HIV reservoirs in resting memory CD4+ T cells, it was shown that their stimulation with a cocktail of pro-inflammatory cytokines, including IL-2, IL-6, and TNF-α (55), and later, IL-7 (56–59), resulted in the activation of virus replication.

31.5

Conclusions and Perspectives

Looking back at the early days when a number of basic paradigms were established may be helpful for the new challenges facing modern investigators dealing with the unsolved problems of the HIV/AIDS pandemic. Will IL-2 based therapy be successful in terms of AIDS-sparing, lifesaving events? How much of its activity is attributable to a direct effect on target cells and how much is the contribution of secondary cascades triggered by a pharmacological use (millions of units per day in cycles of 5 days every 4–8 weeks) of this cytokine? Will other cytokine join IL-2 in clinical trials involving HIV infected individuals? IL-7, IL-15, and GM-CSF, sharing with IL-2 the activation of the Janus kinase/signal transducer activator of transcription (JAK/STAT) pathway and, particularly, the activation of STAT5A and STAT5B (60, 61), have already demonstrated interesting evidence of in vivo activity. Will cytokines such as IL-2 be relevant in the design of strategies aimed at curtailing viral reservoirs or, at least, at controlling their capacity to ignite virus replication in conditions of absent or weak control by ARV therapy?

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In conclusion, the beauty of studying HIV infection in in vitro models, either from a molecular, virological, or immunological perspective, as here exemplified for the study of cytokine, is that most observations will bear relevance to clinical application, if rightly analyzed and interpreted. Acknowledgements. This chapter mostly relies on studies conducted during my stage in the Laboratory of Immunoregulation at NIAID, NIH, Bethesda, MD. I wish to express my sincere gratitude to Tony Fauci and to all my friends and collaborators of that time for what I learned in those years. Whatever I have accomplished thereafter is the consequence of attending a very good school! This study is supported by my grant from the V° National Program of Research Against AIDS of the Istituto Superiore di Sanità, Rome, Italy.

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monocytic lineage. J Acquir Immune Defic Syndr Hum Retrovirol 9:442–449. Angel JB, Saget BM, Wang MZ, Wang A, Dinarello CA, Skolnik PR (1995) Interleukin-10 enhances human immunodeficiency virus type 1 expression in a chronically infected promonocytic cell line (U1) by a tumor necrosis factor alpha-independent mechanism. J Interferon Cytokine Res 15:575–584. Barcellini W, Rizzardi GP, Marriott JB, Fain C, Shattock RJ, Meroni PL, Poli G, Dalgleish AG (1996) Interleukin-10-induced HIV-1 expression is mediated by induction of both membranebound tumour necrosis factor (TNF)-alpha and TNF receptor type 1 in a promonocytic cell line. AIDS 10:835–842. Biswas P, Poli G, Kinter AL, Justement JS, Stanley SK, Maury WJ, Bressler P, Orenstein JM, Fauci AS (1992) Interferon gamma induces the expression of human immunodeficiency virus in persistently infected promonocytic cells (U1) and redirects the production of virions to intracytoplasmic vacuoles in phorbol myristate acetate-differentiated U1 cells. J Exp Med 176:739–750. Biswas P, Poli G, Orenstein JM, Fauci AS (1994) Cytokinemediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor alpha and gamma interferon selectively kill HIV- infected cells? J Virol 68:2598–2604. Alfano M, Poli G (2005) Role of cytokines and chemokines in the regulation of innate immunity and HIV infection. Mol Immunol 42:161–182. Kinter AL, Poli G, Fox L, Hardy E, Fauci AS (1995) HIV replication in IL-2-stimulated peripheral blood mononuclear cells is driven in an autocrine/paracrine manner by endogenous cytokines. J Immunol 154:2448–2459. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M (1993) Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med 118:681–688. Koot M, van Leeuwen R, de Goede RE, Keet IP, Danner S, Eeftinck Schattenkerk JK, Reiss P, Tersmette M, Lange JM, Schuitemaker H (1999) Conversion rate towards a syncytium-inducing (SI) phenotype during different stages of human immunodeficiency virus type 1 infection and prognostic value of SI phenotype for survival after AIDS diagnosis. J Infect Dis 179:254–258. Vyakarnam A, McKeating J, Meager A, Beverley PC (1990) Tumour necrosis factors (α, β) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication. AIDS 4:21–27. Kovacs JA, Baseler M, Dewar RJ, Vogel S, Davey RT JrFalloon J, Polis MA, Walker RE, Stevens R, Salzman NP (1995) Increases in CD4 T lymphocytes with intermittent courses of interleukin-2 in patients with human immunodeficiency virus infection. A preliminary study. N Engl J Med 332:567–575. Chun TW, Engel D, Mizell SB, Hallahan CW, Fischette M, Park S, Davey RT JrDybul M, Kovacs JA, Metcalf JA, Mican

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Chapter 32 HIV-1 Sequence Diversity as a Window Into HIV-1 Biology Milloni Patel, Gretja Schnell, and Ronald Swanstrom

32.1

Overview

Sequence diversity represents a rich source of information about the selective pressures placed on a genetically dynamic organism such as HIV-1. Information can be gleaned by sampling infected subjects cross-sectionally, sampling individuals longitudinally, and comparing the virus present in different compartments within an individual. Patterns of sequence diversity and evolution can provide insights into the nature of the selective pressure and represent an intimate part of the response of the virus to changing virus–host interactions. This chapter discusses research trends in the study of newly transmitted HIV-1, the evolution of co-receptor use, compartmentalization of HIV-1 in the central nervous system (CNS), and differences between HIV-1 subtypes and the neutralizing antibody response. Deciphering the nature of the transmitted virus will contribute to an understanding of the selective pressures at the time of transmission and refine the target of vaccine development. Determining the ways in which the virus is compartmentalized will add to our understanding of virus–host interactions and viral pathogenesis. Part of understanding viral pathogenesis is the evolution of co-receptor use that is associated with advanced disease state. Finally, the natural immune response that prevents disease progression in some patients is partially due to an efficient neutralizing antibody response, and understanding why a particular antibody is broadly cross-reactive will help in designing immunogens that will generate these important neutralizing antibodies.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

32.2 Complexity of Newly Transmitted Virus The HIV-1 population within a chronically infected individual is heterogeneous and complex, and varies in sequence as much as 8% in the env gene (1, 2). However, transmission of virus from a chronically infected person is limited to one or a few variants from the entire viral population, resulting in a genetic bottleneck at the time of transmission (1, 3–6). To study the transmission of HIV-1 viral variants, we focused on cross-sectional examination of primary infection subjects. Primary infection is the earliest stage of HIV-1 infection and occurs in the absence of an adaptive immune response (5–8). The study of transmitted variants is complicated by the early diversification of the virus population. Approximately 8 to 11 weeks post-infection, simian immunodeficiency virus (SIV) diversification begins in response to host immune pressure (9). Thus, the study of transmitted virus requires the correct identification of subjects during primary infection to prevent a bias toward the presence of multiple variants as a result of this diversification. The method we used to examine the complexity of newly transmitted virus is the heteroduplex tracking assay (HTA). The HTA is a gel-based assay that separates viral variants based on sequence differences, and can resolve variants that comprise as little as 3% of the total viral population (1, 4, 5, 10, 11). The HTA has advantages over conventional cloning procedures in that the entire viral population can be sampled at one time, and reproducing the HTA pattern can validate the quality of sampling (5, 11–13). Studies with SIV have shown the transmission of multiple variants under experimental conditions (9). A study by Rybarczyk et al. detected multiple variants by HTA during primary infection in rhesus macaques inoculated with a genetically complex SIVsm strain. A comparison of intravenous versus intrarectal-challenged macaques showed no evidence for selection of specific variants at the mucosal membrane (9). In addition, the timing of SIVsm diversification was correlated with the neutralizing antibody titer (9). 289

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The potential for the transmission of multiple HIV-1 variants has been documented in previous studies (2, 3, 5, 14, 15). However, a link to specific viral or host factors is incompletely understood, and the reported frequency of the transmission of multiple variants varies widely. A study by Ritola et al. assessed HIV-1 viral diversity of the V1/V2 regions of env in a primary infection cohort using HTA. Examination of the V1/V2 region of env detected multiple variants in about half of the primary infection subjects (5). In contrast, a primary infection study by Delwart et al. detected only single HIV-1 variants in men infected via homosexual transmission (3). Studies by Long et al. have shown a correlation between the transmission of multiple variants and the sex of the infected individual, where more than half of women infected via heterosexual transmission had multiple HIV-1 variants and only single variants were found in men infected via heterosexual transmission (14). Because transmission of HIV-1 is a low probability event, we have suggested that multiple variants are present in infected cells, and the transmission of an infected cell could result in the transmission of multiple variants (5). More recently, we have developed evidence that the estimation by HTA of the number of variants transmitted can be an overestimate in this type of cross-sectional analysis. It is likely that multiple variants can be transmitted but not at as high a frequency as previously reported (Schnell, unpublished observation). Our results suggest that the initial diversification of virus in humans occurs earlier than we observed in macaques, suggesting that the higher viral loads in macaques may blunt the initial immune response. Also, this observation indicates how important it is to work with viruses collected as close to the time of transmission as possible to avoid the problem of early genetic diversification.

32.3 Compartmentalization and HIV-associated Dementia Compartmentalized HIV-1 variants have been identified in the CNS and the genitourinary tract of HIV-1 infected individuals (12, 13, 15–19). HIV-1 infection of the CNS occurs shortly after peripheral infection through the trafficking of infected monocytes across the blood–brain barrier (16, 20, 21). In a subset of infected individuals, HIV-1 infection of the CNS leads to compartmentalization and neurological complications, including the development of HIV-1-associated dementia (HAD) or minor cognitive motor disorder (MCMD; refs. 12, 20–23). Invasion of the CNS may provide HIV-1 an opportunity to establish an autonomously replicating viral population in the presence of therapy due to the poor CNS penetration of some antiretrovirals (ARVs; refs. 22, 24–26). Compartmentalized variants in the CNS are genetically distinct from peripheral virus, but the factors important for compartmentalization and the development of neurological disease are not currently known. The HTA is a sensitive assay

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that can be used to study HIV-1 population complexity and dynamics and identify compartmentalized variants within the population (4, 5, 10–13, 27). Examination of viral populations in the CNS frequently relies on studies of HIV-1 in cerebrospinal fluid (CSF), which is thought to contain virus originating from both the periphery and the CNS (19, 23, 28–30). We used HTA to distinguish between viral variants in the CSF that originated from CNS tissue and variants that are shared between peripheral blood and CSF. Viral variants can be considered compartmentalized by HTA if the variants are unique to the CSF, or if their relative abundance significantly differs between the periphery and the CSF (12, 13). Previous studies have found increased HIV-1 compartmentalization in the CNS of individuals with HAD or MCMD (12, 18, 31, 32). A study by Ritola et al. investigated the relationship of CNS compartmentalization and the severity of neurological disease by examining the V1/V2 and V3 regions of env in plasma and CSF from individuals with HAD, MCMD, or no neurological symptoms (12). HTA analysis showed at least some compartmentalized viral variants in the CSF of most subjects; thus, the simple detection of CSF-compartmentalized variants was not correlated with the level of neurological disease. However, the fraction of virus in the CSF that represented CNS-unique variants was present at a higher frequency in the CSF of HAD subjects. The molecular mechanisms that lead to the development of HAD are still unknown. Several studies have suggested that as disease advances, virus present in the CSF shifts to a higher percentage of CNS-derived virus (12, 18, 22, 28, 33). The immunological failure associated with advanced HIV-1 disease may result in increased viral replication in the CNS, leading to a higher fraction of CNS-derived virus in the CSF (12, 28, 33). Perivascular macrophages and microglia in the CNS can be productively infected by HIV-1, and previous studies have suggested that macrophage-tropic viruses are neurotropic (16, 21). In addition, Gonzalez et al. reported that a specific genotype of MCP-1, the MCP-1-2578G allele, was correlated with an increased risk of HAD (34). This specific genotype of MCP-1 was associated with increased inflammation and the ability to upregulate HIV-1 infection, which may increase the risk of developing HAD. The time at which CNS compartmentalization occurs during HIV-1 infection is also unknown. Determining whether compartmentalization initiates during the primary or chronic stages of HIV-1 infection could have major implications for ARV treatment regimens, including the timing of CNSpenetrating drugs (18). A study by Ritola et al. used HTA to examine whether HIV-1 compartmentalization occurs during primary infection using a cross-sectional comparison of the env V1/V2 variable regions for plasma, semen, and CSF (5). Virus was detected in plasma, semen, and CSF from acute primary infection subjects, indicating the rapid expansion of virus in these compartments. However, compartmentalization was not evident between the CSF or semen and the plasma compartments, suggesting that compartmentalization does not

32. HIV-1 Sequence Diversity as a Window Into HIV-1 Biology

occur during primary HIV-1 infection and must develop later in the course of infection.

32.4 Source of Compartmentalized Virus in the CNS HIV-1 invades the CNS shortly after initiating a peripheral infection (20, 21). Once HIV-1 has moved across the blood–brain barrier, it comes in contact with different cell types in the brain parenchyma, including perivascular macrophages, microglia, astrocytes, oligodendrocytes, and neurons (21). Perivascular macrophages and microglia are the only cell types in the CNS that are known to sustain productive HIV-1 infection, although astrocytes may be non-productively infected by HIV-1 (21, 35–37). Our laboratory is interested in characterizing the cellular sources of HIV-1 in the CNS, and determining the mechanisms HIV-1 uses to persist in the CNS. Previous studies examining the kinetics of HIV-1 decay have shown two phases of viral decay in the peripheral blood of subjects initiating highly active ARV therapy (HAART; refs 38–40). The first phase of decay is rapid and results from the death and clearance of short-lived infected CD4+ T cells and free virus (38–40). The second phase of viral decay is slower and of unknown source, but may be the result of clearance of long-lived infected cells like macrophages and/or resting CD4+ T cells (38–40). Several studies examining viral decay rates in the CSF of HIV-1-infected subjects reported similar viral decay kinetics in both CSF and plasma for most individuals, but some subjects showed a slower clearance of virus from the CSF compared to plasma (18, 22, 28, 33). Slower clearance of HIV-1 from the CSF has been associated with the presence of neurological disease (18, 22, 28, 33). A study by Eggers et al. found that HIV-1-infected individuals with severe neurological symptoms show much slower viral decay rates in the CSF compared to plasma after initiating HAART (22). An examination of asymptomatic (no neurological symptoms) subjects initiating HAART by Harrington et al. found that the majority of compartmentalized virus in the CSF is produced by short-lived cells (13). This study utilized the HTA to identify CNS-compartmentalized virus, then measured the viral decay rates for each detected variant. The HIV-1 population in the CSF of these asymptomatic subjects was found to be 11 to 85% compartmentalized (enriched and/or unique to the CSF), and the compartmentalized variants decayed rapidly once HAART was initiated for each subject (13). HIV-1 persistence in the CNS can be described by two pathways based on previous studies (18). The first pathway is based on the continuous trafficking of short-lived infected CD4+ T cells into the CNS. Virus in the CNS is constantly replenished from the periphery, resulting in similar HIV-1 populations in both the periphery and the CSF (12, 18). In this model, virus in both the CSF and plasma should decay rapidly upon the initiation of HAART (18). This type of CNS infec-

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tion has been described in previous studies involving asymptomatic HIV-1 infected subjects (12, 13, 18). The second pathway is based on HIV-1 infection of longlived perivascular macrophages and microglia in the CNS. Virus originates from local CNS tissue, resulting in large differences in HIV-1 populations in the CSF and periphery (12, 18). The second model predicts that virus in the CSF will have slower decay kinetics compared to the periphery due to the slower turnover time of macrophages and microglia (18, 22). Previous studies involving subjects with severe neurological disease that are initiating HAART have described strong compartmentalization between the CSF and plasma, and slower elimination of HIV-1 from the CSF (12, 18, 22, 28, 33). The connection between HIV-1-associated neurological disease and actual viral replication in the CNS has not been established, but is an area of active research.

32.5 Evolution of CCR5 Usage to CXCR4 Usage For reasons that are just beginning to be understood, virus that is most frequently transmitted by any route of infection uses CCR5 as a co-receptor (R5-tropic virus; refs. 41–44). It has been suggested that this is due to the uptake of R5-tropic virus by dendritic cells at mucosal surfaces where most transmission occurs (45, 46). This is further demonstrated in the population that contains a deletion in the CCR5 allele ( 32), where individuals who are homozygous for this allele are usually resistant to infection, and heterozygous individuals have delayed disease progression (47–49). The evolution of virus from CCR5 usage into CXCR4 usage (X4-tropic virus) occurs in 50% of patients infected with subtype B HIV-1, and CXCR4 usage is strongly associated with disease progression (50–53). It is not yet clear whether X4-tropic viruses cause a more rapid decline in T cells or whether X4-tropic viruses grow out when the host is experiencing a rapid decline in T cells due to loss of even modest control of the infection, although both possibilities have been considered (54, 55). Attempts to characterize the progression from R5- to X4-tropism have come up with varied results. The mutations associated with co-receptor switch were at first attributed solely to mutations in V3; however, subsequent data suggests that the story is more complicated (56–59). A study by Bagnarelli et al. suggests that the V3 loop is the only determinant of co-receptor usage in 76% of viruses (56). Another study used CCR5/CXCR4 chimeras to show that R5-tropic virus evolve to acquire broader use of CCR5 (accompanied by an increasing loss of sensitivity to CCR5 ligand RANTES), suggesting that ongoing evolution is not directed to specific V3 amino acids (57). The final question that arises in understanding co-receptor usage is why the switch occurs; unfortunately the answer is not clear. Affinity of gp120 is much higher to CCR5 (4–15 nM) than CXCR4 (200–500 nM) and although CCR5 is expressed

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on many cell types (including activated memory T cells, natural killer T cells, monocytes, macrophages, immature dendritic cells), HIV-1 can only infect cells that co-express CD4 with the co-receptor (60–62). Therefore, R5-tropic virus can only infect 10% of CD4+ T cells, whereas X4-tropic virus can infect 85% of CD4+ T cells (such as naïve T cells; refs. 63, 64). This suggests that co-receptor usage can be driven by appropriate T-cell availability. The co-receptor switch may be important (even with an affinity loss) in increasing host range after depletion of CCR5+ T cells, which is now understood to take place during the acute phase of infection (65–67). However, if CD4+/CCR5+ T-cell depletion takes place so early, how and why does virus persist for so long in a limited host range? There are many intrinsic obstacles to co-receptor switching: X4-tropic virus has diminished replication compared to parent R5-tropic virus; evolutionary intermediates are more sensitive to both CCR5 and CXCR4 inhibitors than parent or final virus; non-random changes in amino acids and glycosylations are required to use CXCR4 as a co-receptor, and R5-tropic virus may release more virus/cell (68, 69). Further questions of the need to change co-receptor arise when we consider the 50% of patients that do not evolve to use CXCR4 (57, 70, 71). How are the viruses in these patients able to utilize CCR5 efficiently enough to cause disease progression? Finally, extended use of a CXCR4 inhibitor (AMD3100) shifted X4-tropic virus back to using CCR5, suggesting that there is a competition between the mixed populations of virus in vivo for the most efficient co-receptor that can be used (72). Current evidence points to a slow evolution to CXCR4 usage through mutations in V3, which may be enhanced or compensated by non-V3 mutations. There have been many studies on the most efficient method of predicting virus co-receptor phenotype using statistical approaches, many of which focus on the V3 region of Env. The original rule uses the net charge of the V3 as an indicator of co-receptor usage, since there is an increase in net V3 positive charge of CXCR4-using Envs (73, 74). The 11/25 rule, with a basic amino acid at position 11 and/or 25 (HXB 306 and 322, respectively), is also strongly associated with an X4-tropic phenotype (75–77). Furthermore, several studies have shown that positions 429, 440, 424, and a cluster of amino acids between 190 and 200 are all involved in the co-receptor switching (78, 79). Jensen et al. created a position-specific scoring matrix (PSSM) specific to subtype B V3 sequences to improve co-receptor prediction that is 84% sensitive and 96% specific (80). PSSM is used to detect non-random distributions of amino acids at adjacent sites associated with empirically determined groups of sequences. Often, PSSM uses background genetic variations as a baseline comparison (or null model) to facilitate comparison of the residues of a sequence fragment to those of a group of aligned sequences known to have the desired property. Using this principle, V3 sequences can be assigned a score; the higher the score, the more closely the sequence resembles those of known X4 sequences. Jensen et al. have also designed PSSM for subtype C V3 sequences to predict co-receptor use (81). These methods have been used extensively to predict

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co-receptor use, but biostatistical modeling is not 100% efficient, because they are not robust for evolutionary intermediates and their accuracy is limited by the volume of X4 sequences available to define different evolutionary pathways.

32.6 CCR5 and CXCR4 Usage Differences Between Subtype B and Subtype C HIV-1 Phylogenetic analysis shows that HIV-1 can be divided into three groups: M (main), N (new), and O (outlier). The M-group (99% of infections) is further subdivided into nine subtypes (A to K) and circulating recombinant forms in which each subtype has an env nucleotide divergence of 35% between subtypes, 20% divergence within subtypes, and up to 8% diversity within one person (1, 82–84). Subtype B is prevalent mostly in North America and Western Europe, and subtype C is the world’s most prevalent strain, found in sub-Saharan Africa, India, and Southeast Asia, accounting for half of the new infections worldwide. As described previously, subtype B virus can evolve from using CCR5 to using CXCR4 in 50% of patients; however, Ping et al. and others have determined that subtype C is less likely to use CXCR4 during disease progression (85–93). Thus, two major subtypes of HIV-1 have evolutionary pathways that are different from each other. The consensus V3 sequence differs in only five amino acid positions between subtypes B and C, suggesting that sequence changes affect structure in a way that limits subtype C HIV-1 from evolving to use CXCR4 efficiently. A recent study by Coetzer et al. suggests that 10% of subtype C isolates can evolve to use CXCR4 (strongly associated with CD4+ T cell count <200 cells/mm2) and sequences that are associated with V3 changes also have increased positive charge (not associated with V3 position 11 and/or 25 as with subtype B), insertions, loss of negatively charged residues, and elimination of glycosylation at N301 (V3 amino acid 6; refs. 73, 94). Most telling was that X4-tropic viruses develop a mutation at Q315 (V3 amino acid 18), usually to Arg (the subtype B consensus amino acid at position 18; refs. 73, 94). This suggests that the presence of R315 plays an important role in the structure of V3 that is capable of switching co-receptor usage. Compared to subtype B R5-tropic virus, subtype C R5-tropic virus may have a slower replication profile (94, 95). Implying subtype C-infected patients harbor lower viral loads during early infection (X4-tropic virus replication is similar for both subtypes; refs. 94, 95). Patients who have progressed to AIDS or are on prolonged ARV therapy do evolve subtype C X4-tropic virus, suggesting that the evolutionary pathway to X4-tropic subtype C virus is different and, perhaps, longer and more difficult than subtype B virus (90, 91). The subtype C virus V3 interaction with CCR5 has been defined by alanine scanning mutations of V3 to determine the effect on co-receptor usage (96). For subtype C: the V3 crown is dispensable for CCR5 usage; R298A and I324A

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(V3 amino acids 3 and 27) have more than 90% decrease in CCR5-mediated fusion; and N301A, N302A, T303A, I327A, and R328A (V3 amino acids 6, 7, 8, 30, and 31) reduce CCR5-mediated fusion modestly. The CCR5 amino acids involved in interacting with subtype C virus have also been investigated. The charged and Tyr residues in the N terminal of CCR5 are important for virus fusion, as are amino acids Q170, Y176, C178, K191, in ECL2. There are many contacts made between subtype C Envs and CCR5, but the interaction of CCR5 with subtype B Envs is not reduced as drastically due to a mutation, suggesting a higher affinity to subtype B Env (97–99). Our group has shown that the V3 stem and C4 are more conserved between subtype B and C; thus, the stem interactions for both subtypes may be similar, whereas the V3 crown–ECL2 interaction may be different between the subtypes (Patel et al., in press). Therefore, we hypothesize that the variability in the V3 regions between subtypes B and C at the amino acid level is responsible for inducing structural differences that allow subtype B virus to evolve from using CCR5 to using CXCR4, but restrict subtype C virus from doing the same. Further evidence that suggests limited subtype C CXCR4 usage is attributed to viral factors, is that subtype B V3 structures can adopt alternative conformations (100). Therefore, subtype C V3 may be conformationally restrained, requiring a more complicated evolutionary pathway to acquire CXCR4 usage. Thus, it can be conceived that with a subtype C infection, R5-tropic virus is more structurally constrained than its subtype B counterpart. Therefore, subtype C X4-tropic virus is only manifested in small numbers late in disease progression.

32.7 Neutralizing Antibodies Against HIV-1 Env An ideal vaccine candidate would elicit neutralizing antibodies in addition to cell-mediated immunity. However, despite large efforts, progress toward an effective immunogen has been unsuccessful. The one large-scale clinical trial thus far was based on gp120 protein (VaxGen) and failed to elicit broadly neutralizing antibodies and was not protective (101). There is poor elicitation of neutralizing antibody in natural infection, most likely due the low immunogenicity of Env (the only target of neutralizing antibody; ref. 102). The trimeric nature of the spike glycoproteins may also occlude many surfaces from antibody binding (103). Neutralizing antibody responses during natural infection do occur, but the high evolution rate of HIV-1 ensures that virus diversification, and thus neutralization escape, is inevitable. A strong neutralizing antibody response is associated with delayed disease progression, and Rybaczyk et al. have shown that the corollary also holds true in the SIV model; lack of a neutralizing antibody response leads to rapid disease progression (9, 104–106). Because broadly neutralizing antibodies are rarely found in natural infection, opportunities to include immunogens as part

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of a vaccine strategy to induce neutralizing antibody responses are limited. The five types of neutralizing antibodies that have been identified are represented by the following monoclonal antibodies: (1) 447-52D, which binds to the crown of the V3 region (G-P-X-R epitope) (107); (2) IgGb12, which binds to the CD4 binding site (108); (3) 17b, which binds to the CD4-induced epitope (109); (4) 2G12, which binds to carbohydrate moieties on the surface of gp120 (110); and (5) 2F5 and 4E10, which bind to the membrane proximal external region (MPER) of gp41 (111, 112). The 2F5 and 4E10 antibodies are the only neutralizing antibodies against gp41. Attempts to elicit other MPER-directed neutralizing antibodies are problematic, because these antibodies are autoreactive and can also bind cardiolipin and double-stranded DNA (113). 2F5 and 4E10 are the most potent neutralizing antibodies in terms of their large breadth and low IC50 (114, 115). Therefore, they have been the focus of the type of neutralizing antibody that would be optimal to generate from a vaccine. Because antibodies with specificity to the 2F5 and 4E10 epitopes are rare in natural infection, the search to induce this type of antibody has been initiated (116). However, our group and other investigators have identified neutralization escape mutants that make the virus resistant to MPER neutralizing antibodies. Furthermore, our group has identified a mutation at position 668 (S→G) in the MPER region that is located between the adjacent 2F5 and 4E10 epitopes that makes virus sensitive to both antibodies (N. Takamune, unpublished observation). In addition, the use of 2F5 and 4E10 as a prophylaxis in conjunction with 2G12 proved efficient in simian SIV trails, but failed when used in chronically infected humans (117–120). One explanation is that 2G12 escape mutants were the first to emerge, and 2F5 and 4E10 did not provide significant selective pressure (no 2F5 or 4E10 escape mutants occurred). Finally, position 240 in the C2 region of Env is another amino acid that is a determinant of neutralization. Wei et al. established that K240T is a mutation that appears early and persists throughout infection, suggesting it confers neutralization escape, most likely due to the addition of a glycosylation site caused by this mutation (103). Position 240 is located close to a neutralization escape mutation found during a study of CNS-derived virus in our laboratory that showed T244K is associated with neutralization escape (Harrington, unpublished observation). The importance of these amino acids in the C2 region remain to be determined; however, their location close to gp41, away from other structures within Env that are associated with neutralization escape, makes 240 and 244 interesting residues to understand.

32.8

Conclusion

The analysis of sequence diversity will remain a potent tool for assessing and validating selective pressure on the virus population. As the evolutionary potential of HIV-1 remains a major obstacle in dealing with the epidemic, the study of sequence

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diversity and evolution will continue to have a central role in the study of HIV-1. In addition, the technical progress and biological insights that are rapidly appearing in the HIV field will help move the study of other infectious diseases forward. Acknowledgments. We are grateful for the long-term support from the NIH and especially the NIAID award R37-AI44667 that allowed us to pursue questions about the biological implications of HIV-1 sequence diversity. We are also grateful to other lab members past and present who contributed to the studies cited in this manuscript.

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Chapter 33 Human Monoclonal Antibodies Against HIV and Emerging Viruses Dimiter S. Dimitrov

33.1

Introduction

Throughout the centuries, viral diseases have killed hundreds of millions of people and continue to do so. More than 100 million died in the past from smallpox caused by the variola virus. Influenza killed about 50 million people during the 1918 pandemic. More than 60 million have been infected with HIV since the first cases of the epidemic were reported in 1981, and more than 20 million have died from AIDS. Although vaccines remain the most cost-effective way to prevent viral infections, in many cases (e.g., HIV) vaccines are not available and, in other cases, available vaccines have to be modified frequently to counteract virus escape (e.g., influenza). Prophylaxis and treatment in such cases are critically important to fight viruses. A number of studies have shown the importance of neutralizing antibodies in recovery and protection from viral infections (1, 2). Sera from humans or animals containing antibodies have been widely used for prophylaxis and therapy of viral and bacterial diseases since the late 1800s (3–6). Serum therapy of most bacterial infections was abandoned in the 1940s after antibiotics became widely available (5). However, polyclonal antibody preparations have continued to be used for some toxin-mediated infectious diseases and venomous bites (3). Serum immunoglobulin (Ig) has continued to be also used for viral diseases where there are few treatments available, although mostly for prophylaxis either prior to an anticipated exposure or following an exposure to an infectious agent (7–9). Antibody products licensed in the United States for prevention or treatment of viral diseases include human Ig for use against hepatitis A and measles; virus-specific polyclonal human Ig against cytomegalovirus, hepatitis B, rabies, respiratory syncytial virus (RSV), vaccinia, and varicella-zoster virus; and the humanized monoclonal antibody (mAb) Synagis (7). Polyclonal Ig has also been used with various success for diseases caused by other human From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

viruses including parvovirus B19 (10–13), Lassa virus (14, 15), West Nile virus (16, 17), some enteroviruses (18, 19), herpes simplex virus (20), Crimean-Congo hemorrhagic fever virus (21), Junin virus (22), SARS-CoV (23, 24), and HIV (25–30). Although serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot-to-lot variation, and uncertain dosing have limited their use (3). mAbs including chimeric animal-human, humanized, and fully human mAbs (hmAbs) possess lower or absent immunogenicity, toxicity, and lot-to-lot variation. The molecular mechanisms of therapeutic efficacy of such antibodies are easier to dissect, and they can be engineered to further improve their therapeutic properties. Recently, some mAbs have shown clinical success. The humanized mAb Synagis (palivizumab), which is still the only mAb against a viral disease approved for clinical use by the U.S. Food and Drug Administration, has been widely used for prevention of RSV infections in neonates and immune-compromised individuals, and very recently has been further improved (31). However, it is not effective for treatment of an already established infection; for example, there were no significant differences in the clinical outcomes between the placebo and the palivizumab groups for children hospitalized with RSV infection (32); in addition, resistance can develop relatively quickly: a recent study found F gene-resistant mutations in an animal model of the RSV infection (cotton rat) 12 weeks after infection including a completely resistant virus (33). The development of hmAbs for prophylaxis and treatment of diseases caused by HIV and emerging viruses is still in an initial stage. This chapter reviews hmAbs with potential for prophylaxis and treatment of diseases caused by HIV-1, SARS-CoV, and henipaviruses (Hendra [HeV]) and Nipah [NiV]). Mostly IgG1 are discussed unless specifically noted otherwise; other isotypes also could be useful but are not so frequently used and other formats including Fabs and single-chain variable region fragments (scFvs) are noted when described. Finally, possible implications for development of effective vaccines are discussed. 299

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33.2

HIV

The demand for new treatment options against HIV is becoming increasingly important as the side effects and the expansion and spread of drug-resistant virus within the infected population limit the clinical benefits provided by available anti-HIV drugs. Despite the promise presented by Synagis and its improved variants (31), developing effective antibodybased therapeutics against HIV presents an especially difficult challenge. Therapies based on small molecule-based drugs eventually fail because of the expansion of a drug-resistant virus population in the infected individual. Antibody-based therapies are not immune to this problem. Further, HIV-1 replicates and spreads within the densely packed cellular environment (reaching about 108 cells per mL) of the lymphoid tissues of the gut, spleen, and lymph nodes. Antibodies may have difficulty preventing the cell-to-cell spread of virus in this seemingly impenetrable lymphoid environment. In fact, passive administration of anti-HIV antibodies as human immune plasma or polyclonal antibody preparations conferred, at best, only modest clinical impact (8, 29). Unfortunately, these trials were complicated by the relative absence of highly effective concentrations of HIV-neutralizing activity in the polyclonal preparations used. Preparations containing high concentrations of hmAbs that exhibited potent HIV-1-neutralizing activity in vitro (nhmAbs), on the other hand, resulted in measurable decrease of plasma virus concentration (34). However, the in vivo potency of this preparation was insufficient to completely block virus replication, and resistant virus rapidly emerged. Despite this discouraging result, these studies suggest that antibodies with enhanced in vivo potency could have a more profound clinical impact. A major question is whether antibody-based therapeutics can provide long-term clinical benefits for patients with established infections that are comparable to or better than drugs currently in clinical use. A specific feature of antibodies compared to other drugs is that HIV has evolved a number of strategies to escape neutralization. Such evasion strategies of the virus against polyclonal antibodies elicited during infection and strategies used by the immune system to generate broadly neutralizing hmAbs have been extensively reviewed (1, 35–40). Thus, a short answer to this question is perhaps it is possible if we can outsmart the virus by engineering potent antibody-based therapeutics against which the virus has not yet evolved protective strategies. Here, we discuss our ongoing efforts to improve the potential clinical utility of already known hmAbs and identify novel, more potent antibodies against HIV.

33.2.1 Anti-HIV Antibodies Elicited by Infection or Immunization HIV entry into cells is initiated by attachment of the viral envelope glycoprotein (Env) to a host cell receptor (CD4). Conformational changes follow, which enable enhanced

exposure of a co-receptor (typically CCR5 or CXCR4) binding site and binding of the viral glycoprotein gp120 to the co-receptor. Subsequent conformational changes finally result in fusion of the viral and cell membranes. In some cases, CD4 is not required and the Env directly interacts with a co-receptor. Because the Env mediates HIV entry and is the only viral surface protein exposed to the surrounding environment, it is a major target for neutralizing antibodies and a potent immunogen (36). Env-specific antibodies are generated as early as a few weeks after productive infection or immunization. They do not typically neutralize current virus isolates but rather neutralize earlier isolates (41). Such antibodies are isolate-specific and lack broad neutralizing activity because the virus has evolved to hide conserved epitopes and escape neutralization by a number of mechanisms. As a result, most of the antibodies generated in natural infection or immunization are non-neutralizing or neutralize few isolates. More than 100 mAbs have been reported as recognizing epitopes on gp120 and gp41, but only a small number exhibit neutralizing activity against primary isolates from different clades, denoted as broadly cross-reactive neutralizing hmAbs (bcnhmAbs).

33.2.2 HIV-1-neutralizing hmAbs Against the Env Using phage display or B cell immortalization, several bcnhmAbs were identified from HIV-infected patients whose sera contained a high titer of such antibodies. Six major classes of such antibodies relevant to the binding location and properties of their epitopes have been identified: (1) antibodies that bind to the region containing the CD4 binding site (CD4bs) on gp120; (2) antibodies binding better to gp120 complexed with CD4 than to gp120 alone (CD4i antibodies); (3) carbohydrate-binding antibodies; (4) gp120 V2 or V3-binding antibodies; (5) gp41 antibodies targeting the membrane-proximal external region (MPER); and (6) antibodies binding to other epitopes on gp41. The best characterized and very potent CD4bs antibody is b12, a hmAb selected from a phage-displayed antibody library constructed from the bone marrow of an HIV-1-infected donor (42, 43). The CD4 binding site is masked by V1/V2 variable loops and further shielded by N-glycan in the region. The limited accessibility of these conserved epitopes in the context of the oligomeric Env could explain why most of the antibodies that are specific for the CD4 binding site bind weakly to Env oligomers and exhibit weak to moderate neutralizing activities against primary HIV-1 isolates. Recently, two novel nmAbs (m14, m18) were identified by sequential antigen panning (SAP) against purified Env ectodomains (gp140s) of another phage-displayed antibody library derived from the bone marrow of three long-term nonprogressors with high titers of bcnhAbs (44, 45). These antibodies cross-react with Envs from primary isolates and exhibit differential neutralizing activity with b12 to isolates from different clades.

33. hmAbs Against HIV and Emerging Viruses

The Env undergoes significant conformational changes after binding to CD4 leading to exposure of structures that contain epitopes or portions of epitopes targeted by CD4i antibodies. The best characterized CD4i antibody, 17b, is only weakly neutralizing as IgG1, but its neutralizing activity increases significantly after its size is reduced to a scFv (46). The neutralizing activity of the most potent and broadly neutralizing CD4i antibody, X5 (47), also increases in many cases with decreasing its size to scFv or Fab. One should note however, that for some isolates and in some assays including assays based on spreading infection in peripheral blood mononuclear cells (PBMCs), IgG1 X5 is more potent than Fab X5 likely due to an increase in avidity. Thus, an interplay between avidity and size could be important in determining the neutralizing activity of CD4i antibodies. Many gp41-specific antibodies have been identified of which three with linear epitopes, 2F5, 4E10, and Z13, exhibit broad neutralizing activity and have been extensively characterized (48). They can bind peptides containing stretches of the MPER of gp41, which is relatively conserved. Using a competitive antigen panning methodology, several new crossclade reactive anti-gp41 mAbs have been identified (m43, m44, m45, m46, m47, and m48) that neutralize a variety of primary isolates in PBMC/infectious virus-based assays and bind to conformational epitopes distinct from those of other anti-gp41 antibodies (49).

33.2.3 Evidence That Antibodies Can Affect HIV-1 Replication in Humans There are several lines of evidence that antibodies can inhibit HIV-1 infections. First, it has been demonstrated that antibodies can exert selective pressure in humans suggesting that they inhibit infection (41). Second, HIV-1 concentration in plasma of patents decreases following administration of hmAbs (34). Third, a triple combination (2G12, 2F5, 4E10) of nmAbs at higher doses can delay viral rebound after cessation of antiretroviral treatment (50). In addition, a number of experiments have shown that antibodies can prevent infection in monkeys (see ref. 37 for a recent review).

33.2.4 Developing Antibodies With Improved Neutralizing Activity Naturally occurring antibodies with potent and broad neutralizing activity could be further improved. Recently, scFv X5 was further improved in potency and breadth of neutralization by using the SAP methodology and a library of X5 mutants; two scFvs, m6 and m9, which exhibited on average two- to threefold lower IC50 than scFv X5 were identified (44). The scFv X5 mutant library was also screened by surface plasmon resonance technique on immobilized Envs. One scFv was selected on the basis of its higher kinetic association rate, and its activity is being evaluated. Because CD4i epitopes may be available for limited time after CD4 binding to gp120 and before their

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interaction with a co-receptor, CD4i antibodies with faster binding kinetics would bind efficiently and are likely to have better inhibitory activity against HIV-1. We have been developing various other constructs including fusion proteins that are being evaluated for neutralizing activity.

33.2.5

Conclusions (HIV)

HIV has evolved a number of strategies to escape host immune surveillance, prominently by modifications of its Env. Thus, naturally occurring whole antibodies against its Env may have little chance of significantly affecting viral replication and disease progression, as also evidenced by the lack of sustained significant effect in the few clinical trials that have tested such antibodies. This is mostly due to the rapid generation of resistant mutants, which remains a fundamental problem not only for antibodies but also for other antiretroviral drugs. Antibodies against components of the entry machinery, engineered antibody fragments and their derivatives, and other antibodybased inhibitors that do not occur naturally and against which the virus has not developed defense mechanisms may be better able to control virus replication, although mechanisms of inhibitory escape such as generation of resistant mutants and difficult access could still be operating. The challenge is to fight these mechanisms and simultaneously ensure a relatively long half-life and biological effector functions. Several directions of research appear promising in this aspect. One direction involves engineering antibody with higher binding affinity to conserved epitopes and with smaller size than the currently known antibodies. Another uses the unique features of some anti-Env antibodies (e.g., the mimicry of receptors) to engineer novel binding entities with high-binding affinity to many isolates. A third consists of engineering novel fusion proteins containing antibody-binding fragments. A fourth relies on using antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and complement, to increase the efficacy in vivo. Yet another direction of research aims to develop antibody-nanoparticle conjugates, or nanoliposomes in particular, that are able to irreversibly inactivate virus and cells expressing viral proteins. The development of novel approaches may also be fruitful in the future, as well as combining existing ones to develop antibody-based HIV inhibitors using other, yet undiscovered principles. Whether these or other research directions will lead to clinically useful antibody-based inhibitors of HIV infections remains unknown; however, even if new engineered antibodies fail to inhibit HIV infections in a clinically useful way, there is still a basis for optimism in this endeavor because the new approaches can prove useful elsewhere. Finally, one should note that all bcnhmAbs have undergone a long maturation process and differ from the germline sequences by tens of mutations. One could speculate that generation of such antibodies in vivo following immunization is very unlikely because of the lack of B cells expressing surface-associated Ig that is close in function to those bcnhmAbs. This may represent

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a challenge in developing effective AIDS vaccines, and further studies are required to find novel approaches for elicitation of bcnhmAbs in vivo.

33.3

SARS-CoV

The SARS-CoV (51–54) caused a worldwide epidemic in 2002 and 2003, and infected more than 8,000 humans with a fatality rate of about 10%. Although there are no recent outbreaks, the need to develop potent therapeutics and vaccines against a re-emerging SARS-CoV or a related virus remains of high importance. SARS-CoV infection leads to generation of potent neutralizing antibodies that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. Antibodies that neutralize the virus in in vitro assays were detected in SARS-CoVinfected patients (55–60), and in mice (61), hamsters (62), and monkeys (63) infected with the virus. These antibodies also protected uninfected animals from SARS-CoV infection, e.g., passive transfer of immune serum to naive mice prevented virus replication in the lower respiratory tract following intranasal challenge (61). Patients infected with SARS-CoV were also treated with convalescent patient plasma containing polyclonal antibodies (64, 65), improvements of the antibody preparations were suggested (24), and batches of virus-inactivated hyperimmune globulins containing five to six times higher titers of SARS-CoV-specific antibodies than convalescent plasma were produced (66). In an amazing pace of research, several groups have recently developed hmAbs to the SARS-CoV spike (S) glycoprotein that neutralize the virus and have potential for therapy and prophylaxis of SARS (reviewed in ref. 67). Recently, an improved method for Epstein-Barr virus transformation of human B cells has been developed based on CpG oligonucleotide (CpG 2006) that increases the B cell immortalization efficiency from 1–2% to 30–100%, and used for selection of hmAbs specific for SARS-CoV proteins (68). One of the selected antibodies (S3.1), which was specific for the S glycoprotein on the viral spikes, was about 500-fold more efficient in neutralization than convalescent serum. S3.1 prevented the cytopathic effect of the SARS-CoV at 300 ng/mL (68), and inhibited entry of pseudovirus with S glycoprotein from Urbani isolate with about the same IC50. However, it did not affect to any significant extent pseudovirus entry mediated by the GD03 isolate S glycoprotein and even enhanced the entry of virus pseudotyped with the S glycoprotein from the palm civet isolate SZ16 (69). In a mouse model of SARS-CoV infection, this antibody prevented viral replication in the lower respiratory tract (at doses of 200 and 800 µg), and reduced it in the upper respiratory tract at the highest dose (800 µg) used. Unfortunately, data for the in vivo neutralizing activity of other nhmAbs selected in this study (68), including the most potent antibody (S215.13), which has a neutralizing concentration (1 ng/mL) 300-fold lower than that of S3.1, have not been

reported. The high neutralizing activities of these two hmAbs in IgG1 format indicates possibilities for their use alone or in combination for prophylaxis and treatment of SARS. Phage display technology has been increasingly used to produce high affinity hmAbs from both naïve and immune libraries. An advantage of using a naïve library is that B lymphocytes from an infected or immunized host are not required. Recently, two human nonimmune scFv libraries containing about 1010 members were developed from B cells of unimmunized donors, and used for selection of antibodies against a purified S fragment containing residues 12 through 672 (70). One of the selected antibodies, IgG1 80R, can neutralize 50% of the virus in a microneutralization assay at a concentration as low as 0.37 nM. It also blocked formation of syncytia, which could contribute to the spread of the virus in vivo, although at significantly higher concentration (25 nM). Its epitope overlaps the binding site of the SARS-CoV receptor ACE2 suggesting a possible mechanism of neutralization by preventing the virus attachment to its receptor (70). When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced to below assay limits (71). One should note that the conditions used for evaluation of the neutralizing activity of different antibodies in this study are not exactly the same as in the study described previously and later; thus comparing the activity of different antibodies should be done with caution unless they are tested side by side under exactly the same conditions. Three neutralizing hmAbs were also selected from another large naïve antibody library (72, 73). They bound a recombinant S1 fragment comprising amino acid residues 318 to 510 that also binds the receptor ACE2—the receptor-binding domain (RBD; ref. 73). The most potent of these hnmAb, IgG1 CR3014, required the residue N479 for binding (73). This antibody exhibited in vitro 50% neutralizing activity at about 1 µg/mL. More importantly, this antibody showed neutralizing activity in ferrets. In one set of experiments, ferrets were inoculated either with virus at two doses (low: 103 TCID50; high: 104 TCID50) or with virus preincubated with the antibody at 0.13 mg/mL for the low dose and 1.3 mg/mL for the high dose. Animals exposed to the virus-antibody mixture had almost undetectable SARS-CoV in the lung, showed no lung lesions on day 4 or 7, and did not shed virus in their throats unlike control animals treated with irrelevant antibody. In a second set of experiments, the antibody at 10 mg/kg was administered 24 hours before challenge with virus and reached 65–84 µg/mL serum concentration in three of the animals (<5 µg/mL in the fourth one). In the three ferrets with high antibody concentration virus shedding in the throat was completely abolished, while in the fourth one it was comparable to that of the control group. The CR3014-treated animals had 3.3 logs lower mean virus titer than the controls and were completely protected from macroscopic lung pathology. Note that the antibody dose used (10 mg/kg) was less than the one (15 mg/kg) used for prevention of RSV infections in infants,

33. hmAbs Against HIV and Emerging Viruses

which is administered once a month. These results suggest a potential use of CR3014 for prophylaxis of SARS-CoV infections in humans if it can reduce the virus replication to the same extent as in ferrets. However, one should note that currently there is no available animal model of the SARSCoV infection that results in death as in humans. Two other hmAbs (201 and 68) were derived from transgenic mice with human Ig genes and evaluated in a murine model of SARS-CoV infection (74). One of these antibodies [201] bound within the RBD of the S protein at amino acid residues 490 through 510, and the other one (68) to a region including residues 130 through 150. In a microneutralization assay based on protection to cytopathic effects the IC50 for 201 was about 0.2 µg/mL (74). Mice that received 40 mg/kg of these antibodies prior to challenge with the SARS-CoV were completely protected from virus replication in the lungs, and doses as low as 1.6 mg/kg offered significant protection. These antibodies have potential as therapeutics and research tools, and further studies are planned to evaluate the nhmAb 201 for potential clinical use (74). We have recently identified a novel cross-reactive potent SARS-CoV-neutralizing hmAb, m396, by using a fragment containing residues 317 through 518 as a selecting antigen for panning of a large human antibody library constructed from the B lymphocytes of healthy volunteers (75). This fragment was previously identified to contain the RBD (76–78), which is a major SARS-CoV neutralization determinant (67, 79–84). It potently inhibited S-mediated cell fusion (IC50 = 0.6 µg/mL), pseudovirus entry (IC50 = 0.01 µg/mL), and replication of infectious virus in mice (complete protection at 0.2 mg per mouse; Zhu et al., unpublished). Interestingly, this antibody also inhibited entry mediated by the S glycoprotein from the 2003/2004 GD03 isolate, which has a 487Thr/Ser mutation compared to the middle/late phase 2002/2003 isolate Tor2, and is not neutralizable by other known potent hmAbs including 80R and S3.1. It bound with high (pM) avidity to the RBD in a Biacore chip and competed with ACE2 suggesting a mechanism of neutralization that involves competition with the receptor for binding to the S glycoprotein (Zhu et al., unpublished). The epitope of this antibody was identified by crystallography and proposed as a possible vaccine immunogen (a retrovaccinology approach for design of vaccine immunogens [85]). Neutralizing antibodies directed to S inhibit SARS-CoV entry either by interfering typically with the S RBD-receptor interactions (70) or by other mechanisms including binding to other portions of S. Recently, a human scFv, B1, was identified, which recognizes an epitope on S2 protein located within amino acids 1023 to 1189 (86). This antibody recognized SARS pseudovirus in vivo and competed with SARS sera for binding to SARS-CoV with an equilibrium dissociation constant, Kd = 105 nM. The B1 also had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein in vitro. Other mechanisms of SARSCoV infection inhibition could include steric hindrance that

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indirectly prevents virus attachment to receptors and binding to entry intermediates. Mechanisms that could operate in vivo (and for lack of data are not discussed here) are related to the antibody biological effector functions conferred by the antibody Fc, e.g., ADCC.

33.3.1

NiV and HeV

NiV and HeV are closely related emerging paramyxoviruses that comprise the Henipavirus genus (87–96). The broad species tropisms and the ability to cause fatal disease in both animals and humans distinguish HeV and NiV from all other known paramyxoviruses (reviewed in ref. 1). They are Biological Safety Level 4 pathogens and are on the NIAID Biodefense Research Agenda as zoonotic emerging category C priority pathogens that could be used as bioterror agents. There are currently no therapeutic modalities for treating NiV or HeV infections, and a vaccine for prevention of disease in human or livestock populations does not exist. Although antibody responses were detected in infections caused by these viruses, the development of hmAbs specific for HeV and NiV have only just been realized. We recently reported the identification of potent neutralizing hmAbs targeting the viral envelope glycoprotein G by using a highly purified, oligomeric, soluble HeV G (sG) glycoprotein as the antigen for screening of a large naïve human phage-display library (97). The selected seven Fabs, m101-7, inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to IgG1 significantly increased its cell fusion inhibitory activity—the IC50 was decreased more than 10-fold to approximately 1 µg/mL. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 µg/mL and 98% neutralization required only 1.6 µg/mL. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation, and less with binding to sG as measured by ELISA and Biacore. M101 and m102 competed with the ephrin-B2, which we and others recently identified as a functional receptor for both HeV and NiV (98, 99), indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other suggesting that they bind to overlapping epitopes that are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine scanning mutants and identified two mutants, P185A and Q191,K192A, which significantly decreased binding to m101, and one, G183A, which decreased binding of m102 to G. These results suggest that m101-7 are specific for HeV or NiV or both, and exhibit various neutralizing activities; they are the first hmAbs identified against these viruses and could be used for treatment, prophylaxis, diagnosis, and as research reagents and aid in the development of vaccine.

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Recently, we matured in vitro m102 to a very potent crossreactive antibody, m102.4, which neutralized both infectious NiV and HeV with an IC50 of 100 ng/mL (Zhu et al., unpublished). We developed a cell line that produces large amounts of IgG1 m102.4. This antibody will be tested in an animal model of henipavirus infection and if successful, which is very likely, it could be clinically useful for humans. Interestingly, the antibodies against SARS-CoV and henipaviruses were identified from naïve libraries, and in contrast to HIV have only few mutations compared to the respective germline sequences. This correlates with the ability of various vaccines to elicit potent cross-reactive neutralizing antibodies against these viruses in contrast to HIV. One could speculate that humans have in their native repertoire antibodies that can bind with relatively high affinity envelope glycoproteins from these emerging viruses but not to the HIV Env. Further experiments are required to test this hypothesis, which if true could lead to novel approaches for design of effective vaccines against HIV and other viruses.

33.3.2 Conclusions (SARS-CoV and Henipaviruses) The hmAbs directed to the SARS-CoV S glycoprotein and the henipavirus G glycoprotein are currently in an advanced stage of development and offer the best hope as potential therapeutics. These antibodies specific for SARS-CoV, HeV, and NiV have potential for further development into a clinically useful product for prophylaxis and perhaps treatment of the diseases caused by these infections. They are very potent, and the viral infections to which they are specific are acute, such that only control or dampening of virus replication for a relatively short period of time (few weeks) is likely to be required after which the host immune system could control virus replication. In addition, these antibodies could be cross-reactive. Thus, the problem of neutralization resistant mutants able to evade their inhibitory activity and the immune response is not as significant as for chronic infections with a high level of virus replication, e.g. HIV infections. A note of caution is that careful examination of candidate antibody therapeutics is required because of the possibility for infection enhancing effects, as e.g., for Ebola, and animal model-dependent effects as well as in some cases, although rare, toxicity. A recent study also reported the possibility that neutralizing antibodies can enhance entry of SARS-CoV by a mechanism that involves antibody interactions with conformational epitopes in the S RBD (69). In addition, it is known that in some cases antibodies that do not neutralize in the assay currently used for evaluation of their in vitro activity could exhibit potent neutralizing activity in vivo; thus new approaches should be developed and antibodies tested also for their effector functions mediated by the Fc including antibody-dependent direct cytotoxicity and complement-mediated immune responses. However, only further exploration of these antibodies and their extensive evaluation in animal models, likely in com-

D. S. Dimitrov

bination with other antiviral drugs (antibodies or small molecules), would allow identification of the best candidates for potential therapeutics. The rapid progress made in the last few years toward the development of potent neutralizing hmAbs against emerging viruses and viruses of biodefense importance is a basis for further more accelerated development of neutralizing antibodies in the next 5 years and their testing in animal models. It is likely to see novel and even more potent antibodies against the SARS-CoV than the currently existing ones. They could be used alone or in combination with the existing antibodies in animal models of viral diseases and for evaluation of toxicity in human clinical trials. The currently available hmAbs against NiV and HeV are likely to be tested in animals. If successful, which is very likely, they can undergo evaluation in human clinical trials. Although the interest of big- and medium-size companies to such antibodies appears to be relatively minor, small companies and start-ups could be interested in developing such antibodies provided there is a continued governmental support by programs like Bioshield. Five years from now, it is likely to have at least several hmAbs of potential clinical use in case of outbreaks or terror attacks. These antibodies could be used in combination with other therapeutics to increase potency and cope with resistance. Several key issues are listed below: • Continuation of research and development funding at the same or accelerated pace is of critical importance for development of potent and clinically useful therapeutic antibodies. • Phage display techniques as well as novel methodologies will be critical for the development of fully human antibodies. • Cloning, expression, and purification of novel antigens for screening of human antibody libraries is of critical importance. • Crystal structures of antibody complexes with virus envelope glycoproteins and their use for further improvement of the antibodies and understanding of their interactions is of critical importance. • Development of appropriate novel animal models that would be of critical importance for the accelerated testing of the therapeutic antibodies is necessary. • Understanding of the pathogenesis and the design of antibodies acting through multiple mechanisms with an increased efficacy in vivo is of critical importance. • Evaluation of combinations of antibodies and other antiviral drugs is of critical importance.

Acknowledgments. This study was supported by the NIH NCI CCR intramural program, the Gates Foundation, the NIH intramural AIDS program (IATAP), and the NIH intramural biodefense program.

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Chapter 35 NIAID HIV/AIDS Prevention Research David N. Burns and Roberta Black

35.1

HIV/AIDS Pandemic

Twenty-five years into the pandemic, HIV infection continues to spread worldwide. It can be likened to a global forest fire that remains out of control. According to The Joint United Nations Programme on HIV/AIDS (UNAIDS), worldwide incidence continued to increase from 4.6 million new HIV infections in 2003 to 4.9 million in 2005 (1). Although the latter estimate has been revised downward somewhat, it is still unclear whether worldwide incidence has peaked (2, 3). Figure 35.1 shows the state of the epidemic as of December 2005. Sub-Saharan Africa remains the epicenter, but HIV continues to spread everywhere, particularly in Central and Eastern Europe and throughout Asia. Despite gains in access to prevention services in some regions, the epidemic continues to seriously affect women and young people. Women represent nearly half of all persons living with HIV, including nearly 60% in Africa, and about half of all new infections are in persons under 25 years of age. In parts of Africa and the Caribbean, young women (aged 15–24) are up to six times more likely to be HIV infected than young men (2). As shown in Figure 35.2, among 15–34 year olds in South Africa, HIV infection has completely swept away gains made in reducing mortality. The 2006 UNAIDS report on the global AIDS epidemic cites several bright spots, but it concludes that “the epidemic continues to outpace the response” (2). In addition to the declines previously reported for Thailand and Uganda, a fall in national HIV prevalence was documented for the Bahamas, Barbados, Kenya, Rwanda, and Zimbabwe as a whole and in urban areas of Burkina Faso and Haiti. However, roughly half of the countries reporting from sub-Saharan Africa failed to reach their 2005 “Declaration of Commitment on HIV/AIDS” target to reduce HIV prevalence among From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

young people (aged 15–24) by 25%, and no low- or middleincome country achieved their goal that 90% of youth aged 15–24 would be able to correctly identify ways of preventing HIV transmission and reject major misconceptions (average percentages achieved: males, 33% [range, 7–50%]; females, 20% [range, 8–44%]; ref. 2). Progress fell far short of the 2005 targets for other risk groups as well, including sex workers (only 10 of 24 lowand middle-income countries reporting these data met their goal of providing prevention services to at least 50% of sex workers), men who have sex with men (less than 10% received any HIV prevention services), infants born to HIVpositive pregnant women (only 9% [range, 1–59%] received antiretroviral prophylaxis to prevent mother-to-child transmission (MTCT), far below the target of 80% coverage), and injection drug users (less than 20% received needle exchange or substitution therapy such as methadone or buprenorphine treatment). In Eastern Europe and Asia, where injection drug use is driving expanding epidemics, this coverage was less than 10% (2). Although the exact causes are uncertain, it is likely that countries that successfully lowered their HIV rates did so not by use of a single risk-reduction intervention such as condoms or abstinence only campaigns, but by implementing multiple interventions simultaneously (e.g., intensive media campaigns; community and peer group education; and condom promotion and distribution). Other factors include the “saturation” of social networks at highest risk of infection and HIV-related mortality (3, 4). Although widespread antiretroviral treatment may eventually have an impact on incidence, projections indicate that this will take decades (5). In the meantime, the resulting decline in HIV-related morbidity and mortality will increase the need for prevention (6). It is therefore not surprising that mathematical models indicate that national programs that combine prevention with treatment can be expected to be much more effective than treatment alone (Figure 35.3). Salomon and colleagues estimated that whereas a scale up of 319

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Figure 35.1. Estimated number of persons living with HIV/AIDS, December, 2005 (See Color Plates).

Figure 35.2. Estimated and projected deaths at ages 15 to 34, with and without AIDS in South Africa, 1980–2025 (See Color Plates).

treatment could avert 3 million new HIV infections by 2020, an integrated approach of prevention and treatment could avert 29 million infections (7). These estimates are based on implementation of currently available prevention interventions (Table 35.1; ref. 8).

35.2

HIV/AIDS Prevention Research

The number of HIV infections that can be prevented will likely increase over the next several years as new biomedical interventions become available. For example, one randomized

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Impact of three scenarios on HIV infection in sub-Saharan Africa, 2003–2020 5. 4.0 Number 3.0 of new HIV infections 2.0 (millions) 1.0 0.0 2003 2005

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2020

Year Baseline

Treatment-centered

Prevention-centered

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Figure 35.3. Impact of three scenarios on HIV infection in sub-Saharan Africa, 2003–2020 (See Color Plates).

Table 35.1. Expanded program of currently available HIV prevention interventions • Mass media campaigns • Voluntary counseling and testing • Peer counseling for sex workers • School-based programs • Workplace programs • Condom social marketing • Public sector condom distribution • Harm reduction programs (including needle exchange and drug treatment) • Peer outreach to homosexual men • Treatment for sexually transmitted infections • Prevention of mother-to-child transmission

clinical trial has demonstrated 61% protection with male circumcision (95% CI: 34–77%) after controlling for condom use, health-seeking behavior, and other behavioral factors (9). The latter included the number of sexual contacts, which increased in the intervention group. Subsequent calculations indicated that implementation of male circumcision in subSaharan Africa could prevent approximately 2 million new HIV infections and 0.3 million deaths over the next 10 years (10). Although one trial is not sufficient basis for implementing large-scale interventions, two other trials are expected to be completed in the coming year. Other interventions currently in clinical trials include female barrier methods, topical microbicides, herpes simplex virus type 2 (HSV-2) suppression, pre-exposure prophylaxis with one or more antiretroviral agents, and chronic highly active antiretroviral therapy. For each new biomedical intervention found to be efficacious, consideration must be given to the impact of behavioral

factors, including incomplete adherence and risk compensation (“disinhibition”). Intervention-specific methods for addressing these factors should be developed and included in the implementation plan of each new intervention. Future studies must also focus on identifying optimal combinations of new and existing HIV prevention modalities for specific populations. The design of these prevention “packages” should be based on the dominant modes of transmission in the population, the stage of the epidemic, and other key factors. Mathematical modeling is likely to facilitate this process, but it cannot eliminate the need for large-scale clinical studies. One can argue that because biomedical interventions are generally easier to implement than behavioral ones, an increase in the number of biomedical options should increase the overall impact of our prevention efforts, gradually moving us in the direction of our ultimate hope for success, a highly effective HIV vaccine. This may not be the case with all biomedical interventions, however, and it is clear that behavioral approaches will remain extremely important. It is imperative to realize that stigma and discrimination are major barriers to the successful implementation of all prevention efforts. Behavioral research can make important contributions in this area. However, structural interventions, leveraged, if necessary, by the World Health Organization, World Bank, Global Fund, and other large international organizations, are essential to reduce these serious obstacles. The commitment of national governments to the “Three Ones” and the ongoing “Declarations of Commitment on HIV/AIDS” are important first steps (11, 12). Legal protections must be enacted and anti-stigma and anti-discrimination media campaigns should be developed and promoted by national and local governments.

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322 Genital viral burden and risk of transmission 5 4 HIV RNA in Semen 3 (Log10 (Log10 copies/ml) copies/ml) 2 Stage

Acute (<3wks)

Estimated 1:50-1:250

Aymptomatic 1:1000-10000

Pre-AIDS

AIDS

1:500-2000 1:100-1000

Transmission Risk

Figure 35.4. Genital viral burden and risk of transmission (See Color Plates).

The support and participation of community organizations and celebrities is also very important. In developed countries, HIV incidence has slowed but the epidemic is far from over. In the US, the number of new infections has plateaued at approximately 40,000 per year (13, 14). This can be attributed, in part, to the large number of newly infected persons who are unaware of their infection. As Figure 35.4 shows, viral loads, and estimated infectiousness, are highest in the first few weeks after initial infection when most persons are unaware that they are infected. In many cases, other sexually transmitted infections (STIs) may also be present, further increasing the risk of transmission (15). It has been shown that the large majority of persons who learn that they are HIV infected will take measures to reduce their transmission to others (16–18). The US Centers for Disease Control and Prevention’s recent recommendation that state and local jurisdictions adopt an “opt out” policy regarding HIV testing is an attempt to reach the substantial proportion of persons who are unaware of their infection (19). This has been strongly supported by public health experts but will remain controversial until confidentiality and civil and human rights issues are fully worked out (20–22).

35.3 National Institute of Allergy and Infectious Diseases (NIAID) HIV Prevention Research NIAID’s HIV/AIDS research is conducted on the main campus in Bethesda, MD, and at medical centers across the country and throughout the world through grants, contracts, and cooperative agreements. This research includes a broad-based basic sciences program, the therapeutics research program, the vaccine program, and non-vaccine prevention research projects. The mission of the Division of AIDS (DAIDS) at NIAID is to help ensure an end to the worldwide HIV epidemic by fostering basic and clinical research. This includes the discovery and development of new treatment, clinical care, vaccines, and

other prevention strategies that are safer, more effective, more acceptable, and more affordable than current interventions. The HIV epidemic is global, and NIAID is committed to partnering with scientists throughout the world to find global solutions. A number of guiding principles have been developed for these collaborations. First, there must be a genuine and substantial benefit to the host country: new or improved infrastructure, training, technology transfer, and/or other benefits. Second, when appropriate, tuberculosis, malaria, STIs, and other infectious disease programs are integrated with the HIV research program. Third, all studies are carried out according to local and international ethical standards. Finally, to the fullest extent possible, all local, national, and international stakeholders are included in the collaboration. Requirements for successful partnerships include political will and commitment; study populations with sufficiently high HIV incidence to determine efficacy (i.e., in a reasonable period of time with a sample size that is feasible to enroll); a hypothesis-generated, protocol-driven research plan that has a clear potential to benefit the participants; and the expertise and resources to recruit and follow volunteers and to perform advanced HIV and other laboratory tests. Other requirements include access for study participants to all necessary clinical care; rigorous scientific review of all proposed protocols; approval of all studies by local and international ethical review boards; careful monitoring of adverse events; and periodic review by a well-qualified data safety and efficacy monitoring board (DSMB). Together with the principal investigators and sponsor, the DSMB must have the authority to determine whether the study should be continued as originally designed, changed, or stopped. The NIAID-sponsored Comprehensive International Program of Research on AIDS (CIPRA) initiative was designed to help sites to develop the capacity to do international quality research. CIPRA projects have taken place at sites in the Caribbean, South America, Africa, Russia, China, and Southeast Asia. Most of these sites have gone on to successfully develop and perform other important research projects. An even larger number of countries and sites have participated in NIAID-sponsored HIV/AIDS clinical trials. Most of these studies were developed and coordinated by the adult and pediatric AIDS Clinical Trials Groups (AACTG, PACTG), the HIV Vaccine Trials Network (HVTN), and the HIV Prevention Trials Network (HPTN). Since 1999, the HPTN has been the primary mechanism by which DAIDS has developed and tested the safety and efficacy of non-vaccine interventions designed to prevent HIV transmission. In 2006, these networks were expanded. The PACTG was reconfigured as the International Maternal-Pediatric-Adolescent AIDS Clinical Trials group (IMPAACT), and two new networks, the Microbicides Trials Network (MTN) and the International Network for Strategic Initiatives in Global HIV Trials (INSIGHT), were formed. The six new and restructured networks will address the following priority areas: drug development, optimization of clinical management, prevention of MTCT, microbicide discov-

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The Microbicide Development Pipeline

Preclinical Virology

Discovery

Preclinical Studies (Critical Path)

Pilot Studies

I II Clinical Studies

III

Deployment

Product recycled In Pipeline

Product Recycled Into Discovery

Successful Microbicide Or Microbicide Strategy

LEAD Lead Identification And Optimization

Product Eliminated

Pipeline Development Multiple activities driven by Milestones that may occur sequentially or simultaneously

DHHS/NIH/NIAID

Figure 35.5. The microbicide development pipeline (See Color Plates).

ery and development, HIV vaccine development, and non-vaccine HIV prevention. A number of new biomedical interventions for HIV prevention are expected to emerge over the next several years from four of these networks, the MTN, IMPAACT, HPTN, and HVTN. The development of new and enhanced behavioral strategies will also remain extremely important, to serve both as complementary interventions and as tools for maximizing the benefits of biomedical interventions. This research is being supported by multiple cross-NIH and cross-agency collaborations. The chief priorities of the MTN are to develop and evaluate microbicides that are safe and effective; to identify correlates of short- and long-term safety; and to optimize acceptability and adherence. IMPAACT’s primary goals are to develop safe, practical, and effective approaches to further reduce perinatal MTCT, to identify methods to interrupt HIV transmission during breastfeeding, and to evaluate the safety and efficacy of vaccines to prevent perinatal and postpartum MTCT. Other priorities of IMPAACT are to assess the safety and pharmacokinetics of new drugs given during pregnancy and the neonatal period, and to evaluate longterm outcomes in newborns exposed to these drugs. The HPTN’s chief objectives are to discover and develop safer, more practical, and effective interventions to halt the spread of HIV, especially in high incidence populations, and to evaluate the worldwide suitability and sustainability of these approaches. Priorities include the treatment and prevention of STIs and other infections that increase the risk of HIV acquisition; optimization of antiretroviral therapy to prevent transmission during acute and established HIV infection and

to prevent acquisition by pre- and post-exposure prophylaxis; and the development of behavioral interventions to reduce HIV transmission and acquisition. The goal of the HVTN is of course to design and develop vaccines that are safe, effective against all HIV subtypes, simple to administer, inexpensive, and induce long-lasting immunity. This will require a better understanding of the immune correlates of protection, HIV genomics and proteomics, and the development of pre-clinical and clinical tools for assessing safety and immunogenicity. These areas are also being addressed by other projects funded by NIAID’s Vaccine Research Program, and by the NIAID Vaccine Research Center and the Center for HIV/AIDS Vaccine Immunology. A general strategy used by the networks is illustrated in Figure 35.5, a schematic of the microbicide pipeline. Grants and contracts awarded for preclinical studies have built-in milestones that must be met to advance to the next stage of development and receive further funding. As indicated in the figure, only a few products are expected to enter clinical trials and even fewer will make it all the way through the pipeline to implementation. To support early microbicide development, the Microbicide Innovation Program was established. It facilitates microbicide discovery and assesses essential biologic requirements for candidate agents. The Integrated Preclinical/Clinical Program for HIV Topical Microbicides supports translational studies to advance microbicide candidates into clinical trials. This is accomplished by integrating basic and applied research with product development. Contracts are utilized to fill any remaining gaps.

D. N. Burns and R. Black

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This approach, which was borrowed in part from industry, has been further expanded by the HVTN. Closely linked to these activities are the Global HIV Vaccine Enterprise, a consortium of organizations committed to accelerating HIV vaccine development through a shared strategic scientific plan (23), the Partnership for AIDS Vaccine Evaluation, a similar collaboration focusing on the evaluation of HIV vaccines, and the NIAID Immune Assessment Laboratory Service, which provides centralized, state-of-the-art immunogenicity testing across all NIAID-sponsored HIV vaccine projects. Biomedical interventions currently being examined by the networks and/or other NIAID-funded research programs include, in addition to HIV vaccines and microbicides, male circumcision, female barrier methods, herpes virus (HSV-2) suppressive therapy, pre-exposure prophylaxis, and others. None of these interventions is likely to be found sufficiently potent to serve as a stand alone prevention strategy in the near future. This includes HIV vaccines, since they are initially expected to only be partially protective. As already noted, future studies must therefore focus on developing optimal combinations of new and existing interventions for specific populations. In addition to safety, efficacy, and cost-effectiveness, these studies must carefully consider acceptability, adherence, risk compensation (disinhibition), and sustainability.

35.4

Conclusion

In summary, in addition to its intense research efforts on the main NIH campus, NIAID is partnering with scientists across the US and throughout the world to find global solutions to the HIV epidemic. HIV vaccine development deserves the highest possible priority, but other biomedical and behavioral interventions that can be applied in the near term are also urgently needed. These are being developed through unsolicited investigator-initiated research, innovation programs, integrated pre-clinical–clinical programs, and clinical trials. The latter efforts are being facilitated by the restructured and expanded clinical trials networks sponsored by NIAID, as well as by multiple cross-NIH and cross-agency collaborations. It is hoped that these efforts will result in safe, effective, affordable, and sustainable HIV prevention “packages” that can be used in the near future to reduce HIV incidence. For further information about NIAID’s HIV prevention research, including new funding opportunities, go to the DAIDS Website at www3.niaid.nih.gov/about/organization/daids/. Other useful Websites include www.clinicaltrials.gov (provides summaries of clinical trials receiving US government funding), http://crisp.cit.nih.gov (abstracts of US government-funded research projects, including preclinical studies), www.fhi.org/en/HIVAIDS/pub/guide/bestpractices. htm (provides a number of current guidelines for HIV prevention), www.cdc.gov/hiv (US Centers for Disease Control and Prevention’s HIV/AIDS Prevention Website).

References 1. The Joint United Nations Programme on HIV/AIDS (UNAIDS) (2005) AIDS epidemic update, December 2005. UNAIDS, Geneva. 2. The Joint United Nations Programme on HIV/AIDS (UNAIDS) (2006) 2006 Report on the global AIDS epidemic. UNAIDS, Geneva. 3. Hallett TB, Garnett GP (2006) Has global HIV incidence peaked? Lancet 368:116–117. 4. Garnett GP, Gregson S, Stanecki KA (2006) Criteria for detecting and understanding changes in the risk of HIV infection at a national level in generalised epidemics. Sex Transm Infect 82:i48–i51. 5. Montaner JS, Hogg R, Wood E, Kerr T, Tyndall M, Levy AR, Harrigan PR (2006) The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic. Lancet 368:531–536. 6. Hayes R, Weiss H (2006) Understanding HIV epidemic trends in Africa. Science 311:620–621. 7. Salomon JA, Hogan DR, Stover J, Stanecki KA, Walker N, Ghys PD, Schwartlander B (2005) Integrating HIV prevention and treatment: from slogans to impact. PLoS Med 2:e16. 8. Stover J, Walker N, Garnett GP, Salomon JA, Stanecki KA, Ghys PD, Grassly NC, Anderson RM, Schwartländer B (2002) Can we reverse the HIV/AIDS pandemic with an expanded response?Lancet 360:73–77. 9. Auvert B, Taljaard D, Lagarde D, Sobngwi-Tambekou J, Sitta R, Puren A (2005) Randomized, controlled intervention trial of male circumcision for reduction of HIV infection risk: The ANRS 1265 Trial. PLoS Med 2:e298. 10. Williams BG, Lloyd-Smith JO, Gouws E, Hankins C, Getz WM, Hargrove J, de Zoysa I, Dye C, Auvert B (2006) The potential impact of male circumcision of HIV in sub-Saharan Africa. PLoS Med 3:e262. 11. The Joint United Nations Programme on HIV/AIDS (UNAIDS) (2005) The “Three Ones” in action: where we are and where we go from here. UNAIDS, Geneva. 12. The Joint United Nations Programme on HIV/AIDS (UNAIDS) (2005) Monitoring the Declaration of Commitment on HIV/AIDS: guidelines on construction of core indicators. UNAIDS, Geneva. 13. Centers for Disease Control and Prevention (2001) HIV/AIDS Surveillance Report, 2001 (vol 13). US Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA. 14. Centers for Disease Control and Prevention (2005) HIV/AIDS Surveillance Report, 2004 (vol 16). US Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA. 15. Cohen MS, Pilcher CD (2005) Amplified HIV transmission and new approaches to HIV prevention. J Infect Dis 191:1391–1393. 16. Weinhardt LS, Carey MP, Johnson BT, Bickham NL (1999) Effects of HIV counseling and testing on sexual risk behavior: a meta-analytic review of published research, 1985–1997. Am J Public Health 89:1397–1405. 17. Marks G, Crepaz N, Senterfitt JW, Janssen RS (2005) Metaanalysis of high-risk sexual behavior in persons aware and unaware they are infected with HIV in the United States: implications for HIV prevention programs. J Acquir Immune Defic Syndr 39:446–453.

35. NIAID HIV/AIDS Prevention Research 18. Marks G, Crepaz N, Janssen RS (2006) Estimating sexual transmission of HIV from persons aware and unaware that they are infected with the virus in the USA. AIDS 20:1447–1450. 19. Centers for Disease Control and Prevention (2006) Revised recommendations for HIV testing of adults, adolescents, and pregnant women in health-care settings. MMWR 55:1–17. 20. Frieden TR, Das-Douglas M, Kellerman SE, Henning KJ (2005) Applying public health principles to the HIV epidemic. N Engl J Med 353:2397–2402.

325 21. Koo DJ, Begier EM, Henn MH, Sepkowitz KA, Kellerman SE (2006) HIV counseling and testing: less targeting, more testing. Am J Public Health 96: 962–964. 22. Kippax S (2006) A public health dilemma: A testing question. AIDS Care 18:230–235. 23. Coordinating Committee of the Global HIV/AIDS Vaccine Enterprise (2005) The Global HIV/AIDS Vaccine Enterprise: scientific strategic plan. PLoS Med 2:e25.

Chapter 30 Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS Edward A. Berger

30.1

Introduction

The initial step of virus infection involves binding and entry of the virion into the host cell. Once inside, the virus co-opts the normal cellular biosynthetic and trafficking machineries to promote synthesis and assembly of viral components and release of new infectious particles that spread the infection to other target cells. Viruses have evolved an extraordinary array of schemes to execute and regulate these replication processes in the face of a comparably intricate arsenal of immune defenses, both innate and adaptive. In this never-ending war of ingenuity and deception between infecting virus and susceptible target cell, the role of the researcher is to harness an inevitably incomplete understanding so as to tip the balance in favor of the host. The virus entry process involves a highly complex cascade of molecular interactions (1). In the case of enveloped viruses, the virion is surrounded by a membrane bilayer (typically one, but in some cases multiple) that must undergo fusion events with particular membranes of the target cell. Two major mechanisms of enveloped virus entry have been defined, the first proceeding by direct fusion between the virion and plasma membranes, and the second involving endocytosis of the virion followed by fusion of the viral membrane with the endosomal membrane. In each case, the fusion event results in delivery of the viral genome into the cytoplasm of the target cell, thereby setting the stage for subsequent steps in the replication cycle. A common feature of both the direct fusion and the endosomal entry pathways is the requirement for interactions between specific viral surface components (typically glycoproteins that catalyze the fusion reaction) and cell surface molecules (receptors that facilitate attachment, endocytosis, and/or triggering steps in the fusion mechanism). From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

We have performed detailed studies on two enveloped viruses that are significant human pathogens: HIV, the direct cause of AIDS (2), and the Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8), which is associated with multiple pathologies of particular significance in the context of AIDS including Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease (3). For each of these viruses, we seek to unravel the molecular mechanisms of the entry process and, when possible, devise novel antiviral strategies to treat and/or prevent infection. We have made extensive use of two types of quantitative assays to study viral entry. The first is a cell fusion assay that measure fusion between “effector” cells expressing the relevant viral glycoproteins, and target cells expressing the appropriate receptors; fusion is quantitated by measuring activation of a reporter gene, usually by using vaccinia expression technology to introduce the bacteriophage T7 RNA polymerase into the cytoplasm of one cell population and a reporter gene (typically the Escherichia coli LacZ gene) linked to the T7 promoter into the cytoplasm of the other cell population (4). The second assay system measures virion entry and involves the use of infectious virus particles (live virions or recombinant virus-like particles) which induce activation of a reporter gene (LacZ, luciferase) upon entry into the target cell. The cell fusion assay has the advantage that the only viral components required are the glycoproteins essential to the fusion process; hence the signal is dependent only on the critical viral glycoprotein-target cell receptor interactions necessary for fusion. The assay thus reveals the minimal molecular components of the fusion machinery, but can potentially overlook requirements for accessory factors that may participate in critical virus entry functions (e.g., in virion attachment). The virion entry assay, by using true cell-free virions or closely related virus-like particles, has the potential to reveal the need not only for the minimal components of the fusion machinery, but also the accessory factors involved in critical steps such as virus attachment; however, it can often be difficult to distinguish molecules and events that constitute the essential entry apparatus from those that provide important 271

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(SDF-1)

(RANTES, MIP-1a, MIP-1b

HIV-1 Variants

CXCR4-specific (X4)

CD4

The Entry Mechanism of HIV

CCR5 & CXCR4 (R5X4)

CCR5-specific (R5)

CXCR4 CD4 T cell line

CCR5

primary CD4 T cell

primary macrophage

Target Cell Types

B. R5 HIV X4/R5X4 HIV

Viral Load

HIV enters target cells by direct membrane fusion, via a tightly choreographed series of steps involving both the external and transmembrane subunits (gp120 and gp41, respectively) of the viral envelope glycoprotein (Env; ref 5). The gp120 subunit is the receptor-binding component of Env, whereas gp41 is the fusogenic entity. The functional Env on the surface of the virion is displayed as a trimer of three gp120–gp41 complexes. Entry is dependent on two specific receptors on the target cell that trigger the Env-mediated fusion event: CD4 (the “primary receptor”) and one of two chemokine receptors (the “co-receptors”) CXCR4, which we first identified by functional cDNA cloning (6), and CCR5 identified soon thereafter by several groups including ours (7, 8). The co-receptors are members of the superfamily of G protein-coupled receptors, characterized by seven transmembrane domains. Their identification has resulted in profound new insights not only into the mechanism of HIV fusion/entry, but also into the broader areas of HIV transmission, HIV pathogenesis, and development of novel antiretroviral agents (7–10). The tropism of an individual isolate for entry into different CD4-positive subtypes is largely determined by the specificity of the corresponding gp120 subunit for usage of CCR5 and/or CXCR4, coupled with the expression patterns of the corresponding co-receptors on different CD4-positive cell types (Figure 30.1A). Variants that exclusively function with CXCR4 (designated X4) readily infect continuous CD4 T-cell lines but not primary macrophages; strains that are restricted to CCR5 usage (designated R5) have the opposite tropism, readily infecting primary macrophages but not CD4 T-cell lines; finally, strains capable of using either CXCR4 or CCR5 (designated R5X4) can infect both target cell types. It is important to note that all HIV-1 isolates can infect primary CD4-positive T cells, because these targets express both CXCR4 and CCR5, although the patterns depend markedly on the state of maturation and activation. Critical relationships have been defined between the phenotype of HIV-1 co-receptor usage and processes of virus transmission and disease progression (Figure 30.1B; refs. 7–9). The virus isolates first detected in the acute phase of the newly infected person almost invariably display the R5 phenotype; this restricted pattern persists throughout the asymptomatic phase of chronic infection that can last many years. CXCR4-using variants (X4 and R5X4) are detected only after transition to the symptomatic phase, when CD4 cell counts rapidly decline

CCR5

CXCR4 (fusln)

30.2 HIV Entry and Neutralization of Infection 30.2.1

HIV-1 Coreceptors

CD4 Count

but indirect accessory activities, and the interpretations can be complicated by the need for post-entry events to trigger reporter gene expression. Combining results from the two assay systems offers the greatest promise for unraveling the complex molecular details of virus entry pathways.

3

wks

6

9

12

yrs

AIDS

Transimission

Time (yrs)

Figure 30.1. Role of HIV-1 co-receptors in viral tropism, transmission, and pathogenesis. See text for details. (A) Selective tropism of different HIV-1 isolates for different CD4-positive target cells. (B) Evolution of HIV-1 coreceptor usage in infected individual.

below a critical level and the virus load in blood and lymph nodes rises dramatically; indeed the appearance of CXCR4using variants is considered a harbinger of serious HIV disease progression, although full-blown AIDS can occur in the absence of detectable CXCR4-using viruses. The molecular determinants underlying the selective transmission of R5 variants and the evolution of co-receptor usage during disease progression are poorly understood, and probably reflect a complex multifactorial combination of physiological selective pressures (11). A particularly remarkable relationship between this pattern of co-receptor usage phenotype and HIV natural infection is the finding that individuals homozygous for a knockout mutation in CCR5 (CCR5 32) are nearly completely resistant to HIV infection (12). To date, CCR5 32 homozygosity remains the only molecularly defined explanation for why some individuals are highly resistant to HIV infection despite repeated exposure, although additional mutations in the chemokine/coreceptor system have been shown to contribute to HIV transmission and disease progression (12).

30. Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS

30.2.2

273

The HIV Neutralizing Antibody Problem

HIV Env has evolved a sophisticated multilayered strategy to protect critical conserved elements involved in receptor binding and membrane fusion from humoral immune surveillance (13, 14). First, the external face of the glycoprotein is extensively glycosylated, thus presenting a poorly immunogenic “glycan shield” (15). Second, the gp120 subunit contains multiple variable loops (V1/V2, V3, V4, and V5) whose sequences continually change to escape antibody recognition. Of particular significance in the V3 loop, which was formerly considered the principal neutralizing determinant and is known to critically influence co-receptor usage phenotype (16). Third, and perhaps most intriguingly, the highly conserved receptor binding domains are protected by tertiary and quaternary structural features that keep them hidden or even unformed prior to receptor engagement. This phenomenon of “conformational masking” (17) is dramatically illustrated by comparison of the X-ray crystallographic structure of gp120 first solved in its CD4-bound form (18) versus its unliganded form (19). The former structure reveals the presence of a so-called “bridging sheet” formed from discontinuous regions in the inner and outer domains of the protein, which forms a highly conserved surface involved in co-receptor binding; the latter structure illustrates that these regions lie far apart in the absence of CD4 and do not form a continuous surface. Thus, CD4 binding induces a major conformational change in gp120 that creates the co-receptor binding site; this change is associated with an extraordinarily high entropic penalty (17), one the greatest ever reported for protein–protein interactions. The implications of this sequential receptor interaction entry mechanism for neutralizing antibody are profound because the conserved co-receptor binding region of gp120 does not exist on the free HIV virion and is formed only after encountering CD4 on the target cell. A number of monoclonal antibodies against this region have been described that recognize the CD4induced bridging sheet on genetically diverse primary HIV-1 strains (13, 14). In fact, such antibodies arise frequently during natural infection (20). However, because the corresponding epitopes are exposed only transiently (after CD4 binding but before co-receptor engagement), both steric (21) and kinetic factors impair the ability of intact immunoglobulins to access the corresponding epitopes as they are formed. Hence, these antibodies are poorly neutralizing (Figure 30.2). While a handful of broadly neutralizing anti-HIV monoclonal antibodies have been isolated over the past two decades, they recognize complex epitopes on gp120 or gp41 distinct from the highly conserved CD4-induced bridging sheet (13, 14, 22).

30.2.3 A Novel Bifunctional HIV-neutralizing Protein Based on Sequential Receptor Interactions The bridging sheet of gp120, highly conserved and critical for interaction with co-receptor, represents a potentially ideal target for neutralizing antibodies, if only the epitopes can be

Figure 30.2. Antibodies against the CD4-induced bridging sheet on gp120. See text for details. (A) The epitope for an anti-bridging sheet monoclonal antibody is unformed prior to CD4 binding. (B) The monoclonal antibody fails to neutralize infection, since steric and kinetic factors prevent it from binding to its epitope that is only transiently exposed, after CD4 binding but prior to coreceptor engagement (See Color Plates).

accessed. The only known mechanism for inducing epitopes in this region is via CD4 binding. This realization led us to conceive of a novel neutralizing bifunctional protein containing a soluble portion of CD4 (sCD4) attached via a flexible polypeptide linker to an antibody against the bridging sheet of gp120 (23). The idea is that binding of the sCD4 moiety would induce formation of the bridging sheet on the free virion; the antibody moiety would then bind to the newly created epitope, resulting in potent neutralization (Figure 30.3A). We had a direct roadmap for specific design of the desired molecule, because the first gp120 X-ray crystallographic structure (18) was of a trimolecular complex containing a gp120 “core” (lacking the variable loops and most of the carbohydrate) bound to a sCD4 fragment (extracellular domains 1 and 2) and an Fab fragment of the 17b monoclonal antibody directed against the conserved bridging sheet. This structure contained all of the elements of interest to design a single chain recombinant protein containing at its N-terminus the sCD4 moiety, attached via a flexible polypeptide linker (L1, 7 repeats of gly4ser) to a single chain variable region fragment (SCFv) of 17b, constructed with the heavy chain V region at its N-terminus linked (L2, 3 gly4ser repeats) to the light chain V region. We designated this protein sCD4-17b (Figure 30.3B). We expressed the bifunctional protein using recombinant vaccinia virus technology and tested its ability to neutralize HIV-1 infection (Figure 30.4; ref. 23). The target cells used in the MAGI assay express CD4 and co-receptors, and also contain the E. coli LacZ gene linked to the HIV LTR; HIV infection results in production of -galactosidase, which can be detected by in situ staining and counting blue (i.e., infected) cells. As shown in Figure 30.4A, sCD4-17b efficiently neutralized infection by the Ba-L isolate with high potency (IC50 ~ 3 nM; 0.1–0.2 µg/mL). By contrast, free sCD4, free 17b, or a mixture of unlinked sCD4 plus 17b had minimal effects over the same concentration range. These results indicate that the potency of sCD4-17b derives from

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struct, whereas the antibodies owe much of their activity to their bivalent structures. Perhaps attaching Fc moieties to sCD417b to produce dimeric Ig-like proteins would enhance the activity even further. Because of the highly conserved nature of both the CD4 binding site and the 17b epitope, we hypothesized that sCD4-17b would show extremely broad neutralizing activity against genetically diverse primary HIV-1 strains. Initial results with the low amounts of protein available proved somewhat disappointing, with only about half of the strains tested showing sensitivity (23); however, recent preliminary studies indicate that most isolates are neutralized over a range of concentrations. If these results are borne out, we are interested in potential applications of sCD4-17b as an antiviral agent, particularly as a potential topical microbicide to prevent HIV infection.

30.3 KSHV Entry and Receptor Identification 30.3.1

Figure 30.3. Bifunctional HIV-neutralizing protein based on sequential receptor binding. See text for details. (A) The sCD4 moiety of the protein binds to gp120 on the free virus prior to its interaction with the target cell; the SCFv moiety then binds and neutralizes infection. (B) Design of sCD4-17b. Top: A direct guide for design of the protein was provided by the first gp120 X-ray crystallographic structure (18), based on a ternary complex containing a gp120 “core,” the first two extracellular domains of sCD4, and the 17b Fab. In the bifunctional neutralizing protein, the L1 linker attaches the C-terminus of the sCD4 moiety to the N-terminus of the 17b heavy chain V region; the L2 linker attaches the C-terminus of the heavy chain V region to the Nterminus of the light chain V region. The resulting protein represents sCD4 attached to a 17b SCFv. Bottom: A schematic of the genetic construct encoding the single chain sCD4-17b protein (See Color Plates).

the bifunctional property of the chimeric protein, whereby a single molecule simultaneously binds to two distinct sites on the same gp120 subunit. The potency of sCD4-17b proved particularly impressive when compared with the activities of the best known broadly neutralizing monoclonal antibodies (23): 3B3 (a derivative of b12, directed against the CD4 binding site on gp120), 2G12 (directed against a complex carbohydrate epitope on gp120), and 2F5 (directed against the membrane proximal external region of gp41; refs. 13, 14, 22). The sCD4-17b protein displayed potency that was at least 10-fold higher than any of these antibodies, despite the fact that it is a monomeric con-

Entry Mechanisms of Herpesviruses

Herpesviruses are surrounded by a complex envelope containing roughly a dozen virus-encoded gene products. Some of these are involved in virus assembly, and a subset participates in the virus entry process. The predominant entry pathway for the most well-studied herpesviruses is direct fusion, although numerous examples of endocytosis-mediated entry have been reported (24). We recently initiated a study of KSHV, a human -herpesvirus associated with serious tumors and lymphoproliferative disorders, particularly in the context of HIV/AIDS. We chose to study the fusion-dependent mechanism, based on findings by others (25) and confirmed by us (26) that recombinant KSHV glycoproteins are capable of mediating cell fusion with a variety of target cells. With a panel of cell types originating from diverse tissues and species, we observed a close correlation between their ability to serve as targets in assays of KSHV glycoprotein-mediated cell fusion and KSHV virion entry; the latter was assessed using a KSHV recombinant virus containing a reporter gene linked to a constitutive cellular promoter. Our results did not support the essential involvement of 3 1 integrin, previously suggested to function as a KSHV entry receptor (27). These results led us to postulate the existence of an as-yet unidentified fusion/entry receptor for KSHV.

30.3.2 Identification of KSHV Receptor by Functional cDNA Library Screening We chose to pursue an unbiased approach to identify a KSHV receptor without making assumptions about the type of molecule likely to be involved. To this end, we devised a method to screen a cDNA library (in recombinant vaccinia viruses) from a cell type that was highly permissive for KSHV fusion, based on the ability of the receptor cDNA to confer fusion-permissiveness to an otherwise refractory target cell type (28).

30. Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS

275

Figure 30.4. Neutralization of HIV-1 infection by sCD4-17b. See text for details. Infection by the Ba-L isolate was measured using the MAGI-CCR5 assay. Virus was preincubated for 30 minutes at 37°C with the indicated agents at the designated concentrations; the mixtures were then added to MAGI-CCR5 target cells. Infectivity was scored 2 days later by staining for -galactosidase using X-gal, which forms a blue precipitate within the cells. (A) The agents tested were supernatant from cells infected with WT vaccinia virus (control) or vCD-3 (encoding sCD4-17b), purified sCD4 containing the first two extracellular domains, and purified 17b IgG. (B) The neutralization activities of sCD4-17b and monoclonal antibodies IgG b12, 2G12, and 2F5 were compared. Results are expressed as a percentage of blue cells compared to the control where virus was preincubated without added protein.

Figure 30.5. Newly identified KSHV fusion/entry receptor xCT. (A) Schematic of xCT, the light chain of the x(c)(–) cystine/glutamate exchange transporter. The essential features indicated are the putative 12 transmembrane domains, the intracellular N- and C-termini, and the disulfide linkage to the 4F2hc heavy chain. (B, C) Demonstration of xCT activity as a KSHV receptor by “gain-of-function” assays in which the recombinant protein was expressed in otherwise non-permissive target cells. In panel B, cell fusion assays were performed with F-515 effector cells lacking (–) or expressing (+) recombinant KSHV gB, gH, and gL, and NIH 3T3 murine target cells lacking (–) or expressing (+) vaccinia-encoded xCT. Fusion was quantitated by measuring -galactosidase activity. In panel C, virion entry assays were performed with K-562 wild type cells (–) or their corresponding xCT stable transfectant cells (+); entry was assessed by flow cytometric measurement of the fraction of cells expressing EGFP.

276

Rare cells infected with a vaccinia virus encoding a functional receptor fused with effector cells expressing KSHV glycoproteins; a novel reporter construct containing multiple markers for physical selection (epitope tags for immunobead enrichment, EGFP for fluorescence selection) was then activated in the fused cells. After several rounds of enrichment, individual vaccinia viruses with fusion-promoting activity (due to their encoding of functional receptor from the cDNA library) were obtained; DNA sequence analysis indicated that they all contained the identical cDNA insert. The KSHV receptor thus identified was a previously known protein called xCT, the light chain of the human cystine/ glutamate obligate exchange transporter x(c)(-) (29). xCT is a predicted 12-transmembrane protein that is expressed at the surface of cells in a disulfide-bonded complex with a protein designated 4F2hc (CD98hc), the so-called heavy chain of this transport system (Figure 30.5A). Several criteria verified that xCT has the properties of a KSHV fusion/entry receptor (28). Gain-of-function experiments demonstrated that xCT expression rendered otherwise nonpermissive target cell types permissive for KSHV cell fusion (Figure 30.5B) and KSHV virion entry (Figure 30.5C). Loss-of-function studies indicated that antibodies against xCT-based peptides specifically inhibited KSHV cell fusion and virion entry with naturally permissive target cells. Finally, a close correlation was observed between the natural KSHV fusion permissiveness of diverse human cell types and their endogenous expression of KSHV mRNA and protein. Taken together, these findings provided strong evidence that xCT functions as a receptor for KSHV glycoprotein-mediated cell fusion and KSHV virion entry.

30.3.3 Potential Significance of xCT for KSHV Pathogenesis The identification of xCT as a KSHV receptor led us to some intriguing speculations about significance for KSHV pathogenesis (28). Cysteine, the intracellular reduced product of cystine, is the rate-limiting substrate for biosynthesis of the major antioxidant glutathione. Indeed, xCT gene expression is strongly upregulated in response to glutathione depletion and oxidative stress, as occurs upon exposure to reactive oxygen species. Recent findings with cultured endothelial cells have suggested that KSHV induces reactive oxygen species along with enhanced virus entry (30). Thus, the identification of xCT as a KSHV receptor suggests a novel pathogenic mechanism whereby the virus might induce physiological conditions that upregulate its own receptor, thereby facilitating its own replication and dissemination. Additional phenomena are given new perspective in the context of KSHV/HIV coinfection. Intracellular glutathione levels are known to be progressively depleted during HIV disease (31), suggesting the possibility that HIV infection might facilitate KSHV replication not only by the obvious route of immunosuppression

E. A. Berger

but also by the novel mechanism of creating physiological conditions that upregulate the KSHV receptor.

30.4

Conclusions

The extraordinary complexities of virus entry mechanisms reflect the epic battles between the invading pathogens and the host defenses. Through molecular understanding of entry, it is possible to gain new insights into the mechanisms by which viruses are transmitted and cause disease. These perspectives in turn offer promise for developing new strategies to prevent and treat viral infections.

References 1. Earp LJ, Delos SE, Park HE, White JM (2004) The many mechanisms of viral membrane fusion proteins. Curr Top Microbiol Immunol 285:25–66. 2. Gallo RC, Montagnier L (2003) Retrospective: the discovery of HIV as the cause of AIDS. N Engl J Med 349:2283–2285. 3. Ganem D (2006) KSHV infection and the pathogenesis of Kaposi’s sarcoma. Ann Rev Path 1:273–296. 4. Dey B, Berger EA (2003) Vaccinia-based reporter gene cellfusion assays to quantitate functional interactions of HIV envelope glycoprotein with receptors. In Current Protocols in Immunology (Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, eds.), pp. 12.10.1–12.10.20. John Wiley & Sons, Inc., New York. 5. Wyatt R, Sodroski J (1998) The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280:1884–1888. 6. Feng Y, Broder CC, Kennedy PE, Berger EA (1996) HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872–877. 7. Berger EA, Murphy PM, Farber JM (1999) Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 17: 657–700. 8. Lusso P (2006) HIV and the Chemokine system: 10 years later. EMBO J 25:447–456. 9. Husman AMD, Schuitemaker H (1998) Chemokine receptors and the clinical course of HIV-1 infection. Trends Microbiol 6:244–249. 10. Pierson TC, Doms RW, Pohlmann S (2004) Prospects of HIV-1 entry inhibitors as novel therapeutics. Rev Med Virol 14:255–270. 11. Margolis L, Shattock R (2006) Selective transmission of CCR5utilizing HIV-1: the “gatekeeper” problem resolved? Nat Rev Microbiol 4:312–317. 12. O’Brien SJ, Moore JP (2000) The effect of genetic variation in chemokines and their receptors on HIV transmission and progression to AIDS. Immunol Rev 177:99–111. 13. Wyatt R, Kwong PD, Desjardins E, Sweet RW, Robinson J, Hendrickson WA, Sodroski JG (1998) The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705–711. 14. Burton DR, Stanfield RL, Wilson IA (2005) Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci USA 102:14,943-14,948.

30. Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS 15. Wei XP, Decker JM, Wang SY, Hui HX, Kappes JC, Wu XY, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM (2003) Antibody neutralization and escape by HIV-1. Nature 422:307–312. 16. Hartley O, Klasse PJ, Sattentau QJ, Moore JP (2005) V3: HIV’s switch-hitter. AIDS Res Hum Retrovir 21:171–189. 17. Kwong PD, Doyle ML, Casper DJ, Cicala C, Leavitt SA, Majeed S, Steenbeke TD, Venturi Μ, Chaiken I, Fung Μ, Katinger H, Parren PWLH, Robinson J, Van Ryk D, Wang LP, Burton DR, Freire E, Wyatt R, Sodroski J, Hendrickson WA, Arthos J (2002) HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature 420:678–682. 18. Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–659. 19. Chen B, Vogan EM, Gong HY, Skehel JJ, Wiley DC, Harrison SC (2005) Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 433:834–841. 20. Decker JM, Bibollet-Ruche F, Wei XP, Wang SY, Levy DN, Wang WQ, Delaporte E, Peeters Μ, Derdeyn CA, Allen S, Hunter E, Saag MS, Hoxie JA, Hahn BH, Kwong PD, Robinson JE, Shaw GM (2005) Antigenic conservation and immunogenicity of the HIV coreceptor binding site. J Exp Med 201:1407–1419. 21. Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, Franti Μ, Binley J, Vivona V, Grundner C, Huang CC, Venturi Μ, Petropoulos CJ, Wrin T, Dimitrov DS, Robinson J, Kwong PD, Wyatt RT, Sodroski J, Burton DR (2003) Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gpl120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10,557–10,565.

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22. Stiegler G, Katinger H (2003) Therapeutic potential of neutralizing antibodies in the treatment of HIV-1 infection. J Antimicrob Chemother 51:757–759. 23. Dey B, Del Castillo CS, Berger EA (2003) Neutralization of human immunodeficiency virus type 1 by sCD4-17b, a singlechain chimeric protein, based on sequential interaction of gp120 with CD4 and coreceptor. J Virol 77:2859–2865. 24. Spear PG, Longnecker R (2003) Herpesvirus entry: an update. J Virol 77: 10,179–10,185. 25. Pertel PE (2002) Human herpesvirus 8 glycoprotein b (gB), gH, and gL can mediate cell fusion. J Virol 76: 4390– 4400. 26. Kaleeba JAR, Berger EA (2006) Broad target cell selectivity of Kaposi’s sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry. Virology 354:7–14. 27. Akula SM, Pramod NP, Wang FZ, Chandran B (2002) Integrin alpha 3 beta 1 (cd 49c/29) is a cellular receptor for Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 108:407–419. 28. Kaleeba JAR, Berger EA (2006) Kaposi’s sarcoma-associated herpesvirus fusion-entry receptor: cystine transporter xCT. Science 311:1921–1924. 29. Verrey F, Closs EI, Wagner CA, Palacin Μ, Endou H, Kanai Y (2004) CATs and HATs: the SLC7 family of amino acid transporters. Pflug Arch Eur J Phy 447:532–542. 30. Wang JF, Zhang XF, Chandran B, Groopman JE (2004) Human herpesvirus-8 (HHV-8/KSHV) induces reactive oxygen species in endothelial cells that facilitate virus infection. Blood 104:174A–175A. 31. Herzenberg LA, DeRosa SC, Dubs JG, Roederer Μ, Anderson MT, Ela SW, Deresinski SC (1997) Glutathione deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci USA 94:1967–1972.

Chapter 42 A Model System for Studying Mechanisms of B-cell Transformation in Systemic Autoimmunity Wendy F. Davidson, Partha Mukhopadhyay, Mark S. Williams, Zohreh Naghashfar, Jeff X. Zhou, and Herbert C. Morse III

42.1 Introduction: Evidence for a Strong Association Between Systemic Autoimmunity and B-cell Lymphoma Numerous studies in humans have linked systemic autoimmunity with benign lymphoproliferation and increased risk of lymphoma (1–8). In most cases, autoimmune disease precedes lymphoma and the lymphomas are predominantly of the B-cell lineage (1–8). The autoimmunity/lymphoma association is strongest for Sjögren’s syndrome (SS), Hashimoto’s thyroiditis (HT), autoimmune hemolytic anemia, and mixed cryoglobulinemia (MC; refs. 4, 7–12). In HT, lymphomas develop in the thyroid from secondary mucosa-associated lymphoid tissue (MALT) characteristic of chronic lymphocytic thyroiditis (LT; refs. 8, 10, 13, 14). MALT is characterized by extranodal follicular structures that may be the site of chronic B-cell activation and eventually B-cell transformation (10). The relative risk of thyroid lymphomas in patients with LT ranges from 67-80 times that of controls (10, 13, 14). Lymphomas also arise from MALT in SS patients (4, 10, 15). A period of prolonged benign lymphoproliferation followed by outgrowth of B-cell clones (pseudolymphoma) characteristically precedes the appearance of dysplastic MALT lymphomas in SS and HT (10). MALT lymphomas are mostly indolent but can progress to higher-grade, largecell lymphomas in both HT and SS patients (10). A very high incidence of B-cell lymphoma as well as other types of malignancies also occurs in patients with autoimmune lymphoproliferative syndrome (ALPS), a disease caused most frequently by dominant heterozygous inheritance of mutations in the death receptor TNFRSF6 (FAS; ref. 16). ALPS develops in childhood and is characterized From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

by pronounced hepatosplenomegaly, lymphadenopathy, the progressive accumulation of benign CD4–CD8– T cells, and autoantibody (autoAb) production (17). Both children and adults are at high risk of developing Hodgkin’s lymphoma (HL) and non-Hodgkin’s B-cell lymphomas (NHLs). The risk of HL and NHL in ALPS patients is estimated at 51and 14-fold greater, respectively, than in the general population (16). The appearance of these lymphomas suggests that post-germinal center (GC) B cells are the predominant targets of transformation in ALPS patients. A similar disease, also characterized by predisposition to B-cell lymphoma, is observed in mice homozygous for recessive inactivating mutations in Fas (lpr) or Fasl (gld; ref. 18). Notably, somatic mutations in FAS also occur in HL and NHL and may be important in lymphoma development and progression in non-autoimmune individuals (19, 20). Patients with systemic lupus erythematosis (SLE) and rheumatoid arthritis (RA) also have an increased risk of B-cell lymphoma, although the relative risk is lower (~twofold) than for individuals with SS, HT, MC, and ALPS (1–6, 21). There is some debate whether lymphoma in SLE and RA patients is predominantly autoimmune disease-related or secondary to immunosuppressive therapy (21). In support of a strong correlation with disease, one recent retrospective study of more than 11,000 RA patients identified high inflammatory activity as the most prominent risk factor for lymphoma (5). The tumors arising in SLE patients are predominantly NHL and in RA patients include HL and NHL (21). Although there is general consensus for a strong linkage between autoimmune diseases involving chronic lymphoproliferation and B-cell lymphoma, relatively little is known about the mechanisms of malignant transformation. Defects in apoptosis resulting from germline mutations in FAS or CASPASE 10 clearly are risk factors for both autoimmunity and B-cell transformation (16, 19, 20, 22). Chronic Ag-driven lymphoproliferation by endogenous or exogenous Ag is another factor that may link systemic autoimmunity and lymphoma.

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42.2 Evidence to Support a Role for Sustained Ag Drive in Lymphoma Etiology Clinical studies indicate that chronic B-cell activation by autoAg or microorganisms may be an important environmental factor in the generation of B-cell lymphomas, especially the indolent MALT-type observed in SS and HT as well as gastric lymphomas (8, 15, 23–26). There are reports of MALT lymphomas in both SS and HT patient populations that produce Ab to immunoglobulin (Ig)G (known as rheumatoid factor [RF]) and exhibit non-random utilization of IgVH and VL genes, suggesting that chronically stimulated, autoreactive B cells may be particularly susceptible to transformation (23–25). There also is a well-established link between chronic infection with Hepatitis C virus (HCV) or Helicobacter pylori and B-cell lymphomagenesis (26–31). Interestingly, studies of the specificities of infection-associated B-cell lymphomas indicate that some are derived from B cells specific for viral or bacterial antigens, whereas others are autoreactive with skewing toward RF production (23–31). Transformation of autoreactive B cells in the context of chronic infection may be explained by molecular mimicry or, alternatively, RF-producing tumors may develop from RF B cells driven to clonally expand by immune complexes (23–25, 28, 31). A particularly strong association between HCV infection and autoimmunity exists for type-II MC (12). HCV infection has been implicated as an etiologic factor in MC, an autoimmune disease that evolves into malignant lymphoma in ~10% of patients (23, 31). Lymphomas from MC patients exhibited a highly restricted use of VH and VL genes, evidence of ongoing Ag selection, and production of Ab with the hallmarks of RF, a common autospecificity in MC (23, 31). Intraclonal variation in IgH and IgL mutation patterns consistent with posttransformation somatic mutation and Ag selection also has been reported in B lymphomas of unknown specificity (32– 34). In some cases, these lymphomas utilized a restricted set of Ig variable region genes, providing further evidence for Ag involvement in tumor growth (33, 34). In addition to supporting tumor growth and survival, continuous Ag drive may be a factor in the progression of follicular lymphomas from low to high grade (34). Overall, there is compelling evidence that sustained stimulation by Ag can culminate in B-cell transformation and, in some instances, also may play a role in tumor survival and progression. B lymphocyte persistence and eventual transformation also may be influenced by abnormal production of factors that promote B-cell activation and survival. Interleukin (IL)-10 and BAFF (also called BLyS, TALL-1, THANK) are two cytokines of particular interest as therapeutic targets as each has established or predicted ties to both autoimmunity and B-cell transformation (35–45).

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42.3 IL-10 and BAFF as Potential Links Between B-cell Hyperactivity, Systemic Autoimmunity and B-cell Transformation IL-10 is an immunoregulatory cytokine with potent antiinflammatory and immunosuppressive effects on myeloid cells and immunostimulatory effects on other cell types (44, 45). Both human and murine B cells can produce and respond to IL-10, although differences are observed between the species (44, 45). In mice, IL-10 prolongs the survival and viability of B cells and promotes the proliferation of the B-1 subset (44, 45). The effects of IL-10 on human B cells are more pronounced and include co-stimulation, enhanced Ig secretion, Ig isotype switching and plasma cell differentiation (44, 45). IL-10 has been implicated in disease pathogenesis in systemic autoimmunity in humans and mice and may be produced in excess by hyperactive B cells (40). Elevated levels of serum IL-10 are associated with SLE, RA, SS, and ALPS and frequently correlate with disease activity (41, 44–48). Autoimmune New Zealand Black (NZB) and NZB/W mice produce abundant IL-10 and interference with IL-10 production or availability blocks autoantibody formation and ameliorates disease (44, 45, 49, 50). Interestingly, IL-10-producing B-1 cells in NZB mice undergo malignant transformation suggesting a possible role for IL-10 in lymphomagenesis (49). In support of this, inactivation of IL-10 genes in NZB decreases B-cell activation and lymphoma incidence (50). A variety of human B-cell malignancies also produce IL-10 and in some cases tumors were shown to be IL-10-dependent for growth (38, 39). BAFF, a member of the tumor necrosis factor cytokine family, also supports B-cell activation and survival and may contribute to the development of systemic autoimmunity and B-cell transformation (42, 43, 51–54). BAFF is produced by monocytes and dendritic cells predominantly, although there is one recent report of BAFF expression by activated human B cells (36, 42, 43, 51, 52). Overexpression of BAFF in Tg mice is associated with lupus-like systemic autoimmunity and in some cases pathology similar to SS with inflammation and destruction of salivary glands and the appearance of B lymphomas in the submandibular gland (53–55). Elevated serum levels of BAFF also are reported in patients with SLE, RA, and SS and correlate with disease severity (42, 43, 56–58). In support of a role for BAFF in disease pathogenesis in mice, treatment with a soluble decoy receptor for BAFF (TACI-Ig) prolongs the survival of NZB/WF1 mice and prevents the development of collagen-induced arthritis (59, 60). In addition to its potential role in lymphomagenesis, BAFF has been implicated as an autocrine growth factor for certain human B lineage tumors including CLL, NHL, and multiple myeloma (MM; refs. 35–37, 61, 62).

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Figure 42.1. PL secrete Ab reactive with nuclear Ag. Staining of Hep2 nucleii with sera from six-month-old BALB-gld mice (positive control) C.B-17-scid mice (negative control), and scid mice bearing the BALB-gld tumors 425 and 2685 (See Color Plates).

42.4 FasL-deficient Mice as a Model System for Studying Relationships Between Systemic Autoimmunity and B-cell Lymphomagenesis Although autoimmune strains of mice including NZB/W F1, C57BL/6-mev/mev, BXSB-Yaa and MRL-faslpr/ faslpr (lpr) frequently exhibit age-related oligoclonal outgrowths of autoreactive B cells, few of these mice survive long enough to develop B-cell lymphomas (63). In contrast, we found that C3H/HeJ and BALB/c mice homozygous for lpr or faslgld (gld) mutations, which have a median lifespan of ~11 months, have a high incidence of B-cell lymphoma (18). Dominant clones are found in ~70% of BALB-gld and ~30% of C3H-gld mice by 10-12 months of age and grow as lethal metastatic lymphomas when transferred into immunodeficient scid mice (18). Both primary and transplanted tumors are predominantly plasmacytoid in morphology and have a remarkably similar phenotype (18, 64). The majority of PL secrete Ig, have undergone Ig isotype switching, and show evidence of somatic mutation of CDR regions and conservation of framework regions of expressed IgH and IgL genes suggesting that they derive from Ag-selected B cells that have experienced a GC-like environment (18, 64). Studies of the specificities of the Ab secreted by PL indicate that a high proportion of tumors are derived from autoreactive B cells specific for nuclear Ag or producing RF and exhibit the skewed IgVH and IgVL gene repertoires and Ig gene rearrangement patterns associated with these specificities (64). Figure 42.1 shows typical nuclear staining with secreted Ig from ANA and anti-dsDNA producing PL. Consistent with our findings, autoreactive B-cell lymphomas secreting RF were reported in T-cell-deficient lpr mice suggesting that the drive for B-cell transformation may be B cellintrinsic and certainly T cell-independent (65). Gene expression profiling of a panel of primary and scid-passaged PL indicated that these lymphomas are highly related, readily distinguishable from other pre- and post-GC B lineage tumors, and have the genetic features of activated cells arrested at an early stage in plasma cell development (64). Relative to normal follicular B cells, PL exhibit decreased expression of Pax-5 and Bcl-6, modestly increased expression of Blimp-1 and unaltered expression of Xbp-1 (64). Consistent with their differentiation towards

Figure 42.2. Increased levels of circulating IL-10 and Il10 mRNA transcript levels in B cells and B cell lymphomas in BALB-gld mice. (A) Levels of IL-10 in serum from 6-month-old +/+ and gld mice of various ages, determined by ELISA. Values represent mean ± SE for 6 to 10 samples. Serum IL-10 levels were significantly increased in gld mice by 2 to 4 months of age. IL-10-producing lymphomas contribute to the age-related increase in serum IL-10 levels in gld mice. (B) Il10 mRNA levels in purified B cells from 5- to 6-month-old gld mice, isolated primary PL and scid-passaged PL cells, determined by real-time RT-PCR using B cells from 6-month-old BALB/c mice as a reference. Values represent mean ± SE for three to seven samples.

plasma cells, the PL have decreased levels of surface Ig, CD45(B220), CD21, and CD23 and variably express CD138 (18, 64). Most PL also express Mac-1 (CR3/CD11b; ref. 18). Among 14,000 genes surveyed, several hundred genes were expressed at levels greater than twofold higher or lower in PL versus normal follicular B cells (64). Some of the differentially expressed genes relate to late-stage B-cell differentiation but others may contribute to tumor growth

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Figure 42.3. BAFF is spontaneously secreted by purified gld and gld, Il10-/- B cells and gld primary PL. (A) Levels of secreted BAFF in twoday cultures of purified B cells from 4-month-old control and mutant mice measured by ELISA. Data represent mean ± SE for three experiments. PL and gld and gld, Il10-/- B cells secreted significantly more BAFF than +/+ and Il10-/- controls (∗ ∗, P values <0.005 for comparisons with controls). (B) Levels of BAFF in serum from five 4-month-old control and tumor-free gld and gld, Il10-/- mice and seven 12-month-old gld and gld, Il10-/- mice with a high incidence of lymphoma. Values represent mean ± SE. At both ages, gld and gld, Il10-/- mice had significantly higher levels of serum BAFF than +/+ and Il10-/- controls (∗, P < 0.05; ∗∗, P < 0.005). (C) FACS analysis of BAFF-R expression on gated CD19+ B cells from indicated strains. Overlays show staining on B cells with rat anti-BAFF-R mAb (solid line) and control rat anti-CD4 mAb (dotted line). The mean fluorescence intensity for BAFF-R staining is indicated for each profile.

and survival. Among the upregulated genes were a number with anti-apoptotic activity, including RhoB, Birc5, Traf1, and Gadd45. The first three are particularly relevant as they are over-expressed in various human lymphomas and presumably contribute to tumor survival through their antiapoptotic effects (66–69). Similar to many human post-GC lymphomas, most PL produce high levels of IL-10 and BAFF mRNA and spontaneously secrete both cytokines (Figures 42.2, 42.3A, and 42.3B). Two other uniformly upregulated genes of interest are Pftk1 and Aldh3b1. PFTK1, a cyclindependent-like kinase, is aberrantly expressed in plasma cells from patients with monoclonal gammopathy and MM and ALDH3, an aldehyde dehydrogenase, is overexpressed in liver and breast cancers (70–73). PL also exhibited significant reduction in the expression of genes involved in tumor suppression, DNA repair and the regulation of cell division, including Rad17, Plagl1, Btg2, Bin1 and Mad1l1. Downregulation of these genes occurs in a variety of non-lymphoid malignancies (74–78) In summary, lymphomas in gld mice develop predominantly from activated autoreactive B cells with the phenotypic and genotypic features of Ag-experienced cells in the early stages of plasma cell differentiation. PL have a novel gene expression profile that confirms their differentiation status and may provide clues for mechanisms of malignant transformation and tumor survival.

42.5 Activated CD21/CD23lo B Cells are the Likely Precursors of PL Both C3H-gld and BALB-gld mice develop progressive autoimmune lymphoproliferative disease characterized by profound lymphadenopathy, splenomegaly, high titers of circulating Ab specific for a variety of nuclear Ag, hypergammaglobulinemia, and immune complex-mediated tissue damage (64). Early in disease, lymphoid hyperplasia is dominated by T lineage cells, particularly B220+ CD4– CD8– T cells. B cells also accumulate abnormally and beyond 4 months of age there is selective expansion of a subset of mature IgM+ CD21-/lo CD23-/lo Mac-1+ CD138– B cells, as well as increased numbers of plasmablasts, and plasma cells (18, 79, 80). Initially, the CD21/CD23lo population is polyclonal but around 5 to 6 months of age, when ~40% of B cells in the spleen and LN are CD21/CD23lo, oligoclonal outgrowths are detectable in some mice (Figure 42.4A and 42.4B). After 6 months of age, the CD21/CD23lo population continues to accumulate and clonal populations become more prominent (Figures 42.4 and 42.5). By 9 to 12 months of age, transformed monoclonal or biclonal CD21/CD23lo Mac-1+ populations are present in a majority of mice (Figure 42.5; ref. 18). Similar progressive clonal expansion of activated B cells is a feature of the pre-lymphomatous stages of B-cell lymphoproliferation in SS and HT patients (81, 82).

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Relatively little is known about the origins or functions of the CD21/CD23lo Mac-1+ B-cell population in gld mice or its role in disease pathogenesis. However, elevated levels of activated mature CD21/CD23lo B cells are found in the circulation of patients with SLE, SS and RA and in joint aspirates from patients with active RA (83–88). The presence of these cells frequently correlates with disease activity, suggesting that they may have clinical relevance (83–88). The fact

that the CD21/CD23lo Mac-1+ B-cell population undergoes clonal expansion and phenotypically resembles PL suggests that it includes pre-lymphomatous cells at risk of malignant transformation. To further explore the relationships among the CD21/CD23lo Mac-1+ B cells, other B-cell subsets and PL, we compared these populations for surface Ag and gene expression. Detailed cytometric analysis of splenic gld B cells showed that the CD21/CD23lo subset was consistently CD38hi CD44hi LFA-1hi Mac-1+ B220+ CD19+ BAFFR+ CD40+ CD40Linter CD11cinter CD29inter ThBinter CD43inter IgDdull B71dull CD5- AA4.1- (Figures 42.6, 42.7, and data not shown). By comparison, the CD21/CD23hi B cells expressed higher levels of ThB (Ly-6D), IgD, B220, and CD40, lower levels of CD43 and CD40L, and were negative for CD11c (Figures 42.6 and 42.7). As would be expected, a small population of AA4.1+ transitional B cells was included in the splenic CD21/CD23lo population (Figure 42.6). The CD21/CD23lo subset also was consistently larger in size than follicular B cells. Overall, the surface phenotype of the CD21/CD23lo B cells distinguishes them from immature transitional B cells, marginal zone B cells, B-1a cells, and resting follicular B cells. Although the cells share some features with peritoneal Mac-1+ CD23- CD43+ B-1b cells, their phenotype and increased size are most consistent with activated mature B cells undergoing differentiation towards plasmablasts. In support of this, morphologic analyses of sorted CD21/CD23lo B cells showed enrichment for immunoblasts and plasmablasts as well as small numbers of plasma cells (Figure 42.8). Moreover, qRTPCR analyses of sorted CD21/CD23lo B cells indicated that the cells have differentiation-associated down-regulation of Pax-5 and Bcl-6 expression but have not proceeded to the stage where Blimp-1, CD138, and Xbp-1 are significantly upregulated (data not shown). The high level of CD40L on CD21/CD23lo B cells is of interest as elevated expression of CD40L is observed on activated T and B cells in SLE patients and other autoimmune mice and chronic CD40/CD40L interactions have been postulated to play a role in disease pathogenesis (reviewed in refs. 89 and 90). Studies of the proliferative responses of sorted gld CD21/CD23lo and CD21/23hi B cells showed that both populations proliferate in response to CD40 croslinking or CpG-rich oligodinucleotides but the CD21/CD23lo population responded poorly to LPS and BCR crosslinking (data not shown). Thus, it is possible that the growth and survival of CD21/CD23lo B cells is influenced by CD40/CD40L interactions among B cells and CpG-directed TLR9-mediated signals induced by interactions with DNA. To further characterize the CD21/CD23lo B cells, we compared gene expression patterns between sorted polyclonal gld CD21/CD23lo and CD21/23hi B cells and +/+ BALB/c B cells by microarray. We identified 149 genes out of 4,000 detected that were expressed at more than twofold higher or lower levels in gld CD21/CD23lo B cells versus +/+ CD21/ CD23hi B cells. Sixty-seven genes discriminated CD21/ CD23lo and CD21/CD23+ gld B cells. Only 34 genes discriminated CD21/CD23hi gld B cells from +/+ follicular B

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Figure 42.6. Comparison of surface Ag expression on splenic CD21/CD23lo (black line) and CD21/CD23hi (solid) B cell subsets from fivemonth-old gld mice. The CD21/CD23lo cells show decreased expression of ThB, IgD, B220, and CD40 and increased expression of Mac-1, LFA-1, CD11c, CD38, CD43, and CD54. The CD21/CD23lo population contains a small proportion of AA4.1+ transitional B cells (See Color Plates).

Figure 42.7. Expression of CD40L is significantly increased on gld CD21/CD23lo B cells. Figure shows expression of CD40L, CD44, and CD29 on +/+ (blue), gld CD21/CD23lo (green), and gld CD21/CD23+ (purple) B cells from 6-month-old mice (See Color Plates).

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cells and 19 of these 34 genes were similarly up or downregulated in the gld CD21/CD23lo subset and may relate to activation. Overall, these data indicate that the CD21/CD23hi gld B-cell population more closely resembles +/+ CD21/CD23+ B cells than gld CD21/CD23lo B cells but shares some gene expression changes with the latter (Figure 42.9). Notably, a subset of the 95 genes selectively upregulated in the CD21/ CD23lo population versus +/+ B cells also was upregulated in oligoclonal CD21/CD23lo B cells, PL, and PL cell lines. These discriminating genes included RhoB, Birc5, Traf1, Gadd45, Baff, Il10, Itgb1, Derl3, Pftk1, Aldh3b1, Hamp1, and S100A8. PCR verification of expression of Il10, Baff and Pftk1 is shown in Figure 42.10. The similar pattern of gene

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Hspca Rybp Ptgal Mapkl Ifitm3 SioDa1 Ltb4dh 1700013H19R1k Trk Blrc5 lfih 1 Ezh2 Rfc4 H3f3b Ndufc2 Cmnn Dui Nipsnapl Mk167 Nek3 Cd3g Derl3 1810027O10Rik Alox5ap 1195881 Prdxl Tcebl Acp5J 1209445 Smc211 Co19 Ssr3 TSc22d3 903011K21Rik Rock 1 5520402M19RiK Tmcm23 Purn2 K1f5b Ei64ebp2 Usp25 Ceml Txndel Saib 1 Cnn 3 Ctps 2 Ccnt 2 Ube3a 1810038L18Rik Catnb Eis2 Chordel 2810409H07Rik Tmcm33 Dnajal Hsp105 Car2 Dnajbl Mac2a D430028C21Rik Prei3 4930535B03Rik Banp BC018601 Clqp Cxcr1 2810021B07$Rik Clq8 Hmoxl Nap111 Ahey11 Grp58 Cyfip 1 Anape10 Sfra9 Zdhhei2 Scoc Mphosph6 Spcsl Traf1 D8Entd23e Slc33nl M6prbp1 1810057B09Rik 1110021H02Rik Mrpa18c 4632413K17Rik Birclb Adssl 1 2610029101 Rik Slp1 Rgs1 Ly6a Lam5 End2 H10032O16Rik Rnasel Sfrs6 2010004B12Rik 1210829 F13al Gng12 Gadd45g 2610042O14Rik Plscr 1 0610011D08Rik Ly96 Scx11 Eaf2 Irgb1 Lman 1 Ev12a 5830443L24Rik Rhob Arib4b Thrap3 1700012B14Rik Igtp Blvrb Nek7 9130213B05Rik Zfhx1b 9430098E02Rik Capn2 D6Wau176e Myo5e Tlr7 1500012A13Rik Irgav Hsdiib1 Tslpr Rab4b Mdflc Gadd45b Ccdpl S100a8 Ccr6 Bc111a Snca Zfp318 Gnas Bel6 Cd22 Ms4a1 Gga2

CD21-/CD23-_2

-2.93 -0.53 0.19 0.99 1.20

Figure 42.9. Hierarchical clustering of gene expression data for 149 genes that distinguish sorted gld CD21/CD23lo B cells from CD21/CD23hi BALB/c follicular B cells as determined by t -test (P < 0.000001). Lanes 1 to 3 (left), gld CD21/CD23lo B cells; lanes 4 and 5 (center), BALB/ c CD21/CD23+ B cells; and lanes 6 to 8 (right), gld CD21/CD23+ B cells. Band color intensity ranges from red (high level expression) to blue (low level expression) (See Color Plates).

these populations also express BAFFR and IL-10R (Figures 42.2, 42.3, and data not shown). Thus, BAFF and IL-10 potentially may function as autocrine growth and survival factors for lymphomas and their precursors. IL-10 also may suppress anti-tumor immune responses.

20 16 12 8 4

BAFF

gld 6 mo CD21/23 −

gld 6 mo CD21/23 +

gld 3 mo CD21/23 −

gld 3 mo CD21/23 +

PL

gld 6 mo CD21/23 −

gld 6 mo CD21/23 +

gld 3 mo CD21/23 −

PL

gld 3 mo CD21/23 +

gld 6 mo CD21/23 −

gld 3 mo CD21/23 −

IL-10

gld 6 mo CD21/23 +

PL

1

NB

0

gld 3 mo CD21/23 +

Fold change

24

Pftk1

Figure 42.10. Quantitative RT-PCR analysis of expression of Il10, Baff, and Pftk1in sorted B cells and PL. Values represent mean ± SEM for five separate samples normalized to a value of 1 for BALB/ c B cells. Comparisons are for sorted gld CD21/CD23lo and CD21/ CD23hi B cells and PL.

expression in the PL and CD21/CD23lo B-cell population provides further evidence that PL likely develop from clones of CD21/CD23lo B cells. Studies are in progress to determine whether enhanced expression of these genes relates to dysregulated growth and survival or is a function of differentiation towards plasma cells. The enhanced expression of Il10 and BAFF is of interest since both cytokines are implicated in the pathogenesis of systemic autoimmune disease and also can function as autocrine growth factors for malignant B cells (42, 43, 51–54). Serum levels of BAFF and IL-10 are elevated in pre-lymphomatous gld mice and highest in tumor-bearing mice (Figures 42.2 and 42.3). Both BAFF and IL-10 are spontaneously secreted by CD21/CD23lo B cells and most primary and transplanted PL and PL cell lines and

42.6 IL-10 Is Not Essential for the Development of Autoimmunity or B-cell Lymphomas in gld Mice To further investigate the importance of IL-10 in the development of autoimmunity and B-cell lymphomas, we generated BALB- gld/gld, Il10-/- ( gld , Il10-/-) mice. Comparisons of gld and gld , Il10-/- mice ranging in age from 2 to 12 months revealed that IL-10 is redundant for the development of ALPS and B-cell lymphoma but does play a role in controlling inflammatory lesions involving epithelium. The IL-10 deficient mice developed splenomegaly and lymphadenopathy with similar kinetics and severity to BALB-gld mice and there was no evidence for exacerbation of the mild kidney disease normally observed in this strain (data not shown). In addition, the proportions of mice producing autoAb and the titers of serum autoAb specific for nuclear Ag or dsDNA also were similar in both strains (data not shown). As in gld mice, activated CD21/ CD23 lo B cells accumulated progressively with age in the spleens and LN of gld , Il10 -/- mice, and by about 7 months of age were the dominant B-cell subset (Figure 42.11). Similarly, tumors with the features of PL developed in the

W. F. Davidson et al. 90 80 p = 0.007

70 60 50 40 30 20

3-4 mo

5-6 mo

gld Il-10 -/-

gld

gld Il-10 -/-

gld

0

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gld

% of B cells that are CD21/23 lo

392

7-8 mo

Figure 42.11. IL-10 is not required for the accumulation of CD21/ CD23lo B cells. Percentage of CD21/CD23lo B cells among all B cells in the spleens of gld (black bars) and gld, Il10-/- (open bars) mice at the ages indicated. IL-10 deficiency is associated with an increased rate of accumulation of CD21/CD23lo B cells between 4 and 6 months of age. Values represent mean ± SE for 8 to 17 mice.

IL-10-deficient mice with comparable frequency, kinetics, and growth characteristics to those in gld mice. Figure 42.12A shows the presence of dominant B-cell clones in spleen in 10 gld, Il10-/- mice with advanced lymphomas. These lymphomas closely resembled primary gld PL in surface phenotype and gene expression profile (Figure 42.12B and 42.12C). Notably, PL from gld, Il10-/- mice also spontaneously produced high levels of BAFF mRNA transcripts (Figure 42.12B). The possibility that BAFF functions as a dominant autocrine growth and survival factor for pre-lymphomatous B cells and PL is currently under investigation. Although inherent IL-10 deficiency had no notable effects on the development of non-malignant lymphoid hyperplasia, autoimmunity or lymphomagenesis, it worsened normally mild inflammatory lesions associated with epithelium. A low proportion of aging BALB-gld mice develop blepharitis, an inflammatory condition of the eyelid, as well as skin lesions that predominantly affect the ears. In the absence of IL-10, both conditions were accelerated and exacerbated and a higher proportion of mice were affected (data not shown). Consistent with our findings, autoimmune dermatitis was more severe in MRL mice deficient in both Fas and Il10 (91). Thus, IL-10 may be an important modulator of dermatitis associated with systemic autoimmunity.

Figure 42.12. Gld, Il10-/-mice develop plasmacytoid lymphomas that are similar in phenotype and gene expression to gld PL. (A) Southern blot of genomic DNA from spleens of 10 gld, Il10-/-mice with primary lymphomas showing prominent clonal IgH gene rearrangements. Lane C shows location of germline band in BALB/c kidney. (B) Quantitative RT-PCR of genes that distinguish PL from other plasma cell tumors. Gene expression is normalized to normal BALB/c B cells (NB). Levels of transcripts for eight genes are similarly increased or decreased in isolated primary PL from gld and gld, Il10-/-mice. Values represent mean ± SE for four tumors. (C) FACS profiles for a typical primary gld, Il10-/-PL. Solid line shows staining of CD19+ cells with indicated Ab and dotted line staining with non-reactive control Ab. CD21/CD23lo Mac-1+ B220+ CD5- phenotype is similar to gld PL.

42. Autoimmunity and B-cell Lymphoma

42.7

Conclusions

The development of B-cell lymphomas associated with systemic autoimmune diseases such as SS, HT and MC frequently is preceded by the appearance of oligoclonal outgrowths of pre-lymphomatous B cells. The signals responsible for driving the expansion of these clones and the mechanisms leading to their eventual transformation are poorly understood. Here, we present evidence that oligoclonal populations of activated CD21/CD23lo B cells also accumulate prior to the appearance of lymphomas in murine ALPS and likely include tumor precursors. This pre-lymphomatous population provides a valuable model for studying mechanisms of survival and transformation in chronically activated B cells. Comparisons of the pre-lymphomatous B cells and PL indicate that the two share a novel surface phenotype indicative of activation and have overlapping gene expression profiles. The distribution of surface markers suggests that the tumors and their precursors may receive non-overlapping survival signals from a variety of sources including cell–cell and cell–matrix interactions involving adhesion molecules and integrins (LFA-1, CD44, Mac-1, CD11c, CD29) and autonomous CD40/CD40L interactions. BAFF and IL-10 also may contribute to cell survival and growth via deregulated autocrine or paracrine loops, although we have established that IL-10 is redundant for the accumulation and transformation of CD21/CD23lo B cells and the survival of PL. Chronic activation of pro-survival pathways in pre-lymphomatous B cells and PL may explain the enhanced production of the anti-apoptotic molecules, TRAF-1, RhoB, GADD45 and Birc5a in these populations. Two genes, Pftk1 and Aldh3b1, each constitutively overexpressed in PL and their precursors as well as certain human malignancies also may be important downstream effectors of cell survival and growth. Currently, we are investigating the relative contributions of cell adhesion molecules, CD40/CD40L and BAFF/BAFFR interactions, and dysregulated expression of Pftk1 and Aldh3b1 to the survival and growth of PL and their precursors. Eventually, these studies may lead to the development of improved targeted therapies for chronically stimulated autoreactive B cells and their transformed counterparts.

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Chapter 39 A Role for Complement System in Mobilization and Homing of Hematopoietic Stem/Progenitor Cells M. Z. Ratajczak, R. Reca, M. Wysoczynski, M. Kucia, and J. Ratajczak

39.1

Introduction

During inflammation or other stressed situations related to tissue/organ damage, hematopoietic stem and progenitor cells (HSPC) are released from the bone marrow BM; refs. (1–21). This is important evidence for the existence of a close link between inflammation and stem cell trafficking. It is widely accepted that the α-chemokine stromal derived factor (SDF)-1–seven transmembrane span Gαi proteincoupled receptor CXCR4 axis plays a crucial role in the retention and homing of HSPC in BM as well as in their egress (mobilization) from BM into peripheral blood PB; refs. (6, 7, 10, 11, 22–24). We recently noticed that complement (C) is activated in BM during conditioning for hematopoietic transplantation by myeloablative radio-chemotherapy as well as in all protocols that are currently employed for mobilization of HSPC (25–27). These observations suggest the involvement of C in homing and mobilization of HSPC. This hypothesis is supported by data from our laboratory demonstrating C as a regulator of SDF-1-mediated homing of HSPC into BM as well as a regulator of their egress/ mobilization from BM into PB (25, 26, 28). We present evidence that cleavage fragments of the third component (C3) of the C system—C3a and desArgC3a—play an important role in enhancing the responsiveness of HSPC to an SDF-1 gradient (4, 28, 29). Thus, a new concept emerges where the trafficking of HSPC is tightly connected with the activation of C and innate immunity.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

39.2 The Role of C in Stem Cell Trafficking 39.2.1 Retention, Mobilization, and Engraftment of HSPCs First, we will present the current understanding of mechanisms that govern retention, mobilization and engraftment of HSPC. In particular, mobilization and engraftment of HSPC could be envisioned as mirror images of a similar biological process (Figure 39.1).

39.2.1.1

Retention of HSPC in BM

It is well known that BM contains functional niches in which HSPC reside and self-renew (30, 31). It is also known that HSPC home to and engraft within these niches after transplantation (32–34). Two distinct types of niches have been described: an endosteal niche (35, 36) and an endothelial niche (37). The retention of HSPC in BM is controlled by an adhesive interaction between adhesion receptors on HSPC, endosteum, endothelium, and stroma (38. These receptors include integrins (VLA-4, VLA-5, Mac-1; refs. 39–42), selectins (L-selectin; refs. 43 and 44), CD44 (e.g., glycoform of CD44: HCELL; refs. 43 and 45), members of the immunoglobin superfamily, tyrosine kinase receptor–c-kit (46, 47) and the chemokine receptor CXCR4 (6, 11, 48). It has been postulated that an interaction between stroma/endosteum-secreted SDF-1 and CXCR4 expressed on HSPC as well as stromal cell adhesion molecule VCAM-1/CD106 and its HSPC counter-receptor integrin α4β1 (VLA-4) is critical for the homing and retention of HSPC in the BM (38). The most critical pathway involved in the retention of HSPC within BM is the CXCR4–SDF-1 chemotactic axis (Figure 39.1; refs. 6, 7, 10, 11, 22, and 24). The α chemokine SDF-1 plays a key role during ontogeny of the hematopoietic system by inducing the migration of primitive HSPC from the fetal liver to the 357

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Figure 39.1. Homing of HSPC into BM and their mobilization into PB. Homing of HSPC to BM after transplantation and mobilization of HSPC from BM into PB depends on the activity of the SDF-1-CXCR4 axis. Conditioning for transplantation by chemotherapy (e.g., cyclophosphamide [CY]) and/or total body irradiation (TBI) increases via hypoxia inducible factor (HIF)-1a the concentration of SDF-1 in the BM microenvironment. This results in chemoattraction of CXCR4+ HSPC. During the reverse process, mobilization, a mobilizing agent (e.g., G-CSF): (i) increases the concentration of proteases in the BM microenvironment, degrading SDF-1 and CXCR4 and (ii) decreases SDF-1 expression at the mRNA level. This results in the release of CXCR4+ HSPC into PB. Both these processes are modulated by C3 cleavage fragments (C3a, desArgC3a and iC3b) whose BM-concentration increases both during conditioning for transplant by CY/TBI or stimulation of mobilization by G-CSF.

BM during development. SDF-1 has been shown to promote engraftment and subsequent hematopoietic reconstitution by adult HSPC (6, 11, 22). In vitro, SDF-1 is a potent chemoattractant for primitive BM CD34+CD38– cells including HSPC that express CXCR4 (28, 49, 50). The chemotactic effects of SDF-1 are mediated by the G-protein-linked receptor CXCR4, which upon ligand binding activates integrin-mediated firm adhesion and transmigration of HSPC through the BM endothelium (28, 50). SDF-1 also enhances secretion of metalloproteinases by HSPC that are critical for the transendothelial migration of these cells into the BM microenvironment (28, 51). Furthermore, recent studies showing expression of C receptor CR3 (αMβ2-integrin; CD11b/CD18) on HSPC that binds the C3 cleavage fragment, iC3b, and the significant enhancement of granulocyte-colony stimulating factor (G-CSF)-induced mobilization by administration of blocking antibodies against β2-integrins, indicated involvement of CR3 in the homing/retention of HSPC in BM (41, 42, 52, 53). Supporting this, our recent studies in CR3-/- mice revealed that, compared to their wt littermates these mice are more sensitive to mobilization by suboptimal doses of G-CSF (data not published). Taken together, these data suggest that CR3 also participates in the retention of HSPC in BM.

39.2.1.2

Mobilization of HSPC to PB

It is known that under steady state conditions a small pool of HSPC leaves BM niches and circulates in the PB, and it is believed that this circulating pool functions to maintain

a balance in the total HSPC population widely distributed among various marrow sites (7, 8). The normal low level of continual HSPC release into PB can be significantly raised by administration of G-CSF, cyclophosphamide (CY), cytokines (interleukin IL-1, IL-3, IL-7, IL-12), chemokines (e.g., IL-8, Gro-β) and glycans (e.g., zymosan, fucoidan; refs. 26, 54–57). This process is called “mobilization,” and is now the major method used for obtaining HSPC for hematopoietic reconstitution of patients in need of a hematopoietic transplant. It is accepted that mobilization by either G-CSF or CY transforms the BM into a highly proteolytic environment due to the accumulation of granulocyte-derived neutrophil elastases that directly cleave (i) SDF-1, (ii) the N-terminus of CXCR4, and (iii) VCAM-1 (54, 58, 59). Also, it was recently reported that G-CSF directly inhibits BM-expression of SDF1 at the mRNA level (60). All of these events together lead to the release of HSPC from the BM (Figure 39.1). After mobilization, the BM needs time to “recover” from this GCSF- or CY-triggered local tissue injury. In the process of marrow regeneration, the marrow-concentration of SDF-1 as well as expression of VCAM-1 on BM fibroblasts has first to be restored. In addition, functional CXCR4 has to be reexpressed on the surface of HSPC. After the secretion of SDF1 is restored and VCAM-1 is again expressed, HSPC may return to the BM and home into their hematopoietic niches. Interestingly, at the same time we noticed an increase in the BM concentration of C3 cleavage fragments (Figure 39.2;

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Figure 39.2. Cleavage of C3 into biologically active fragments. Cleavage/activation of C3 in BM is initiated by a C3-convertase to generate fluid-phase C3a and stromal cell-bound C3b. Both C3a and C3b have a short half-life. Fluid C3a is rapidly degraded to des-ArgC3a and bound C3b is proteolyzed to iC3b. While C3a mainly activates the C3aR, des-ArgC3a binds to another unidentified receptor X? iC3b tethers HSPC by interacting with CR3.

refs. 25–27). Thus, during regeneration both SDF-1 and C3 cleavage fragments (C3a, desArgC3a and iC3b) are up regulated in BM tissue.

39.2.1.3

Homing of HSPC After Transplantation

The early stages of BM seeding, which precede proliferation/ differentiation, are collectively termed homing. Homing is the mirror image process to mobilization and can be divided into several overlapping steps. CXCR4+ HSPC infused into PB have to respond to a chemotactic SDF-1 gradient in BM, attach to BM endothelium, transmigrate through the basal membrane in a metalloproteinase-dependent manner, and finally home to the niche where they have subsequently to survive, expand, and proliferate. Thus, homing of HSPC to BM niches is the first step in the engraftment process before HSPC self-renew and differentiate into precursor cells for all of the hematopoietic lineages. We have provided evidence that SDF-1-dependent homing of HSPC into BM is orchestrated by C3 cleavage fragments (25, 28, 29). The expansion of transplanted HSPC is directly responsible for the final reconstitution of the BM tissue. Since more differentiated HSPC have more limited self-renewal potential, they contribute only to short-term engraftment after transplant. Short-term engraftment can be assessed by evaluating hematopoietic recovery from lethal irradiation after transplants or formation of the so-called spleen colonies in irradiated recipients (colony-forming units spleen [CFU-S] assay; ref. 25). Long-term engraftment, which is most important for longterm survival after transplantation, is achieved by the most primitive HSC which have long-term repopulating capacity. Later, we discuss how all of these processes mentioned previously—retention, homing, mobilization, and expansion of HSPC—are co-regulated by C3 cleavage fragments.

39.2.2 Role of C in Inflammation and Tissue Injury The C system is a recognized component of the innate immunity that evolved in invertebrates as a mechanism involving the capture of microorganisms by macrophages (61, 62).

Nevertheless, discoveries about the C system over the past 10 years have indicated that it has many functions that are not directly related to host defense. In particular, tissue injury and inflammation initiate C activation and release of the chemotactic peptides C3a and C5a (63, 64). These peptides represent very early signals of tissue injury. The C is activated by tissue damage such as a burn or ischemia/reperfusion (I/R) injury (65, 66). The mechanisms responsible for C activation by damaged tissue are many-fold. Sometimes there is a loss of normal membrane regulatory proteins that prevent C activation by normal tissue (e.g., CD46 and CD55) leading to activation of the alternative pathway. With I/R injury, a neoantigen becomes exposed on hypoxia-damaged tissue that reacts with a natural IgM antibody activating the classical pathway of C (66). C activation releases C3a, which recruits mast cells (as well as basophils and eosinophils), and C5a, which recruits neutrophils, monocytes, and macrophages. Furthermore, C3b is deposited at the inflammatory site and quickly converted to iC3b, the major ligand for CR3 expressed by all granulocytes, macrophages, mast cells, and NK cells, as well as subsets of B cells and T cells. This iC3b is bound to the injured tissues via a covalent ester or amide bond and functions to tether CR3bearing cells to the site. With granulocytes and macrophages, CR3 attachment to iC3b deposited onto microorganisms stimulates phagocytosis and degranulation. Our recent research demonstrates that C is activated and bioactive peptides are released within the BM-environment both during pharmacological mobilization (e.g., after administration of G-CSF; ref. 26) and conditioning for hematopoietic transplantation by radio-chemotherapy (25). In this respect, we consider conditioning for transplantation by radio-chemotherapy as a model of I/R insult to the BM tissue. This concept is discussed in the following paragraphs.

39.2.3 C3 is Secreted by BM Stroma Cells and Activated/Cleaved During Marrow Injury In our recent work we have demonstrated that C is an important link between inflammation/tissue organ damage and hematopoiesis and C is activated/cleaved in BM after irradiation, exposure to cytostatics or G-CSF infusion, and that it increases the responsiveness of HSPC to an SDF-1 gradient (25, 26, 28). C3 is present in blood (1 mg/mL) that flows through the BM. Using a sensitive ELISA assay, it was also shown that human BM stromal fibroblasts secrete C3 (28). It is also well known that BM macrophages synthesize and secrete all C components. Thus, C3 is a physiological constituent of the BM environment (61, 62). As shown in Figure 39.2, two groups of C3 cleavage fragments are distinguished: fluid phase (C3a, des-ArgC3a) and cell- or extracellular matrix-bound (C3b, iC3b) fragments. C3a and C3b are the first cleavage products of C3 and each has a short half-life in plasma. C3a is processed by serum carboxypeptidase N (SCPN) to desArgC3a (long half-life), and C3b is cleaved into iC3b (long half-life) by factor I. Accordingly, while C3a activates the classical C3aR, des-ArgC3a selectively activates a

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still unknown receptor (Receptor X?). We noticed that HSPC express abundant amounts of C3aR and it has been reported that they also express CR3 in abundance (28, 41). Thus, since C3 is cleaved/activated to C3a and des-ArgC3a (liquid phase) and iC3b (solid phase) in BM during conditioning for transplantation or mobilization, they may directly interact with C3aR+ or CR3+ HSPC, which is important for modulating SDF-1 dependent trafficking of these cells.

39.2.4 C3aR Increases Incorporation of CXCR4 Into Membrane Lipid Rafts and This Increases Responsiveness of the CXCR4 Receptor to an SDF-1 Gradient We noticed that HSPC respond much better to SDF-1 gradient in particular to threshold levels of this chemokine, in the presence of C3 cleavage fragments (Figure 39.3; ref. 28). We termed this “priming effect” and became interested in identifying the molecular basis for this phenomenon. It has been recently demonstrated that CXCR4 requires association with membrane lipid rafts for proper signaling function (67). These membrane domains are rich in sphingolipids and cholesterol, which form a lateral assembly in a saturated glycerophospholipid environment. The raft domains are known to serve as moving platforms on the cell surface, and are more ordered and resistant to nonionic detergents than are other areas of cell membranes. These domains also act as good sites for crosstalk between various cellular proteins. Lipid rafts have been shown to be important for T-cell polarization and chemotaxis (68, 69). It has been recently reported that small GTPases such as Rac-1 and Rac-2, that are crucial for engraftment of hematopoietic cells after transplantation, are present in lipid rafts of migrating HSPC (70, 71). We hypothesized that the C3 cleavage fragments (C3a, C3a and iC3b) by activating their corresponding recepdesArg tors (C3aR, putative desArgC3a binding receptor X, and CR3,

respectively) on the surface of hematopoietic cells may facilitate the association of CXCR4 with lipid rafts resulting in better SDF-1 binding and signaling (Figure 39.4). In fact our previous work revealed that several small molecules (e.g., fibronectin and fibrinogen fragments, hylauronic acid) that are present in supernatants from leukopheresis products may modulate the responsiveness of HSPC to an SDF-1 gradient by enhancing, as we assumed, the incorporation of CXCR4 into membrane lipid rafts. Our recent research on human hematopoietic cells primed by fibrinogen and fibronectin confirmed that the sensitization/priming of cell chemotaxis to an SDF-1 gradient is dependent on the cholesterol content in the cell membrane and the incorporation of the SDF-1 binding receptor, CXCR4, and the small GTP-ase Rac-1 into membrane lipid rafts. This co-localization of CXCR4 and Rac-1 in lipid rafts facilitated GTP binding/activation of Rac-1 (6). Based on these observations, we asked if C3a and C3ades-Arg may also prime (increase) the responsiveness of HSPC to an SDF-1 gradient similarly as fibronectin and fibrinogen fragments by increasing the incorporation of CXCR4 into membrane lipid rafts. Thus, the hypothesis to be investigated was that the priming effect of C3a and C3ades-Arg was dependent on lipid raft formation. Accordingly, chemotaxis assays were performed with low doses of SDF-1, either alone or with a priming concentration of C3a or C3ades-Arg. To determine the contribution of lipid rafts, cells were pre-incubated for 30 min before chemotaxis in 2.5 mM hydroxypropyl-β-cyclodextrin to extract cholesterol from the membranes and to perturb lipid raft formation. These studies with hematopoietic cells suggested that the priming effect of C3a and C3ades-Arg on SDF-1 chemotaxis was blocked by this raft-disrupting agent. To provide more evidence that the priming effect of these small C3 fragments is dependent on lipid raft formation, confocallike and Western blot analysis were carried out to assess the CXCR4 incorporation into membrane lipid rafts in hematopoietic cells primed by C3a and C3ades-Arg. As expected, CXCR4 in hematopoietic cells stimulated by C3a and

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Figure 39.3. Priming effect: increases in HSPC responsiveness to an SDF-1 gradient in the presence of other molecules. (A) HSPC respond better to low/threshold doses of SDF-1 in the presence of some molecules such as C3 cleavage fragments (C3a, desArgC3a and iC3b). (B) In a chemotactic assay more cells transmigrate from the upper transwell chamber to the lower transwell chamber if before chemotaxis to SDF-1 they are primed, pre-incubated with C 3 cleavage fragments (C3a, desArgC3a and iC3b).

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Figure 39.4. The hypothetical role of C3 cleavage fragments (C3a and desArgC3a) and iC3b in sensitizing the responsiveness of HSPC to an SDF-1 gradient. C3 is activated in BM in response to marrow damage (e.g., after conditioning for transplantation by TBI or chemotherapy). Cleavage/activation of C3 in the BM microenvironment is initiated by a C3-convertase to generate C3a and C3b fragments. Subsequently, C3a is quickly cleaved by carboxypeptidases to C3a. Similarly, C3b is converted to iC3b by Factor I. Although desArg C3a and C3b have short half-lives, the C3a inactivation product, C3a and C3b-derived iC3b have long half-lives. While the seven desArg transmembrane-span G-protein coupled C3aR binds C3a, another non-identified receptor binds both C3a and desArgC3a (depicted as X receptor). Both C3b and iC3b interact with the integrin-type receptor CR3. We postulate that C3a, desArgC3a and iC3b fragments increase the responsiveness of HSPC to SDF-1 gradients by enhancing incorporation of CXCR4 into membrane lipid rafts and thus effecting its better association with downstream signaling proteins. Furthermore, because iC3b is deposited on BM stroma cells during C3 cleavage the interaction of iC3b with CR3 is also important for tethering of HSPC in the BM microenvironment.

C3ades-Arg became incorporated into cell membrane lipid rafts. Figure 39.4 depicts the role of C3 cleavage fragments in lipid raft formation (25). The type of C3 receptors involved in this process needs further clarification. Generally, it is widely accepted that while the seven transmembrane-spanning G-protein coupled C3aR binds anaplylatoxin C3a, its degradation product C3abinds to another unidentified receptor and the sevendesArg transmembrane span orphan receptor C5L2 was postulated to be a possible candidate for this latter interaction (25, 72). However, it is not clear at this point whether this receptor X would be another member of the seven transmembrane span receptor family, similar to C3aR or a different type of molecule. We can expect in the near future that this hypothetical receptor will be identified and cloned. Finally, we noticed that the solid phase C3 cleavage fragment, iC3b, which as discussed above, seems to play a role as a tethering molecule in BM and may also prime the responsiveness of HSPC to an SDF-1 gradient and increase the incorporation of CXCR4 into the membrane

lipid rafts. This supports the presence of a direct crosstalk between integrin type CR3 receptor and CXCR4. Thus, the C3 cleavage fragment-mediated CXCR4 incorporation into membrane lipid rafts is crucial for SDF-1-mediated hematopoietic cell motility as reflected by the formation of membrane ruffles and filopodia, as well as migration. These processes are regulated by small Ras-like GTPases (Rho, Rac, and Cdc42) and PI3K, which can reorganize actin. The actin cytoskeleton needs to be dynamic for cell shape change, alterations in cell contact, and cell motility. The priming of HSPC by C3 cleavage fragments to increase their responsiveness to SDF-1 as proposed by us, may provide a new strategy to improve the seeding efficiency/homing of HSPC. This could be particularly important in CB transplants, where the number of cells available for transplantation is usually limited.

39.2.5 CR3 Tethers Hematopoietic Progenitor Cells (HPC) to iC3b Deposits on Irradiated Stroma Human CD34+ and murine Sca-1+ HSPC express CR3 (41, 42), and conditioning of mice for HSPC transplant by irradiation results in the deposition of iC3b on cells within the marrow (27). On the other hand, treatment of mice with antiCR3 prior to G-CSF produces a significant enhancement in HPC mobilization, suggesting that CR3 may function to retain HSPC in the marrow and prevent their uncontrolled egress into PB. The ligand for CR3 that is blocked by anti-CR3 treatment is unknown, but a similar enhancement of HSPC mobilization was not induced by anti-ICAM-1, leaving iC3b and fibrinogen/fibrin as the other possible candidates among the known protein ligands for CR3 (53). Preliminary data point to iC3b as the ligand, as enhanced HSPC mobilization was also observed in C3-deficient mice that are unable to generate the putative iC3b ligand to retain the CR3-bearing HPC in the marrow (26). Furthermore, the enhanced HPC mobilization generated by anti-CR3 in wt mice could not be elicited in C3-deficient mice. These data, in combination with the evidence for iC3b deposition within the G-CSF-stimulated marrow, suggest that iC3b is the ligand that retains stimulated HSPC in the marrow via CR3. Indeed, we have recently shown in vitro that iC3b-coated BM stroma is able to tether HPC via CR3 (73). In addition, we also observed that iC3b has another potential important function in the engraftment of HSPC, namely, increasing the responsiveness of HSPC to an SDF-1 gradient (28).

39.2.6 The Need for New Strategies to Improve Mobilization, Homing, and Expansion of HSPC The number of transplanted HSPC and their seeding efficiency are important factors that decide the final clinical outcome following hematopoietic transplantation. The most common strategy to enhance engraftment is transplantation of higher numbers of HSPC. In the case when HSPC from mobilized PB are employed it is related to the mobilization efficiency.

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Unfortunately, it is difficult to achieve good mobilization in some of the patients who are termed poor mobilizers. Therefore, a novel strategy aimed to enhance mobilization by employing C3aR antagonist could be employed in the clinic to increase the number of mobilized HSPC in leukopheresis products (26). The number of HSPC available for transplantation may be also limited because of the source of stem cells employed for transplantation as seen in the case of cord blood (CB). The current strategies to increase the number of HSPC by in vitro expansion are also not efficient in expanding the most primitive HSC. Thus, optimization of stem cell mobilization and enhancing of homing (seeding) efficiency after transplantation are important goals that could significantly improve clinical outcomes during transplantation. In this respect, our recent studies demonstrating a novel role for C3 in hematopoiesis provided the basis for developing new strategies to optimize homing/engraftment of HSPC. The potential utilization of ex vivo HSC priming by C cleavage fragments before transplantation to improve their homing/engraftment is a relatively simple procedure that could be of significant clinical benefit. Furthermore, current strategies used to expand HSPC do not allow for the expansion of the most primitive cells. A possible explanation for these difficulties is the fact that expansion protocols do not use all of the physiological compounds that are present in hematopoietic niches and are crucial for self-renewal of HSPC (e.g., Wnt, Notch). Of note, the currently available expansion protocols also lack activated C3, which is an important component of the hematopoietic microenvironment during BM regeneration. Accordingly, C3 is missing in heat-inactivated sera used for cell culture and is not present in artificial sera. We hypothesize that the activation of C3 during expansion of BM fibroblasts may allow for better expansion of HSPC.

39.3

Conclusion

During inflammation or tissue/organ damage, HSPC are released from BM. This is important evidence for the existence of a close link between inflammation/tissue injury and stem cell trafficking. Our recent research demonstrated that C3 is synthesized in the BM, and that BM conditioned for transplantation by radio-chemotherapy or during G-CSF-mediated mobilization is a site of C activation releasing C3a/des-ArgC3a and depositing iC3b onto damaged stroma (25, 26). Thus, C activation provides in BM one of the earliest signals of tissue injury. Furthermore, our research has shown that HSPC express C3aR and CR3 and respond to both C3a/des-ArgC3a and iC3b–C3 cleavage fragments (28, 29). At the molecular level, C3a/ des-ArgC3a and iC3b stimulation of HSPC appears to prime the responsiveness of HSPC to SDF-1. In addition, HSPC bind through CR3 to the iC3b deposited onto BM stroma. Thus, stromal cell-bound iC3b is likely to be the tethering ligand for CR3+ HSPC. Based on this, it is hypothesized that

C3a/des-ArgC3a and iC3b play important roles in HSPC trafficking, their retention in BM and their homing to BM after hematopoietic transplantation.

Acknowledgments. This work was supported by an NIH grants R01 DK074720 to MZR.

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Chapter 38 A Tapestry of Immunotherapeutic Fusion Proteins: From Signal Conversion to Auto-stimulation Mark L. Tykocinski, Jui-Han Huang, Matthew C. Weber, and Michal Dranitzki-Elhalel

38.1

Introduction

Immunotherapeutic proteins entering the clinic have largely featured single primary functional units. Therapeutic antibodies (Abs) and solubilized receptors have dominated this early protein biologics pipeline, with blockade of immune cell surface and soluble molecules as their primary functional activity. Cytokines have also emerged as viable immunomodulators for therapeutic applications, and lead candidates have been drawn from a growing panoply of cytokines capable of modulating an ever more segmented immune cellular repertoire. Yet, this early stream of immunotherapeutic proteins provides only a glimpse of the rich tapestry of protein design possibilities. From single amino acid to functional mini-cassette substitution, there are a myriad of primary sequence engineering options that can be invoked for enhancing therapeutic efficacy and tailoring functional properties of natural proteins. Moreover, beyond the re-design of individual natural proteins, there is the further possibility of fusing more than one protein, in whole or in part, to combine existing functional properties, or even to sometimes create entirely new ones. Even if only a minute fraction of the proteome’s A × B two-protein fusion combinations prove to be functionally meaningful, this would translate into a substantial catalogue of multi-functional therapeutic agents. Multi-functional therapeutic fusion proteins have been designed with a variety of functional endpoints in mind. Perhaps the most common type of fusion has linked proteins of interest to an immunoglobulin Fc region, frequently that of IgG1 (1–4). Such Ig fusions have been generated mostly to prolong the systemic half-lives of the respective proteins, to facilitate their ex vivo enrichment, and in some instances to mediate Fc-associated effector activities. Another recurFrom: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

rent theme is protein chimerization directed towards driving multimerization, with the rich set of options encompassing constitutive dimerization (e.g., via fusion with leucine zippers [5]), drug-inducible dimerization (e.g., via fusion with FK506 binding protein, with dimerization driven by the cell-permeable ligand AP1510 [6]]), trimerization (e.g., via fusion with isoelucine zippers [7, 8]), tetramerization (e.g., via fusion with streptavidin or modified leucine zippers [9– 11]), and hexamerization (e.g., via fusion with the collagen domain of ACRP30 [12]). Protein chimerization has also been commonly directed toward payload delivery. In this instance, each fusion protein combines a targeting moiety (e.g., Ab, lipocalin, ligand derivative, receptor derivative, mimetic peptide, membrane-associating bacterial toxin) with an effector component (e.g., toxin, RNase, caspase, enzyme, ligand, antigen, T-cell epitope). Examples of such chimeric proteins in the immunotherapeutic realm include Ab·toxin/ caspase/cytokine/costimulator (8, 13–17), cytokine·toxin (13, 18), ligand·angiogenin RNase (19), mimetic peptide·cytokine (20), bacterial toxin·antigen (21), Ab·MHC-antigen (22), and solubilized receptor·antigen (23) fusions. Fusion proteins have been designed for yet other ends, such as cell-to-cell or cell-tostroma adherence (e.g., bi-specific Abs with appropriate bridging capacities [13]), signal blockade (e.g., soluble receptor derivatives [24]), viral entry blockade (25), dual cell signaling (e.g., coupled ligands [26, 27]), and signal potentiation (e.g., coupled ligand:receptor pair [28, 29]). Yet these various functional endpoints (protein stabilization, multimerization, payload delivery, cellular and stromal adherence, signal blockade, viral entry blockade, dual cell signaling, signal potentiation) represent a mere beginning for immunotherapeutic fusion protein design. Our laboratory and others have begun to envision new goals for protein chimerization. The repertoire now encompasses innovative ends such as molding the cell surface molecular repertoire, converting intercellular signals, and fostering cellular auto-signaling and signal reinforcement. Some newer fusion protein classes that enable such functions will now be discussed. 349

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350

38.2

Costimulator and Coinhibitor Paints

A number of years ago, we invoked the term protein painting to connote the exogenous incorporation of proteins onto cellular surfaces. Protein painting provides a means for changing the face of a cell and modifying its cross-talk with neighboring cells. Although gene transfer has been the dominant approach for effecting such cell surface engineering, painting of exogenous proteins offers distinct advantages over endogenously expressing them for some therapeutic and experimental applications (30). For example, protein transfer makes sense in settings where persistent expression may be problematic, fine control of expression levels is advantageous, multiple proteins are to be expressed in a titratable way, and cells are difficult to transfect. Thus, protein painting has emerged as an important ancillary tool within the cellular engineer’s molecular toolbox. Over the years, our laboratory has elaborated on the protein painting concept in the context of T-cell immunomodulation. Keying in on the antigen-presenting cell (APC):T cell interface, the aim has been to modify APC-to-T cell intercellular messaging, by altering the intercellular (trans) signal display on APC surfaces. Viewed from the APC side, primary options include introduction of either immunoregulatory proteins onto their surfaces, with the alternative goals of either potentiating protective T cells or disabling pathogenic ones. The pallete of protein paint options continues to enlarge as mechanistic studies provide insights into the protein players mediating APC:T cell dialogue. Furthermore, the heterogeneity of APC, both professional and nonprofessional, as well as the phenotypic and functional subset diversity of those T cells that they engage, will inform progressively more refined selection of protein paints that might enable selective teasing apart of distinct APC:T cell pairs. Dating back to the late 1980s, our laboratory has been innovating protein transfer strategies, with APC engineering in mind. Our early efforts along these lines have been reviewed elsewhere (30–32). As a starting point, we capitalized on the unique membrane reincorporability of native glycophosphatidyl-inositol (GPI) proteins (33–36). This special property stems from the fact that such proteins, unlike regular transmembrane proteins, can form pseudo-micelles in solution, allowing for depletion of membrane solubilization detergents and add-back of detergent-depleted GPI protein complexes to cells. When added back to cells in this way, the GPI proteins spontaneously reincorporate into cell membranes. We and others cDNA cloned a paradigmatic GPI protein, human decay-accelerating factor (DAF; refs. 37 and 38), identified a GPI modification signal sequence at the carboxyl terminus of DAF, and then proceeded to demonstrate that any protein can be produced as an artificial GPI protein derivative by appending the DAF GPI modification signal sequence to that protein’s carboxyl terminus (39, 40). Once purified, such artificial GPI proteins, like native GPI proteins, are membrane reincorporable, and thus constitute GPI protein paints.

One limitation of GPI protein paints is that they are by definition produced as membrane proteins that must then be purified from membrane extracts. This necessarily translates into lower yields and cumbersome purification procedures, as compared to recombinant soluble proteins. To address this limitation of GPI protein paints, we subsequently developed an alternative protein painting strategy that circumvents these limitations. Taking advantage of the ease of producing soluble Fcγ1 fusion protein derivatives and the large number of them reported in the literature, we devised a protein transfer procedure for coating Fcγ1 fusion proteins onto cell surfaces (41). According to this procedure, membrane incorporable palmitated protein A (pal-protA), generated by chemical palmitoylation of protein A, serves as an anchor for Fcγ1 fusion proteins at cell surfaces, in a sense constituting a molecular velcro. The pal-protA and Fcγ1 fusion protein can be either applied to cells consecutively, or pre-mixed and then applied as a protein conjugate paint. The fusion protein paints highlighted so far (artificial GPI proteins, pal-protA:Fcγ1 conjugates) are but a subset of the protein transfer options available. Some represent variations on the pal-protA:Fcγ1 conjugate theme. For example, the novel chelator lipid, NTA-DTDA, can be invoked as a membraneincorporating anchor, with its NTA groups serving to secondarily trap his6-tagged costimulators (42). Another approach is to chemically modify cell membranes in a way that confers to them small molecule anchors that can latch onto appropriately modified proteins (e.g., cell surface biotinylation for binding of avidin-conjugated proteins [43, 44]). This latter approach entails generalized chemical perturbation of the cell surface, and this can be bypassed through the combined use of surfacetargeted Ab·avidin fusion proteins and biotinylated proteins of interest (45). The functionality of GPI and pal-protA:Fcγ1 protein paints has been validated by now for a diverse set of proteins and in a number immunologically-relevant functional settings. Efficient T-cell activation relies on coordinate trans signaling along two parallel molecular axes: the major histocompatibility complex (MHC)/antigen-to-T cell receptor (TCR) axis (signal #1) and the costimulatorto-costimulator receptor axis (signal #2). We and others have applied protein transfer to both of these molecular axes. Thus, GPI derivatives of MHC class I (46, 47), MHClike, lipid antigen-presenting CD1 (48), and costimulators (49– 51) exhibit the intercellular T-cell stimulatory functions of their native (non-GPI-modified) counterparts. Similarly, costimulator·Fcγ1:pal-protA complexes painted onto APC surfaces costimulate just as expected (41). These proof-ofprinciple ex vivo studies additionally served to showcase the power of painting-enabled quantitative ligand transfer, as one exploits the capacity to finely titrate surface expression of MHC or costimulator paints. One dividend has been mechanistic discovery. Costimulator painting was invoked to establish the existence of costimulator receptor activation thresholds, paralleling previously documented TCR activa-

38. Immunotherapeutic Fusion Proteins

tion thresholds (41). Another dividend has been in vivo applicability and therapeutic spin-offs. This has included the demonstration that intra-tumoral tetra-costimulator protein transfer is feasible, and indeed can elicit effective systemic anti-tumor immunity (52). Some time ago, we introduced the term coinhibitor to denote a protein that, when expressed on an APC surface, can send an inhibitory trans signal to T cells, in opposition to APC-resident costimulators (53). APC with enforced expression of coinhibitors were designated as artificial veto cells (AVC), because they mimicked natural veto cells in their capacity to inhibit responding T cells in an antigen-specific fashion. Natural veto cells had been described for some time in the literature (54, 55), and so the new twist was that artificial ones could be engineered by introducing defined exogenous protein components onto APC surfaces. In a sense, the AVC is a cellular Trojan horse, masquerading as an APC but conveying to the target T-cell molecular signals that trigger its destruction. Significantly, from the perspective of the present discussion, we showed that AVC can be engineered not just by gene transfer, but also by protein transfer. The first coinhibitor paint was GPI-modified CD8 (56, 57). When CD8 is artificially anchored on APC surfaces, it is capable of sending inhibitory signals to responding T cells through its cognate receptor on these cells, MHC class I, which can mediate inhibitory back-signaling on T cells (58, 59). A subsequent study showed that CD8 delivered to APC surfaces by other means, for example, via an Ab·CD8 fusion protein, could similarly generate AVC, validating our original observation (60). Over the subsequent years, AVC have surfaced under different names (61, 62) and with alternative coinhibitors (e.g., Fas ligand [63] and TRAIL [64]). A central message is the plasticity of APC, their amenability to engineering by both gene and protein transfer, and their convertibility to inhibitory AVC. Armed with MHC, costimulator and coinhibitor paints, there are numerous APC and AVC engineering options. To deploy them optimally for therapeutic ends, much will still have to be learned about the interplay between costimulators and coinhibitors (65), and how specific T-cell subsets might be best targeted. One pivotal question will be: what is the optimal surface display for an APC or AVC for targeting particular types of lymphoid responders? In the case of AVC, one might frame the challenge as being how to decipher the codes that control entry through the T-cell apoptotic door. A confounding factor in such analyses is the possibility that inter-species differences in molecular function will emerge, exemplified by the report of a signaling molecule that functions alternatively as a costimulator versus a coinhibitor in a species-dependent fashion (66). While protein painting emerged in these ways as a robust concept, it is restricted to situations amenable to ex vivo or local in vivo delivery. This limitation beckoned the development of in vivo paints that could be administered systemically, home to cells in situ, and in this way coat them with desirable signaling molecules. This pointed to new classes of therapeutic fusion proteins.

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38.3 Trans Signal Converter Proteins (TSCP) As one kind of in vivo paint, we explored fusion proteins that can deliver trans signaling components to cells of interest. The cell-targeting moiety within such fusion proteins can be any one of a number of molecular recognition units, and soluble receptors emerged as an attractive first choice. CTLA-4·Ig, an early immunotherapeutic fusion protein to enter the clinic (24, 67), has as its primary functional unit a solubilized receptor unit capable of binding to the B7 costimulator and blocking its ability to activate the CD28 costimulator receptor on T cells (68, 69). We therefore elected to work with CTLA-4 as a targeting (and costimulator blocking) moiety. Of note, while CTLA4·Ig can block many T-dependent responses in vivo, this agent, on its own, cannot reproducibly establish a permanent state of peripheral tolerance (70). This makes it interesting to explore CTLA-4 fusion protein derivatives that might be more efficacious and lead to actual deletion of targeted T cells. The CTLA-4 derivative we generated was CTLA-4·FasL, designating it as a TSCP (71). Our concept of trans signal conversion evolved from the seminal idea of combining costimulator blockade and coinhibition within a single fusion protein. The choice of FasL(CD95L) as the coinhibitor component was prompted by data suggesting that blocking B7 signaling potentiates FasL-driven inhibition (65, 72). We posited that a CTLA-4·FasL fusion protein would be able to: (1) passively block B7 costimulation (via CTLA-4); (2) actively send an inhibitory trans signal (via FasL); and (3) home to (paint) B7+ target cells in vivo (via CTLA-4), generating deletional APC (i.e., AVC) in situ. Since CTLA-4·FasL is in effect converting an activating trans signal into an inhibitory one, it is mediating trans signal conversion. Our data substantiated this mode of action for CTLA-4·FasL. We demonstrated that this costimulator receptor·coinhibitor fusion protein, once anchored on B7+ cells, effectively induces apoptosis of Fas-sensitive Jurkat T cells and potently inhibits the proliferation of human peripheral T cells (up to 1,000fold higher than CTLA-4·Ig), not just in response to polyclonal activators, but also to alloantigen (73, 74). We built upon these ex vivo findings by showing that CTLA-4·FasL inhibits alloantigen-specific responses in vivo (74), and this in vivo efficacy was supported by others who demonstrated that CTLA-4·FasL expressed via an adenoviral vector prevents the development of autoimmune diabetes (75) and prolongs cardiac allograft survival (76). The trans signal conversion concept is robust, and we are actively designing additional immunoinhibitory costimulator receptor·coinhibitor TSCP that incorporate alternative costimulator receptor and coinhibitor components. In principle, this should enable more refined targeting of different APC:T cell combinations, taking into account the heterogeneity of APC and T-cell subsets and their differing contributions to pathogenesis in various autoimmune and alloimmune diseases. Costimulators that might be targeted in this way include 4-1BB ligand, OX40

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ligand, GITR ligand, and various others, which are directed towards T cells of different subsets and activation states. Moreover, there is also the prospect of reversing the functional goal and designing TSCP that would activate, rather than inhibit, T cells. In this case, one might combine a coinhibitor receptor with a costimulator within a TSCP fusion protein. In configuring TSCP, Ab components, such as single chain Fv (scFv), might be substituted for solubilized receptors as more flexible signal blocking elements. The feasibility of our original suggestion along these lines (30, 31) has been supported by a spectrum of scFv·costimulator, Ab·cytokine, and Ab·chemokine fusion proteins that have emerged (16, 17, 77–80). Looking forward, by turning to alternative Ab or scFv components that not only bind to, but also block their target trans signaling protein, such fusions would by definition acquire trans signal converter status.

38.4

Cis Loop-Back Proteins (CLAP)

Having established the functionality of TSCP, the notion emerged that a given TSCP might have additional functional capabilities. For instance, while TSCP are designed to alter intercellular signaling, blocking a ligand on one cell and triggering an alternative receptor on a second cell, what happens when the two are also present on the same cell? This consideration prompted a consideration of the possibility of auto-stimulation between ligand and receptor when side-by-side on a given cell. For instance, in the case of CTLA-4·FasL, there is the theoretical potential for auto-triggering in contexts where B7 costimulators are neoexpressed on Fas receptor-positive cells, a situation which occurs in a number of autoimmune and infectious disease settings (81, 82). We designed a unique fusion protein, CD40·FasL, to probe this concept more directly (83, 84). Chimeric CD40·FasL incorporates the combined capacities to bind to two surface receptors on activated T cells, CD40 ligand (CD40L; CD154) and Fas receptor (CD95). CD40·FasL, once tethered to the cell surface via one of its ends should be able to transmit a signal via its other end. Hence, in principle, simultaneous triggering from both ends is possible, with the intriguing potential for auto-inhibition if such dual triggering occurs on the same cell itself. The choice of CD40: CD40L and FasL:Fas signaling axes to be co-targeted was prompted by the fact that CD40L and Fas are both upregulated on T cells post-activation, providing a way of targeting activated T cells selectively in the context of certain disease settings, such as autoimmune flares. Furthermore, there is data suggesting that back-signaling through CD40L reinforces inhibitory Fas signaling, and promotes apoptosis of activated T cells (85). Thus, we posited that a chimeric CD40·FasL would combine a number of interesting properties that serve to reinforce each other: (1) By engaging surface CD40L, CD40·FasL

anchors FasL to the cell membrane, thereby potentiating its pro-apoptotic activity—in this respect, it parallels other fusion proteins that have been devised for appending FasL and other TNF family proteins to membranes (8, 13, 44, 86– 88; 2) CD40·FasL drives CD40L back-signaling, rendering T cells more sensitive to activation-induced and Fas-mediated apoptosis; (3) CD40·FasL blocks CD40:CD40L-mediated costimulation, with the potential to additionally function as a TSCP, in for instance converting T cell-to-B cell intercellular signals. Several lines of evidence supported auto-inhibition as the operative mechanism for CD40·FasL (84): (1) CD40·FasL is cytotoxic to Fas receptor-positive cell lines of different cell lineages; (2) CD40·FasL’s function is potentiated when CD40L is expressed on target cells; (3) intercellular contact is not a prerequisite for CD40·FasL inhibition, with neither soft agar suspension nor cell dilution impeding this fusion protein’s inhibitory activity; and (4) introduction of exogenous CD40 into the system interferes with CD40·FasL inhibition. Taken together, these data are consistent with a loop-back inhibitory mechanism within individual activated (CDHOL+ and Fas+) T cells. Significantly this type of fusion protein provides a unique way to confine immunoinhibition to activated T cells, and the loop-back mechanism for this particular CLBP invokes propagation of two intracellular signaling cascades in parallel. There is also the possibility that CD40·FasL could be used for selective killing of cancer cells abnormally expressing both CD40L and Fas receptor (89, 90). The concept of T-cell suicide was suggested some time ago, prompted by the potential for FasL:Fas receptor signaling on individual activated T cells suspended in soft agar or serially diluted (91). CD40·FasL represents a novel fusion protein with the capacity to artificially elicit this T-cell suicide phenomenon. Cis auto-stimulation between neighboring membrane-anchored molecules may be a more general phenomenon, based upon findings from our laboratory (discovery of auto-costimulation, that occurs when a costimulator and its cognate receptor are arrayed side-by-side on the same membrane [92]) and others (suggestion that NK receptors can interact with neighboring MHC class I molecules [93]). For that matter, in any situation where membrane-anchored ligand and receptor are co-expressed on the same cell type, and paracrine signaling between cells of that type has been inferred, the further possibility of loop-back signaling might also be entertained. Clearly, fusion proteins could be designed that build upon this auto-stimulation theme in many different directions. One can envision other CLBP beyond CD40·FasL. For example, inhibitory CLBP might be designed that target other activation-induced surface molecules that are specific to other T-cell activation stages or are associated with specific pathological states. Alternatively, CLBP might be configured to activate, rather than inhibit T cells or other immune cells. These and other spin-offs from the loop-back signaling concept can now be explored.

38. Immunotherapeutic Fusion Proteins

38.5

Mining the Fusion Protein Concept

The potential to draw upon the proteome to mix and match protein elements into composite proteins in a highly flexible way creates an almost staggering set of new fusion protein design opportunities. The protein fusion concept is but in its infancy, in terms of both the protein components that can be fused and in the way such proteins can be combined and deployed in biological settings. For each of the functional fusion protein classes set forth above (protein paints, TSCP, and CLBP), numerous combinations of protein building blocks are possible that go well beyond the paradigmatic fusion protein showcased for that class. Thus, one could potentially develop mini-libraries of fusion proteins for each class. Moreover, additional fusion protein classes can be conceived, as one thinks imaginatively of the spectrum of functional possibilities. The functional richness of the tapestry of fusion proteins is likely to go even further. Even a relatively straightforward two-component fusion protein can manifest functional abilities that go beyond the primary ascribed functions of its component elements, and thus be more than the sum of the parts. New functions elicited by chimerization could stem from a variety of factors, such as unique topologies and associations elicited at cell surfaces through molecular bridging. While fusion proteins are designed to function individually, there is the possibility of deploying them in creative combinations. For example, in the case of TSCP, which are each designed to convert a message conveyed between two interacting cells, one might envision combining TSCP in ways that alter or even generate new cellular webs, that bring together and modulate multiple cell types to achieve a given functional endpoint. TSCP could also be administered in conjunction with CLBP in ways that allow them to functionally reinforce each other, by coordinately changing intercellular linkages (via TSCP) and modifying participating cellular elements within cellular networks (via CLBP). The notion of cellular networks, with cytokines (94) and immunosuppressive factors (95) as connectors, is now part of immunological discourse. There is the intriguing possibility of leveraging membranefocused fusion proteins, such as TSCP and CLBP to build or elaborate upon such networks. The focus here has been on fusion proteins that are soluble or can be suspended in solution and added back to cells. Of course, there is a growing repertoire of engineered transmembrane fusion proteins, expressed via gene transfer. This encompasses fusions that incorporate components from MHC (96–100), costimulator (101), and TCR (102, 103) proteins in varying ways. The implications of such designer membrane proteins for therapeutic cell engineering are considerable (104). While the interface between APC and effector T cells has so far constituted the primary target for immunotherapeutic fusion proteins, it is tempting to look to other immune cell targets and intercellular interfaces. Among the myriad possibilities are not only other members of the T-cell phenotypic and

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functional continuum, but also non-T cells, both lymphoid and non-lymphoid alike. Thus, while the focus has been on effector T cells, the possibilities go much further, not only to other immune cells, but beyond the immune system.

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356 78. Schrama D, Straten P, Fischer WH, McLellan AD, Brocker E-B, Reisfeld RA, Becker JC (2001) Targeting of lymphotoxin-alpha to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue. Immunity 14:111–121. 79. Sabzevari H, Gillies SD, Mueller BM, Pancook JD, Reisfeld RA (1994) A recombinant antibody-IL2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice. Proc Natl Acad Sci USA 91:9626–9630. 80. Xu X, Clarke P, Szalai G, Shively JE, Williams LE, Shyr Y, Shi E, Primus FJ (2000) Targeting and therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibodyinterleukin 2 fusion protein. Cancer Res 60:4475–4484. 81. Azuma M, Yssel H, Phillips JH, Spits H, Lanier LL (1993) Functional expression of B7/BB1 on activated T lymphocytes. J Exp Med 177:845–850. 82. Podojil JR, Kohm AP, Miller SD (2006) CD4+ T cell expressed CD80 regulates central nervous system effector function and survival during experimental autoimmune encephalomyelitis. J Immunol 177:2948–2958. 83. Dranitzki-Elhalel M, Huang J-H, Sasson M, Rachmilewitz J, Parnas M, Tykocinski ML (2005) CD40·FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism. J Am Soc Nephrol 16:232A. 84. Dranitzki-Elhalel M, Huang J, Sasson M, Rachmilewitz J, Parnas M, Tykocinski M (2007) CD40·FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism. Int Immunol 19:355–363. 85. Blair PJ, Riley JL, Harlan DM, Abe R, Tadaki DK, Hoffmann SC, White L, Francomano T, Perfetto SJ, Kirk AD, June CH (2000) CD40 ligand (CD154) triggers a short-term CD4+ T cell activation response that results in secretion of immunomodulatory cytokines and apoptosis. J Exp Med 191:651–660. 86. Samel D, Muller D, Gerspach J, Assohou-Luty C, Sass G, Tiegs G, Pfizenmaier K, Wajant H (2003) Generation of a FasLbased proapoptotic fusion protein devoid of systemic toxicity due to cell-surface antigen-restricted activation. J Biol Chem 278:32,077–32,082. 87. Gerspach J, Muller D, Munkel S, Selchow O, Nemeth J, Noack M, Petrul H, Menrad A, Wajant H, Pfizenmaier K (2006) Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug. Cell Death Differ 13:273–84. 88. Assohou-Luty C, Gerspach J, Siegmund D, Muller N, Huard B, Tiegs G, Pfizenmaier K, Wajant H (2006) A CD40-CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95. J Mol Med 84:785–797. 89. Storz M, Zepter K, Kamarashev J, Drummer R, Burg G, Haffner AC (2001) Coexpression of CD40 and CD40 ligand in cutaneous T-cell lymphoma (mycosis fungoides). Cancer Res 61:452–454. 90. Pham LV, Tamayo AT, Yoshimura LC, Lo P, Terry N, Reid PS, Ford RJ (2002) A CD40 Signalosome anchored in lipid rafts leads to constitutive activation of NF-kappaB and autonomous cell growth in B cell lymphomas. Immunity 16:37–50.

M. L. Tykocinski et al. 91. Dhein J, Walczak H, Baumler C, Debatin K-M, Krammer PH (1995) Autocrine T-cell suicide mediated by APO-1 (Fas/ CD95). Nature 373:438–441. 92. Zheng G, Chen A, Weber M, Tykocinski M (2003) T cells painted with B7-1 and 4-1BB ligand auto-costimulate themselves. FASEB Exp Biol Abstr 414.3. 93. Scarpellino L, Oeschger F, Guillaume P, Coudert JD, Levy F, Leclercq G, Held W (2007) Interactions of Ly49 family receptors with MHC class I ligands in trans and cis. J Immunol 178:1277–1284. 94. Harris JE, Nuttall RK, Elkington PT, Green JA, Horncastle DE, Graeber MB, Edwards DR, Friedland JS (2007) Monocyteastrocyte networks regulate matrix metalloproteinase gene expression and secretion in central nervous system tuberculosis in vitro and in vivo. J Immunol 178:1199–1207. 95. Kim R, Emi M, Tanabe K, Arhiro K (2006) Tumor-driven evolution of immunosuppressive networks during malignant progression. Cancer Res 66:5527–5536. 96. Margalit A, Sheikhet HM, Carmi Y, Berko D, Tzehoval E, Eisenbach L, Gross G (2006) Induction of antitumor immunity by CTL epitopes genetically linked to membrane-anchored beta2-microglobulin. J Immunol 176:217–224. 97. Lee L, McHugh L, Ribaudo RK, Kozlowski S, Margulies DH, Mage MG (1994) Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active beta 2-microglobulin domain. Eur J Immunol 24:2633–2639. 98. McCluskey J, Germain RN, Margulies DH (1985) Cell surface expression of an in vitro recombinant class II/class I major histocompatibility complex gene product. Cell 40:247–257. 99. Burrows GG, Chang JW, Bachinger H-P, Bourdette DN, Offner H, Vandenbark AA (1999) Design, engineering and production of functional single-chain T cell receptor ligands. Protein Eng 12:771–778. 100. Uger RA, Chan SM, Barber BH (1999) Covalent linkage to beta2-microglobulin enhances the MHC stability and antigenicity of suboptimal CTL epitopes. J Immunol 162:6024–6028. 101. Kowolik CM, Top MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, Smith DD, Forman SJ, Jensen MC, Cooper LJN (2006) CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res 66:10,995–11,004. 102. Zhang T, Barber A, Sentman CL (2006) Generation of antitumor responses by genetic modification of primary human T cells with a chimeric NKG2D receptor. Cancer Res 66:5927–5933. 103. Pinthus JH, Waks T, Kaufman-Francis K, Schindler DG, Harmelin A, Kanety H, Ramon J, Eshhar Z (2003) Immunogene therapy of established prostate tumors using chimeric receptor-redirected human lymphocytes. Cancer Res 63:2470–2476. 104. Kershaw MH, Teng MWL, Smyth MJ, Darcy PK (2005) Supernatural T cells: genetic modification of T cells for cancer therapy. Nat Rev 5:928–940.

Chapter 41 B-cell Dysfunctions in Autoimmune Diseases Moncef Zouali

41.1 B Lymphocytes Play Multiple Roles in the Autoimmune Pathologic Process The role of B cells in the pathogenesis of autoimmune disease has been amply documented in experimental models. In lupusprone MRL-lpr mice, for example, elimination of B cells resulted in a complete abrogation of glomerulonephritis, vasculitis, and skin disease (1). Reversibly, B cells of genetically manipulated lupus-prone mice that have no αβ+, and no CD4+, T cells were still able to mediate efferent tissue damage by secreting autoantibodies. In the (NZBxNZW) F1 lupus-prone mouse model, pre-B cells obtained from the embryonic liver and transferred to SCID mice secreted anti-double-stranded (ds)DNA autoantibodies and produced a lupus-like disease (2). Similarly, administration of a human anti-dsDNA autoantibody to SCID mice caused hyaline thrombi and proteinuria (3). Another striking example of autoantibody effector activity has been described in a mouse strain transgenic for a Tcell receptor (TcR) that recognizes a ubiquitously expressed self-antigen (4). In this system, mice develop an arthritis that is driven almost entirely by immunoglobulins (Igs). The target of both the initiating T cells and the pathogenic Igs was identified as glucose 6-phosphate isomerase, a glycolytic enzyme. Initially, characterization of the genes encoding human and murine autoantibodies led to the conclusion that the corresponding B cells were produced through an antigen-dependent, T-cell-mediated process (5–7). However, B cells also can contribute in a T-cell-independent manner to autoimmune disease expression, at least in certain experimental models. While they may mediate efferent tissue damage by secreting autoantibodies, their role in autoimmunity is beyond Ig production. A striking observation comes from studies of the MRL-lpr lupus-prone model. Mice with B cells that cannot secrete Abs From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

still developed nephritis and vasculitis (1). This finding indicates that, independent of autoAb, B cells are essential for disease expression. As other studies have indicated, they can play several different roles in the autoimmune pathologic process (8). First, by acting as highly efficient APC, B cells can support the activation and autoreactivity of T cells. In rheumatoid arthritis (RA), for example, the T-cell activation is depending on B cells, and other antigen-presenting cells (APCs) cannot substitute for the maintenance of T-cell activation (9). Second, activated B cells can be the source of cytokines and membrane-associated molecules that provide cognate help and support the activities of autoreactive T cells. In the BXSB lupus mouse, for example, the autoimmune disease is much worse in males than in females due to a gene called Yaa, for Y chromosome autoimmune accelerator. B cells from male BXSB mice are more responsive to CD40 signaling than B cells from female mice (10). In addition, unlike female mice, male BXSB mice have a large proportion of B cells expressing functionally active CD154, a CD40 ligand. Third, B cells appear to regulate the development of T cells. This conclusion stems from the description of two subsets of effector B cells, Be1 and Be2, secreting cytokines in a similar way to Th1 and Th2 cells (11). The cytokines produced by Be1 and Be2 cells contribute to the in vitro differentiation of naive T cells into a Th1 or Th2 type. In a model of arthritis, B cells producing IL-1α arise after stimulation of arthritogenic splenocytes with type II collagen and agonistic anti-CD40 mAb (12). Transfer of these IL-10-secreting B cells to syngeneic mice controls the pathogenic Th1 response, inhibits the onset of collageninduced arthritis (CIA), and treats ongoing disease. The role of IL-10-producing B cells in regulating Th2-driven chronic intestinal inflammation also supports a regulatory function for B cells in T-cell-dependent chronic inflammatory disorders (13). Thus, B cells may have a strong impact in conditioning T-cell differentiation during an autoimmune process. Finally, further support for the important role of B cells in autoimmunity comes from in vivo studies demonstrating that autoantigen-specific B cells, when present in the repertoire, are 377

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the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells (14). Studies of human autoimmune diseases also provide ample evidence for the important role of B cells in autoimmune diseases (15–17). Even autoimmune disorders initially thought to involve T cells also require B-cell contributions. Thus, multiple sclerosis (MS) is a chronic, demyelinating inflammatory disease of the central nervous system (CNS). Its etiology is still unknown, but it is generally assumed that autoreactive T cells homing into the CNS play a major role in disease pathogenesis. However, in addition to T lymphocytes that are dominantly detected in the cerebrospinal fluid (CSF) during inflammation of the CNS, B cells may account for up to 25% of the CSF-infiltrating leukocytes, and increased numbers of CSF B cells have been associated with a more rapidly progressing clinical course in MS patients (18). Whereas the antigenic specificity of the B cell repertoire inside the CNS is unknown, molecular analysis has shown the accumulation of cells carrying mutated Ig variable (V) genes in the CSF (19–22). These findings support the idea that a germinal center (GC)-like reaction may take place during the autoimmune attack against CNS structures. This concept is supported by the existence of a large number of clonally related V sequences from the CSF of individual patients, indicating that different B cell clones have originated from the diversification of a single or a few progenitor B-cell clones (19). Recently, it was shown that each B-cell subset, which takes part in the GC reaction, can be detected in the CSF of MS patients (23). The clearest evidence that B cells recapitulate a GC reaction inside the CNS arises from the detection of CD38high, CD77+, and bcl2– centroblasts. Indeed, centroblasts are found exclusively inside the secondary lymphoid organs, but not in the peripheral blood. Another remarkable feature of CSF B cells is the striking increase of CD27+ IgD– memory B cells compared to the peripheral blood (23, 24). Memory B cells detected inside the CSF are activated, as suggested by the upregulation of costimulatory molecules on their surface and the capacity to produce pro-inflammatory cytokines, such as IFN-γ. Moreover, memory B cells express very high levels of the chemokine receptors CCR1, CCR2, and CCR4 in comparison with paired peripheral blood memory B cells. These receptors are likely to be involved in the process of B-cell homing and/or retention inside the inflamed CNS. In several autoimmune diseases, formation of ectopic lymphoid follicles has been demonstrated in the target organs and is thought to sustain local T-cell priming and B-cell maturation. In the case of muscular dystrophy, there is an increased proportion of CD138+ and CD19– plasma cells in the CSF as compared to individuals with other inflammatory neurological diseases (23). However, there is a lack of Ig gene sequence overlap between clonally expanded B cells and plasma cells detected simultaneously in the CSF from individual MS patients (25). A similar discrepancy has been observed in

M. Zouali

synovial fluid B cells and plasma cells from RA patients. Remarkably, ectopic follicles containing follicular dendritic cells, proliferating B cells, and plasma cells are present in the inflamed meninges of a subset of patients with secondary progressive MS, suggesting that ectopic lymphoid structures may provide the appropriate microenvironment for B-cell differentiation in the CNS (26). It is likely to be driven by cytokines and chemokines regulating T-cell and B-cell homing, and positioning in secondary lymphoid organs. Among these, lymphotoxin-α, CXCL12, and CXCL13 have been detected in the CNS of MS individuals. Expression of BAFF, a cytokine involved in B cell survival and proliferation, has also been demonstrated in MS lesions (27).

41.2 Multiple Mechanisms Contribute to B-cell Tolerance to Self To provide the host with an arsenal of specific antibodies capable of neutralizing foreign antigens and mobilizing various defense mechanisms, B lymphocytes must develop receptors that can interact with exogenous antigens and signal cell activation. To prevent undesirable autoimmune reactions, the production of antigen-specific B cells must be simultaneously balanced by negative regulation of cells that express B-cell antigen receptors (BcRs) specific for endogenous components. This unique behavior is the result of several checkpoints that act in a stepwise and orderly fashion throughout the B-cell development. In the early ontogeny, antigen receptor genes are randomly recombined and expressed on the surface of the B cell. Generation of the initial repertoire in pre-B cells by VDJ recombination is an antigenindependent process, and B cells expressing Igs specific for both foreign and self-antigens are generated. Those lymphocytes that complete heavy (H)- and light (L)-chain assembly and H/L chain pairing express a fully mature BcRs on their surface and enter an antigen-regulated phase (28). Studies of B-cell responses in vitro revealed that, while the intrinsic response of immature-stage B cells to strong BcRinduced signals is apoptotic cell death, mature B cells are able to progress through the cell cycle and begin clonal expansion. More specifically, the type of BcR clusters that form on the cell surface of stimulated immature cells determines whether the cells become cell cycle arrested and unresponsive, or deleted by apoptosis. Relatively small clusters induced with soluble antigen, mAbs, or low concentrations of polyclonal Abs appear to induce unresponsiveness. Large receptor clusters induced by multivalent, membrane-bound Ag, hypercross-linked mAbs or high concentrations of polyclonal Abs appear to induce apoptosis (29). Importantly, the ultimate fate of B-cell interaction with Ag is dependent upon the balance between intrinsic and extrinsic signals, including the microenvironment at which Ag is first encountered. For example, signals generated through CD40 on immature B cells are capable of blocking the BcR-induced apoptosis and redirecting the signal toward proliferative responses (30).

41. B Lymphocytes and Autoimmunity

Evidence for several tolerance-inducing mechanisms used to tolerize self-reactive B cells has come from in vivo studies using mice transgenic for IgH- and L-chain genes specific for either a self-antigen or a transgenic self-antigen (31–33). In the presence of self-antigen expressed on the cell surface, deletion of autoreactive B cells appears to occur at, or shortly after, the pre-B to immature B-cell transition. However, if the antigen is soluble, autoreactive B cells survive, but are unresponsive to subsequent antigen stimulation, a state termed anergy. Further studies in transgenic mice have clearly shown that clonal anergy is not the only way to extinguish selfreactivity among immature B lymphocytes to strong signals through their BcRs. In certain circumstances, immature B cells are not eliminated by self-Ag, but instead down-regulate their receptors and undergo a second round of Ig L-chain rearrangements. This process, called receptor editing, provides an opportunity for the immature B cell to edit its self-reactive BcR and to exit into the periphery (34–36). Thus, potentially autoreactive B cells are tolerized in the BM before they give rise to immunoreactive mature B cells. Once precursors develop into mature, naive B cells, they emigrate to secondary lymphoid organs, where they circulate awaiting activation by antigen. Engagement of the BcRs on mature B cells leads to positive growth responses, including cell activation and proliferation. The BcR also appears to play a role in mature B cells before they encounter antigen by providing a general survival signal. Its continued presence is required for the maintenance of long-lived mature B cells, even in the absence of cognate Ag (37). Importantly, inasmuch as B cells continue to encounter new self-Ags as they exit into the periphery, additional tolerance mechanisms must be activated to avoid the emergence of undesirable autoreactivity (38).

41.3

Regulation of B-cell Survival

Following migration to peripheral lymphoid organs, the selection and survival of B cells is controlled by a variety of signals, including co-stimulatory factors, cytokines, chemokines, and non-hematopoietic elements, and the longevity factors APRIL (a proliferation-inducing ligand) and BAFF (B-cell activating factor). Also called TALL-1, THANK, BlyS or zTNF4, BAFF is a member of the TNF super-family that includes TNF-α, FasL, CD40L and CD30L (39). It is secreted by monocytes, macrophages and DCs, and the amount produced is increased by CD40L or IL-10. It binds primarily to B cells, but also to activated T cells. Together with the longevity factor APRIL, BAFF influences B-cell maturation and survival at several levels. B cells express three receptors for BAFF: BAFF-R, transmembrane activator and calcium-modulator and cyclophilin ligand [CAML] interactor (TACI), and B-cell maturation antigen (BCMA). These receptors belong to the TNFR family, but, in contrast to most other members of the family, they do not have an intracellular death domain. While expression of BCMA is restricted to CD19+ B cells, TACI and

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BAFF-R are also detectable on T cells (40). In marked contrast to BAFF-R, which appears to be selectively expressed on B cells and is highly specific for BAFF, TACI and BCMA also bind APRIL. In vitro, BAFF is a weak stimulant and a potent co-stimulant of B-cell growth (41). Like CD40L, soluble BAFF stimulates proliferation of human B cells in synergy with other B cell activators, augments Ig production, and upregulates expression of cell-surface molecules involved in B-cell effector functions (40). When coupled with BcR ligation, BAFF allows survival and differentiation of transitional type 2 (T2) and marginal zone (MZ) B cells found in the spleen and subject to negative selection of autoreactivity (42). Signaling via the BAFF-R enhances the processing of NF-κB2 protein p100 to p52, activates NF-κB, and makes a key contribution to prolonging the life of maturing B cells by inducing the anti-apoptotic factors Bcl-2 and Bcl-xL. BAFF was found to improve survival of human memory B cells in vitro without inducing cell division (43), consistent with its anti-apoptotic and non-mitogenic effects reported previously for murine B cells (42, 44). By increasing the number of generated Ig-secreting cells (ISCs), it also augmented the levels of IgM and IgA, but not IgG. The effect appears to be mediated by stimulating CD38+ ISCs derived from isotypeswitched memory cells, rather than inducing isotype switching by IgM+ memory B cells. An additional mechanism whereby BAFF may promote survival of activated human B cells comes from the observation that BAFF can induce IL-10 secretion by a human B cell line (45). BAFF, and to a lesser extent APRIL, are expressed in secondary lymphoid organs that support extra-follicular Ab responses. Studies of genetically mutated mice revealed that BAFF, in both trimeric membrane-bound and soluble forms, affects the mature B-cell repertoire, but not progenitor B cells in the bone marrow. Mice deficient in BAFF have a block in maturation of transitional type 1 (T1) to T2 B cells (46, 47). When the BAFF-R gene is mutated, as occurs in A/WysnJ mice, peripheral B cells are absent (48), indicating that BAFF signaling is required for production or maintenance of the mature B-cell repertoire. Neutralization of BAFF and APRIL by BCMA or by TACI-Ig will profoundly deplete recirculating and MZ B cells, indicating that longevity factors are required for continued survival of this B cell subset. While BCMA and BAFF-R appear to act as amplifying receptors, TACI seems to exert an inhibitory role. Initially, the impairment of signaling through TACI has been reported to be associated with reduced type 2-independent (TI-2) and, to a lesser extent, T-dependent Ab responses (49). Conversely, TI2 responses are enhanced in mice over-expressing APRIL or BAFF. Additionally, B cells lacking TACI were found to be hyper-responsive in vitro. As a result, TACI-/- mice manifest lymphoproliferation and fatal lupus-like AID (46), suggesting a dominant inhibitory role for TACI in maintaining B-cell homeostasis. Thus, unlike BAFF-R and BCMA, TACI is an inhibitory receptor that regulates B-cell function in vivo. It may

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function by outcompeting BAFF-R for binding to BAFF or by directly antagonizing BAFF-mediated signaling. It, therefore, appears that BAFF-R controls B-cell maturation, and TACI controls B-cell homeostasis and T-cell-independent immune responses, whereas the role of BCMA is still unknown. Also under investigation is the function of the recently identified APRIL-specific receptor, APRIL-R.

Further evidence for the view that elevated levels of BAFF are sufficient to cause autoimmunity comes from studies of (NZBxNZW) F1 and MRL-lpr/lpr autoimmune models. In diseased mice, BAFF serum levels were increased, and the augmentations correlated with disease progression (40), suggesting that overproduction of BAFF may be associated with the development of certain AID.

41.4

41.5 B-cell Survival in Human Systemic Autoimmune Disease

B-cell Longevity and Autoimmunity

In peripheral organs, self-tolerance can be broken due to intrinsic defects that can lead to increased B-cell longevity or to alterations in the expression of genes that regulate B-cell survival. Initial data suggested that, by promoting survival of immature lymphocytes, overexpression of BAFF may allow the autoreactive B cells to escape deletion, a prediction that was confirmed by transgenesis. Mice transgenic for BCMA exhibit enlarged spleens and lymph nodes, and high numbers of sIgM+ and IgD+ antigen-responsive mature B cells that express an activated phenotype, with increased expression of major histocompatibility complex class II molecules (40, 41). Strikingly, the transgenic mice also show the presence of autoantibodies, including anti-DNA antibodies. As the animals age, they display overt signs of lupus nephritis with proteinuria, enlarged glomeruli containing fibrin deposits and cellular infiltrates that precede kidney failure. Similarly, mice transgenic for BAFF have increased numbers of B-1 cells and effector T-cells, and develop autoimmune-like manifestations, such as autoantibodies and Ig deposition in the kidney (50). The changes observed in transgenic mice also are seen following administration of recombinant BAFF to normal mice, with massive expansion of the T2 and MZ B-cell subsets (42), and upregulation of the anti-apoptotic genes A1 and bcl-xL in mature B cells, but not in immature B cells (51). Since increased numbers of MZ B cells have been reported in other autoimmune murine models and because MZ-like B cells are thought to be the target of negative selection in the spleen (8), excess BAFF-mediated survival signals might compromise the ability of autoreactive B cells to respond to censoring death signals. Further studies disclosed that, as they age, BAFF transgenic mice develop a secondary pathology reminiscent of Sjögren syndrome (SJS; ref. 52). This is remarkable because, in some patients, human SJS can also develop secondary to systemic lupus erythematosis (SLE; ref. 53). It is of further interest that the salivary glands from BAFF transgenic mice contained a subpopulation of B cells with a MZ-like phenotype (B220+ HSA+CD21highCD1high) that could derive from the expanded MZ population present in the spleen of BAFF Tg mice (52). These cells may have aberrantly acquired homing receptors, enabling them to circulate and migrate to other lymphoid locations (42, 54). Thus, transgenic over-expression of BAFF is sufficient to cause the development of two AID in vivo, SLE and SJS, indicating an important role for BAFF-mediated signaling in the development and long-term survival of autoreactive B cells.

In humans too, there is deregulation of BAFF expression or activity in systemic autoimmunity (38, 52, 55–58). As compared to healthy individuals, the BAFF serum levels are significantly elevated in patients with SLE. The increase in all classes (µ, γ, and α) of anti-dsDNA Abs in SLE patients with high levels of BAFF argues that BAFF might be a primary factor which is acting directly on B cells to drive production of anti-dsDNA Abs in a T-cell-independent fashion. This conclusion is supported by animal studies showing that transgenic expression of BAFF is sufficient to trigger autoAb production without significant involvement of T cells. In SJS, BAFF levels also correlate with those of autoAbs, suggesting that BAFF may play a role in activating autoreactive B cells (58). In RA, the BAFF and APRIL levels are locally produced in inflamed joints (59). Remarkably, the elevated serum levels of BAFF were associated with aberrant production of IL10 by B cells and monocytes from SLE patients (43). Since IL-10 is a potent inducer of BAFF production by human myeloid cells, it is possible that the elevated BAFF levels are secondary to the augmented levels of serum IL-10 in these patients. It is possible that the combined effect of increased IL-10 and BAFF and/or APRIL contribute to the disease by enhancing the survival of plasmablasts in the affected tissues, by breaking tolerance during B cell development (39), or by selectively triggering B cell tolerance loss driven by dsDNA or other antigens. It will be important to probe BAFF- and APRIL-associated polymorphisms in human immune-mediated rheumatic diseases.

41.6 Autoimmunity and BcR-mediated Signaling In addition to the role of longevity factors in production of an autoimmune response, other observations point to alterations in B-cell signaling in shifting the balance of the immune system toward autoreactivity (16). In experimental animals, inactivation of genes encoding a number of BcR-associated signaling molecules gives rise to autoimmune syndromes (30). Even a single point mutation in the gene encoding the protein tyrosine phosphatase results in autoimmunity. Remarkably, genetic elimination of B cells, but not T cells, ablates the

41. B Lymphocytes and Autoimmunity

381

B Lymphocytes * Proximal Deficits

T Lymphocytes * Proximal Deficits

-↑ P and activity of Lyn

- ↓ CD45 activity

-↑ P and activity of SHP-1

- ↓ Absence of the ζ chain

-↓ of CD22

- ↑ P and activity of Lck

2+

- ↑ [Ca ]i - Abnormal CD45 activity * Intermediate Deficits - Not studied

- ↑ Tyrosine P - ↑[Ca

2+

]i

* Intermediate Deficits

- ↓ Activity of PKA-I & PKA-II - ↓ Catalytic activity of PKC - ↓ AP-1 - ↓ p65-RelA subunits of NF-κB - ↑ pCREM binding to IL-2 promotor

* Distal Deficits - Not studied

* Distal Deficits

- ↓ P of ERK catalyzed by MAPKK - ↓ DNAase methyl transferase activity - ↑ PKR-dependent phosphorylation

Figure 41.1. Signaling alterations in B and T lymphocytes from patients with SLE (16, 30, 71).

lymphoproliferative disorder and the lupus-like autoimmune syndrome (60). Importantly, alterations in BcR-mediated signaling pathways also exist in patients with autoimmune diseases, such as SLE (61, 62). The impaired signaling is not limited to B cells as altered signaling molecules and pathways have been reported in T cells from SLE patients, providing a unifying mechanism for the production of autoreactivity in autoimmune diseases (Figure 41.1).

41.7

Therapeutic Implications

As summarized earlier, studies of longevity factors indicate that pathways within this group of receptors play key roles in regulating B-cell activation and tolerance, and provide promising therapeutic targets. Not only they do provide critical positive second signals that augment and sustain B cell responses, but they also contribute key negative second signals that down-regulate B-cell responses. The observations that BAFF and APRIL participate in survival and/or expansion of B cells that produce pathogenic autoAbs suggest that longevity factors may provide an effective therapeutic target in systemic autoimmunity (55). Since BAFF is capable of interacting with the orphan TNF receptor BCMA, a soluble BCMA-Ig fusion protein was prepared by fusing the BCMA extracellular domain to the Fc portion of human IgG1 (48). Injection of the fusion protein constructed into (NZBxNZW) F1 mice led to a dramatic reduction in the total number of peripheral B cells. It behaved as a specific antagonist for BAFF by delaying the frequency of proteinuria and increasing survival. But it did not modify the autoantibody levels (40). In addition to

BCMA, data obtained with TACI, another BAFF receptor, are encouraging. Treatment of nephritic mice with TACI-Ig, used as a decoy receptor, induced a reduction in the levels of activated B cells with alleviation of kidney injury (47). It also reduced symptoms and even prevented disease in mouse models of SLE and CIA (47, 63, 64). The potent activity of TACI on ongoing disease may be related to its sustained production by DCs. By providing constant high levels of soluble TACI ligands, DCs sustain pathological B-cell activation and survival, characteristic of systemic autoimmune diseases. Their short-term in vivo blockade preferentially targets the T-cellindependent (TI) Ag-activated B-cell compartment (65), suggesting that TACI-Fc treatment in vivo inhibits TI-Ag specific plasmablast differentiation. The demonstration that blockade of BAFF function with soluble forms of BAFF receptors can decrease disease severity and prolong the survival in animals with systemic autoimmunity suggests rational therapeutic approaches to AID. As discussed above, patients with SLE exhibit elevated serum levels of BAFF (52, 55, 56), but also IL-10 aberrantly produced by B cells and monocytes (66). Because IL-10 has been found to be a potent inducer of BAFF production by human myeloid cells (67, 68), it is possible that elevated levels of serum BAFF in SLE are secondary to the elevated levels of serum IL-10 in these patients. This implies that, in addition to BAFF, targeting IL-10 (66) may have therapeutic benefit for the outcome of SLE, RA and SJS. In patients with systemic autoimmune disorders, especially RA and SLE, a number of clinical trials are underway to evaluate the clinical efficacy of targeting B cells (69, 70). In lupus, targeting BAFF indicates that specific blockade of

382

this longevity factor might be sufficient to suppress systemic autoimmunity. Targeting CD20 represents another promising avenue for the treatment of refractory lupus in both adults and children. While the clinical data add weight to the importance of B cells in the pathogenesis of lupus, new targets for B cell depletion therapy are being investigated. In experimental models, combining CD19 and CD20 antibodies was more effective than either treatment alone.

Acknowledgments. This work was supported by INSERM institutional grants.

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41. B Lymphocytes and Autoimmunity 33. Goodnow CC, Crosbie J, Adelstein S, Lavoie TB, Smith-Gill SJ, Brink RA, Pritchard-Briscoe H, Wotherspoon JS, Loblay RH, Raphael K, et al(1988) Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676–682. 34. Tiegs SL, Russell DM, Nemazee D (1993) Receptor editing in self-reactive bone marrow B cells. J Exp Med 177:1009–1020. 35. Gay D, Saunders T, Camper S, Weigert M (1993) Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med 177:999–1008. 36. Radic MZ, Zouali M (1996) Receptor editing, immune diversification, and self-tolerance. Immunity 5:505–511. 37. Lam KP, Kuhn R, Rajewsky K (1997) In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90:1073–1083. 38. Zouali M (2002) B cell diversity and longevity in systemic autoimmunity. Mol Immunol 38:895–901. 39. Mackay F, Browning JL (2002) BAFF: a fundamental survival factor for B cells. Nat Rev Immunol 2:465–475. 40. Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, Xu W, Parrish-Novak J, Foster D, Lofton-Day C, Moore M, Littau A, Grossman A, Haugen H, Foley K, Blumberg H, Harrison K, Kindsvogel W, Clegg CH (2000) TACI and BCMA are receptors for a TNF homologue implicated in Bcell autoimmune disease. Nature 404:995–999. 41. Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, Tschopp J, Browning JL (1999) Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med 190:1697–1710. 42. Batten M, Groom J, Cachero TG, Qian F, Schneider P, Tschopp J, Browning JL, Mackay F (2000) BAFF mediates survival of peripheral immature B lymphocytes. J Exp Med 192:1453– 1466. 43. Avery DT, Kalled SL, Ellyard JI, Ambrose C, Bixler SA, Thien M, Brink R, Mackay F, Hodgkin PD, Tangye SG (2003) BAFF selectively enhances the survival of plasmablasts generated from human memory B cells. J Clin Invest 112:286–297. 44. Do RK, Hatada E, Lee H, Tourigny MR, Hilbert D, Chen-Kiang S (2000) Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune response. J Exp Med 192:953–964. 45. Xu LG, Wu M, Hu J, Zhai Z, Shu HB (2002) Identification of downstream genes up-regulated by the tumor necrosis factor family member TALL-1. J Leukoc Biol 72:410–416. 46. Schiemann B, Gommerman JL, Vora K, Cachero TG, ShulgaMorskaya S, Dobles M, Frew E, Scott ML (2001) An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293:2111–2114. 47. Gross JA, Dillon SR, Mudri S, Johnston J, Littau A, Roque R, Rixon M, Schou O, Foley KP, Haugen H, McMillen S, Waggie K, Schreckhise RW, Shoemaker K, Vu T, Moore M, Grossman A, Clegg CH (2001) TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease. impaired B cell maturation in mice lacking BLyS. Immunity 15:289–302. 48. Thompson JS, Bixler SA, Qian F, Vora K, Scott ML, Cachero TG, Hession C, Schneider P, Sizing ID, Mullen C, Strauch K, Zafari M, Benjamin CD, Tschopp J, Browning JL, Ambrose C (2001) BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293:2108–2111.

383 49. Bulow GU, vonDeursen JM, vonBram RJ (2001) Regulation of the T-independent humoral response by TACI. Immunity 14:573–582. 50. Laabi Y, Strasser A (2000) Immunology. Lymphocyte survival— ignorance is BLys. Science 289:883–884. 51. Hsu BL, Harless SM, Lindsley RC, Hilbert DM, Cancro MP (2002) Cutting edge: BLyS enables survival of transitional and mature B cells through distinct mediators. J Immunol 168:5993–5996. 52. Groom J, Kalled SL, Cutler AH, Olson C, Woodcock SA, Schneider P, Tschopp J, Cachero TG, Batten M, Wheway J, Mauri D, Cavill D, Gordon TP, Mackay CR, Mackay F (2002) Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjogren’s syndrome. J Clin Invest 109:59–68. 53. Zouali M (2005) Taming lupus. Sci Am 292:58–65. 54. Zeng D, Lee MK, Tung J, Brendolan A, Strober S (2000) Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/ NZW mice. J Immunol 164:5000–5004. 55. Zhang J, Roschke V, Baker KP, Wang Z, Alarcon GS, Fessler BJ, Bastian H, Kimberly RP, Zhou T (2001) Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J Immunol 166:6–10. 56. Cheema GS, Roschke V, Hilbert DM, Stohl W (2001) Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases. Arthritis Rheum 44:1313–1319. 57. Roschke V, Sosnovtseva S, Ward CD, Hong JS, Smith R, Albert V, Stohl W, Baker KP, Ullrich S, Nardelli B, Hilbert DM, Migone TS (2002) BLyS and APRIL form biologically active heterotrimers that are expressed in patients with systemic immune-based rheumatic diseases. J Immunol 169:4314–4321. 58. Mariette X, Roux S, Zhang J, Bengoufa D, Lavie F, Zhou T, Kimberly R (2003) The level of BLyS (BAFF) correlates with the titre of autoantibodies in human Sjogren’s syndrome. Ann Rheum Dis 62:168–171. 59. Tan SM, Xu D, Roschke V, Perry JW, Arkfeld DG, Ehresmann GR, Migone TS, Hilbert DM, Stohl W (2003) Local production of B lymphocyte stimulator protein and APRIL in arthritic joints of patients with inflammatory arthritis. Arthritis Rheum 48:982–992. 60. Hermiston ML, Tan AL, Gupta VA, Majeti R, Weiss A (2005) The juxtamembrane wedge negatively regulates CD45 function in B cells. Immunity 23:635–647. 61. Liossis SN, Kovacs B, Dennis G, Kammer GM, Tsokos GC (1996) B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events. J Clin Invest 98:2549–2557. 62. Huck S, Le Corre R, Youinou P, Zouali M (2001) Expression of B cell receptor-associated signaling molecules in human lupus. Autoimmunity 33:213–224. 63. Siegel RM, Lenardo MJ (2001) To B or not to B: TNF family signaling in lymphocytes. Nat Immunol 2:577–578. 64. Wang H, Marsters SA, Baker T, Chan B, Lee WP, Fu L, Tumas D, Yan M, Dixit VM, Ashkenazi A, Grewal IS (2001) TACI-ligand interactions are required for T cell activation and collagen-induced arthritis in mice. Nat Immunol 2:632–637. 65. Balazs M, Martin F, Zhou T, Kearney J (2002) Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17:341–352. 66. Beebe AM, Cua DJ, Waal Malefyt R de(2002) The role of interleukin-10 in autoimmune disease: systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Cytokine Growth Factor Rev 13:403–412.

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Chapter 43 Breach and Restoration of B-cell Tolerance in Human Systemic Lupus Erythematosus (SLE) Iñaki Sanz, R. John Looney, and J. H. Anolik

43.1

Introduction

43.1.1 B Cells as Central Pathogenic Players in SLE A central pathogenic participation of B cells in SLE has been demonstrated by numerous animal and human studies and is illustrated by the abundance of disease associated-autoantibodies. It is quite apparent, however, that B cells also contribute to autoimmune diseases through diverse antibody-independent functions in addition to more conventional antibody-mediated mechanisms (1, 2). The latter type includes the ability of antiRNP antibodies to form immune complexes that induce interferon (IFN)-α production from dendritic cells (3). The former type includes antigen-presentation, T-cell activation and polarization, and dendritic cell modulation (2, 4–22). Several of these functions are mediated by the ability of B cells to produce immunoregulatory cytokines, chemokines and lymphangiogenic growth factors and by their critical contribution to lymphoid tissue development and organization, including the development of ectopic tertiary lymphoid tissue that is likely to play a major pathogenic role in multiple autoimmune diseases in addition to SLE, rheumatoid arthritis (RA) and Sjogren’s syndrome (Table 43.1; refs. (23–30). Of note, B cells profoundly influence T-cell activation and infiltration of target organs in SLE and RA (kidneys and synovium, respectively; refs. (4, 31–33). However, the actual mechanism whereby B cells induce or mediate disease is likely to be different in different conditions and remains to be fully elucidated. More recently, the pathogenic potential of B cells has been further demonstrated by the effectiveness of B-cell depletion in the treatment in SLE as well as in other multiple autoimmune diseases, some of which had not been considered as B From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

cell-mediated (2, 34). Of great interest, our own SLE studies with Rituximab have confirmed the observation in animal models that the absence of B cells in human SLE has significant therapeutic benefit irrespective of autoantibody levels (35).

43.1.2 B-cell Tolerance as a Critical Factor in Autoimmunity Whatever the ultimate participation of autoreactive B cells may be in a particular autoimmune disease, B-cell tolerance must be first breached in order to recruit them into the pathogenic process. Similarly, B-cell depletion therapy (BCDT) has the potential to ameliorate disease by inhibiting autoantibody production or by interfering with other B-cell pathogenic functions. However, such improvement will be necessarily transient unless the absence of B cells can successfully restore immunological tolerance and reverse the pathogenic process. Of course, this concept does not exclude a contribution of nonautoreactive polyclonal B cells (perhaps stimulated through innate immune receptors and/or cytokines such as IFN-α or BLyS) at a later time in the disease course which in turn might contribute to disease pathogenesis through the production of pro-inflammatory cytokines or inhibition of Treg cells (36). It is therefore essential to understand human B-cell tolerance and to be capable of assessing this critical property in patients treated with BCDT. As demonstrated in animal models, B-cell tolerance is established at multiple checkpoints throughout B-cell development and it is enforced largely by negative selection (deletion, editing or anergy), although positive selection and “sequestration” into the B1 and marginal zone (MZ) compartments has also been described (37). Even in animal models, the precise mechanisms of tolerance breakdown are less well defined and our knowledge of this phenomenon in human disease is quite incomplete. Recent work, however, has shed some light on this fundamental question. Thus, we initially showed that an important checkpoint operates in healthy subjects to censor autoreactive 397

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I. Sanz et al. Table 43.1. Antibody-independent functions of B cells ATα-dependent

Formation of the T-cell zone Formation of the marginal zone Maturation of FDC networks Homeostasis of dendritic cells Recruitment of germinal center DC cells Lymphoid neogenesis T cell functions

LTα-independent

Dendritic cell functions

Cytokine production Chemokine production

Priming of naïve CD8 cells Priming/expansion of naïve CD4 cells T-cell anergy Activation of autoreactive CD4/CD8 cells Promotion of Th2 differentiation Induction of Th1 differentiation Recruitment of FTH (follicular helper) T cells Inhibition of Tregulatory cells Maduration and migration of DCs Activation of DCs Inhibition of DC-induced T cell immunity Attenuation of IL-12-induced Th1 differentiation IL-1, IL-4, IL-6, IL-8, IL-7, G-CSF, GM-CSF, IL-10 IL-12, TNFα, LTα, TGFβ, BMP-6/7, VEGF-A MIP-1α, MIP-1β, IL-16

Lymphangiogenesis and lymph node expansion

(9G4) B cells in the mature naïve compartment thereby preventing their expansion into the memory compartment (38). This checkpoint has also been documented in other experimental systems (39). We also showed that the regulation of 9G4 B cells at this checkpoint is specifically defective in SLE (discussed later). Of note, additional checkpoints that work at earlier stages of B-cell development have been reported by other investigators. Interestingly, defects in these checkpoints are shared by patients with SLE and RA (40, 41).

43.1.3 Experimental Approaches to the Study of Human B-cell Tolerance Two major obstacles must be overcome in order to understand B-cell tolerance. First, it is necessary to identify a population of autoreactive B cells of well defined properties that are relevant to the disease process and whose frequency is large enough to permit accurate measurements. We have approached this problem by studying an autoreactive B-cell population bearing surface antibodies that express the 9G4 idiotype (heretofore referred to as 9G4 cells and 9G4 antibodies respectively), that satisfies these requirements (42). Elevated serum titers of 9G4 IgG antibodies are highly specific for SLE, correlate with disease activity and organ involvement and participate in pathogenic anti-DNA antibody responses. 9G4 cells represent 5 to 10% of all naïve B cells in healthy subjects, yet are virtually absent from the IgG memory and plasma cell compartments. Therefore, we postulated that autoreactive 9G4 cells must be strictly censored in the peripheral compartment of healthy subjects. This hypothesis was proven correct through the analysis of normal tonsil and spleen specimens, which helped us demonstrate that physiological censoring of 9G4 cells is accomplished by

preventing these cells from participating in productive germinal center (GC) reactions and that this is largely accomplished in the early phases of the GC reaction (38, 42, 43). We also postulated that normal censoring should be subverted in patients with SLE. Proving this point required solving the second obstacle, namely the need for access to secondary lymphoid tissue in autoimmune patients. This limitation was overcome through the pioneering use of tonsil biopsies for such purpose (43). The widespread utilization of this approach should contribute to major advances in our understanding of autoimmune diseases. In our hands, tonsil biopsies provide from 20 to 60 million B cells in addition to enough additional tissue for detailed histological studies. This procedure is safe and has the critical advantage that it allows investigators to obtain lymphoid tissue in a controlled and systematic fashion in the patients of choice. Using tonsil biopsies, we have demonstrated that indeed faulty GC censoring of 9G4 B cells occurs specifically in SLE and is not shared by RA patients (Figure 43.1; ref. 43). Thus, approximately 25% of all productive GC in SLE tonsils are filled with proliferating 9G4 B cells as compared to virtually none in healthy subjects or in RA tonsils. This defect results in a 10- to 25-fold expansion of 9G4+ B cells within the IgG memory compartment. Furthermore, despite the striking absence of 9G4+ plasma cells in healthy controls, 9G4 antibodies are expressed in up to 30% of PBL plasmablasts in active SLE. Together with previous demonstrations of the high specificity of 9G4 serum antibodies for SLE (>95% and comparable to anti-ds DNA antibodies) and of the association of serum 9G4 antibody levels with disease activity and organ involvement in SLE, our results clearly establish the relevance of studying the fate of 9G4 B cells to assess B-cell tolerance in SLE.

43. Breach and Restoration of B-cell Tolerance in Human Systemic Lupus Erythematosus (SLE)

399

Figure 43.1. Physiological censoring of 9G4 B cells by exclusion from germinal center (GC) reactions. Breakdown of these mechanisms in SLE and restoration by BCDT. (A) Flow cytometry of tonsil B cells from healthy subjects and SLE and RA patients (38, 43). IgD and CD38 identify naïve (N), GC, and memory cells (M). A comparison of total and 9G4 B cells demonstrates clearly that 9G4 cells failed to acquire a GC phenotype and are greatly underrepresented within the post-GC IgD-negative memory compartment. Such strict censoring of 9G4 cells has been consistently observed in more than 20 normal tonsils and in RA tonsil biopsies. In sharp contrast, we have observed a dramatic expansion of 9G4 B cells in the GC and memory compartments of all SLE patients in whom tonsil biopsies have been performed to date. A remarkable exception to this rule is our recent observation that proper censoring of 9G4 B cells is restored in a subset of SLE patients in whom complete clinical remission has been sustained for up to 5 years after treatment with Rituximab. A representative example is shown in the figure. (B) The ability of 9G4 B cells to form productive GC in SLE but not in normal subjects or RA patients (not shown) can be also documented by immunofluorescence staining of secondary follicles with IgD, CD38 and 9G4 (See Color Plates).

Table 43.2. Immunological profiles of SLE patients treated with full dose Rituximab (N = 11) Baseline ENA autoantibody + Peripheral blood B-cells <5/µl post-Rituximab Mean time to repopulation (>10/µL) Total memory B-cells 12 months post-Rituximab Total memory B-cells 3–5 years post-Rituximab IgG memory B-cells 3–5 years post-Rituximab IgM memory B-cells 3–5 years post-Rituximab

43.1.4

Group A (N = 3)

Group B (N = 7)

0 (0%) 3 (100%) 7.8 3.4 ± 0.7% 6.3 ± 0.9% 3.6 ± 0.5% 2.7 ± 0.4%

7 (100%) 6 (86%) 3.6 (p < 0.001) 22.1 ± 11.5%, p < 0.0001 30.5 ± 6.9%, p < 0.000005 18.3 ± 5.8%, p < 0.0005 3.2 ± 1.4%, p = 0.2

B-cell Depletion in the Treatment of SLE

The therapeutic utilization of B-cell-depleting agents such as the anti-CD20 monoclonal antibody Rituximab offers great promise and hope for the treatment of multiple autoimmune diseases. Moreover, the study of B-cell-depleted patients allows investigators to understand the role of B cells in autoimmunity by analyzing the clinical and immunological consequences of the absence of B cells. Several studies, including our own, have demonstrated that B-cell depletion induces significant clinical benefit in SLE and RA and WG. We have shown that such benefit can be attained in SLE without the addition of high-dose steroids or cyclophosphamide (35). Furthermore, our studies have shown that in SLE, clinical improvement (occurring within two months) precedes the decline of serum autoantibody levels (such as anti-dsDNA and 9G4), which are sustained for prolonged periods of time by preexisting longlived plasma cells. These observations strongly support the notion of antibody-independent pathogenic roles for B cells initially advanced in the mouse (4).

We have identified two major types of responses in SLE patients treated with full-dose Rituximab in the absence of significant doses of corticosteroids or cytotoxic agents (Figures 43.2 to 43.4; refs. 44–46): • Group A: Patients with complete remission (including biopsy-proven reversal of type IV nephritis and disappearance of ANA), which has been maintained without concomitant immunosuppression for the duration of follow-up (three to five years). These patients achieve profound B-cell depletion and their serum anti-DNA and 9G4 autoantibodies normalize over three years (average of 16 months). • Group B: Patients with transient clinical responses with an average time to flare of seven months. In these patients, initial B-cell depletion is of similar intensity but of shorter duration than in group A (see later) and autoantibody levels did not significantly decline over time. Although the initial degree of B-cell depletion was comparable in these two groups, they nonetheless can be sharply separated by their autoantibody and B-cell reconstitution profiles (Table 43.2).

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I. Sanz et al.

Figure 43.2. Two types of response in SLE after B-cell depletion therapy. Representative examples of two types of clinical and immunological response attained in SLE patients treated with Rituximab are shown. The graph represents the number of total CD19+ B cells/µl of peripheral blood (See Color Plates).

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Figure 43.3. B-cell repopulation after BCDT recapitulates ontogenic development. (A) Flow cytometric analysis of PBL B cells from healthy adults or neonates as compared to SLE patients treated with Rituximab (representative examples are shown from patients with type A or B responses). Staining with CD24 and CD38 discriminates naïve, transitional (T1 and T2), and memory B-cell subsets. Typically, healthy adults display a predominance of naïve B cells. In contrast, neonatal blood is characterized by abundant transitional cells. SLE patients with transient response to BCDT (group B) are characterized by quick reaccumulation of memory B cells. In contrast, patients with prolonged response (group A) are characterized by scarcity of memory cells and expansion of transitional B cells. Of note, as in the example shown, such “neonatal” pattern may be observed even five years after BCDT, a striking length of time when one considers that an “adult” repertoire with a normal component of memory cells is formed within the first two to three years of life (75). (B) Peripheral blood B cells from healthy

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Figure 43.3. (continued) donors were stained with CD24 and CD38 as before to identify transitional (T1, T2), naïve (N), and memory (M) cells according to their surface phenotype. These subsets were further analyzed for their ability to extrude dyes such as Rhoda mine 123 (R123; ref. 76). Healthy controls demonstrate typical separation of these populations by surface phenotype (dot plots) and intracellular staining (histograms) with the negative peak representing mature naïve B cells (red line). SLE patients after BCDT (group A) show expansion of transitional cells with scarcity of memory more than a year after therapy. This deficiency is still seen at 28 months (histograms). Interestingly, the vast majority of cells in these patients (including cells with a surface phenotype overlapping with naïve B cells) appear to be immature on the basis of their inability to extrude R123. This situation is illustrated by the red histograms to the right (See Color Plates).

Group A is distinguished by a more limited autoantibody repertoire at baseline with positive anti-dsDNA but negative anti-ENAs (extractable nuclear antigens - Ro, La, U1-RNP, Sm); in contrast all short-term responders (Group B) had at least 1 ENA at baseline (p = 0.002). With regard to B-cell repopulation, Group A is characterized by slower re-expansion than Group B. Strikingly, up to 80% of all B cells in these patients display a transitional phenotype and the memory B-cell compartment remains significantly shrunk for up to three to five years after treatment (Figures 43.3 to 43.4). In essence, the repertoire of these patients closely resembles the neonatal B-cell compartment suggesting that they are undergoing a slow de novo immune reconstitution without reemergence of autoimmunity as indicated by the disappearance of autoantibodies (despite the maintenance of normal serum

total immunoglobulin levels), and by the normalization of the levels of autoreactive 9G4 B cells (44–46). In contrast, patients in Group B are characterized by a quick normalization of memory B cells during their faster B-cell recovery raising the possibility that reconstitution may be driven by the expansion in a lymphopenic environment of residual memory B cells enriched for autoreactivity. Interestingly, IgM+ memory B cells failed to normalize for prolonged periods of time even after re-expansion of switched IgG+ cells in Group B. This observation suggests that GC reactions and/or post-GC B cells may be more refractory to B-cell depletion than MZ B cells. Alternatively, these results could be explained by invoking differential repopulation kinetics between these two memory subsets. Factors responsible could include more profound or more prolonged disruption of the anatomical MZ, decreased

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Figure 43.4. B-cell repopulation profile in SLE patients treated with Rituximab. Groups A and B are defined as before and compared to normal controls for their abundance of PBL memory CD27+ B cell subsets (either IgM/IgD+ or IgD– switch memory). Both subsets are underrepresented in patients from group A even 5 years after treatment. Interestingly, the recovery of the IgM+/IgD+ memory subset appears to lag behind the switched memory subset in patients from group B (See Color Plates).

competitiveness for survival factors, such as BLyS or intrinsic properties of MZ B cells. Of note, repopulation of the MZ does not occur in rats undergoing B-cell depletion from birth with anti-µ antibody (47). Of additional interest is the notion that B1 cells may contribute at least in part to the composition of the human unswitched memory B-cell subset (48). In this scenario, the contraction of unswitched memory cells observed after B-cell repopulation could be due to decreased de novo generation of B1 cells in adult individuals. The slow repopulation of IgM+ memory cells may be of clinical consequence as the loss of this population correlates with recurrent lower respiratory tract infections in patients with common variable immunodeficiency (49).

43.1.5 Restoration of B-cell Tolerance After Prolonged B-cell Depletion As previously discussed, tracking the fate of autoreactive 9G4 B cells provides a very useful measurement of B-cell tolerance. We have previously shown that 9G4 cells are censored in healthy subjects and prevented from expanding in productive GC reactions (38, 43). Thus far, such censoring has only been found to be faulty in patients with SLE but not in other autoimmune diseases (38, 43). This has been documented by flow cytometry as in Figure 43.5 as well as by histological analysis of tonsils obtained either from healthy subjects or from patients with SLE or RA (38, 43). Importantly, the presence of 9G4+ GC appears to be universal to all SLE patients thus far analyzed, presumably reflecting a breakdown of tolerance common to all patients with established disease irrespective of disease activity. Recently, however, we have had the opportunity to perform tonsil biopsies in the SLE patients

who have experienced complete and sustained remission after treatment with Rituximab (Group A). Strikingly, these patients show a near total absence of 9G4 B-cells in the GC (Figure 43.1). Thus, they represent the only SLE patients in whom this absence has ever been observed. Collectively, we believe that our data strongly suggest that proper GC censoring of autoreactive B cells has been restored by B-cell depletion in this subset of patients.

43.2

Discussion

The results presented strongly suggest that prolonged B-cell depletion induces sustained clinical remission in a subset of SLE patients. In turn, the study of autoreactive 9G4 cells strongly suggests that B-cell tolerance may be restored by prolonged B-cell absence and that such restoration of tolerance may be at the center of clinical improvement. While the actual immunological consequences of the absence of B cells remain to be understood, two not mutually exclusive mechanistic models can be proposed to explain the benefit of B-cell depletion (Figure 43.5): 1. Tolerance model: B-cell depletion induces restoration of B-cell tolerance. This model assumes that tolerance is broken by the impact on genetically susceptible B cells of environmental and/or stochastic events during the generation of the B-cell repertoire (50). It also postulates that upon reconstitution, these factors may not converge again and as a result, previously defective tolerance checkpoints will now be operative in censoring autoreactive B cells. 2. Regulatory model: B cells play important immunomodulatory roles through antigen-presentation and the production of

43. Breach and Restoration of B-cell Tolerance in Human Systemic Lupus Erythematosus (SLE)

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Figure 43.5. Immunological consequences of B-cell depletion therapy. Schematic representation of the B-cell recovery profile observed in SLE patients experiencing prolonged remission after treatment with Rituximab. These patients display prolonged expansions of immature/ transitional cells and decreased abundance of mature B cells including naïve cells, MZ, and post-GC B cells. Multiple intrinsic and extrinsic B-cell variables, some of which are indicated in the figure, could contribute to this impaired maturation. The integrity of the lymphoid architecture after BCDT remains to be systematically analyzed. However, our results indicate that memory cell contraction remains despite normal architecture with active GC several years after treatment (Anolik et al., manuscript submitted) (See Color Plates).

cytokines and chemokines. They also play fundamental roles in the organization of the lymphoid architecture and in the orchestration of cellular interactions to a large extent through the expression of membrane LTα1β2 (thereby controlling the formation of the T-cell zone, the MZ and the maturation of FDC networks as well as the homeostasis of lymphoid tissue dendritic cells; refs. 2, 26, 28, 51–53). It can therefore be postulated that the prolonged absence of B cells is likely to induce critical changes in lymphoid organization and in other immune cells prominently including T cells. With respect to T-cell effects the following roles could be suggested for B cells in SLE: • Activation of autoreactive CD4 and CD8 T cells. A corollary of these functions is that B-cell depletion could attenuate T-cell activation. It is also plausible that upon reconstitution predominantly transitional/naïve B cells devoid of co-stimulatory ability could contribute to induce T-cell tolerance (54–56). Of interest, significantly decreased frequencies of CD4+CCR7–effector memory T cells has been reported in abstract form in association with fast clinical improvement in SLE patients treated with Rituxan (57). In the only work published thus far, the numbers of CD40L+ CD4+ T cells decreased significantly as early as one month post-therapy (in the presence of profound B-cell depletion; ref. 58). Of note, the continuous decline of CD40L+ CD4+ T cells over time correlated with the attainment of clinical remission.

• Promotion of Th2 differentiation. This effect can be mediated through CD40, ICOS-L or OX40L and by production of IL-10 (12, 14, 17, 20, 21, 59–61). It follows that in the absence of B cells, a dominance of IL-12 producing DCs among APCs might favor a Th1 shift. It has been postulated, however, that Rituximab-induced down-regulation of CD80/86 on B cells might induce a Th2 shift in SLE (62). Whether this is indeed the case and whether it correlates with a favorable clinical response remains to be determined as the view of SLE as a purely Th2 disease is rather simplistic (63, 64). • Recruitment, co-localization and modulation of the helper phenotype of CXCR5+ FTH (follicular T helper) cells (12, 65, 66). Of note, increased expression of ICOS on FTH cells induces hyperactive GC, breakdown of B-cell tolerance, autoantibody production and a lupus-like phenotype (67). Therefore, the absence of B cells might inhibit the recruitment and activity of FTH cells and repress autoimmunity. • Blocking of expansion of Treg cells (36, 68). Accordingly, the absence of B cells would be expected to induce expansion of Treg cells. The regulatory model could also work through effects on pathogenic cytokines of central importance for SLE such as IFN-α (69). This effect could be mediated by decreased production of interferogenic autoantibodies capable of stimulating the production of IFN-α by DC (69, 70). Conversely, the

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persistence of anti-ENA antibodies would result in sustained IFN production and both type I IFN and anti-ENA immune complexes could play an important role in expanding memory B cells, especially autoimmune B cells, thereby contributing to clinical relapse. These effects could be central to explaining our observation that patients expressing these antibodies are much less likely to experience prolonged remission. We believe that mechanisms from both models need be invoked in order to explain our observations in Group A of Rituximab-treated patients. Indeed, one of the central issues we labor to understand is why the full maturation of the newly emerging B-cell repertoire is delayed in these patients. It is informative in this regard to consider the analogy with the similarly slow development typically seen in early life and after bone marrow/stem cell transplantation (71, 72). Indeed, in these situations the delayed development or reconstitution of memory B cells are likely to be explained at least in part by the immaturity/disruption of the lymphoid architecture and other immune cells (due to neonatal immaturity and to the ablative preparative regimens used for transplantation, respectively). Yet, if this should be the case in Rituximabtreated patients, one would have to conclude that these profound effects have been mediated by the absence of B cells. In turn, it is likely that the very consequences triggered by B-cell depletion (lymphoid disruption, diminished T-cell help, reduction in DC function) may in turn contribute to delaying the maturation of the newly emerging B-cell compartment. Similarly, the recent demonstration that Tregs may directly suppress B cells (73, 74), suggests that expansion of Tregs upon B-cell depletion could in turn contribute to downregulating newly emerging B cells. A systematic study of the immunological consequences of B-cell depletion and other B-cell targeted interventions will undoubtedly result in a better understanding of B-cell tolerance, the pathogenic roles of B cells in different diseases, and the mechanistic basis for success or failure of these therapeutic interventions. Of great importance is that such studies will also help understand any potential untoward effects of BCDT including infections, elimination of preexisting protective B- and T-cell memory, and undesired Th cell polarization. The knowledge gained will contribute greatly to our ability to monitor and predict clinical responses and adverse effects.

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Chapter 49 Correlates of Immunity Elicited by Live Yersinia pestis Vaccine Vivian L. Braciale, Michael Nash, Namita Sinha, Irina V. Zudina, and Vladimir L. Motin

49.1

Introduction

Yersinia pestis is the causative agent of plague, one of the most severe bacterial infections in the history of mankind. Y. pestis has been squarely placed on the category A select agent list because of its potential to be used as an agent of biowarfare and bioterrorism (1). Most human plague cases usually present as one of three primary forms, i.e., bubonic, septicemic, or pneumonic, with the latter two having a high mortality rate (2). Currently, no plague vaccine exists in the United States, although until 1999, a formaldehyde-killed, whole-cell vaccine was available for military and laboratory personnel. This vaccine required a course of injections over a period of six months and was effective against bubonic plague. However, the protection was short-term and annual boosters were required; additionally, the incidence of side effects, such as malaise, headaches, elevated temperature, and lymphadenopathy, was high (in ~10% of those immunized with vaccines); and the vaccine was expensive. Moreover, the protection of killed, whole-cell vaccine against the pneumonic form of plague was uncertain (3). A live-attenuated vaccine is available in the former Soviet Union (SU) countries. This vaccine Y. pestis EV, line NIIEG, represents a non-pigmented version of the virulent strain isolated in Madagascar. The vaccine strain is attenuated due to deletion of the 102-kb locus Pgm, which includes the hemin-storage region Hms and a cluster of genes encoding the siderophorebased yersinia bactin biosynthetic/transport systems (2, 4). A comparison of killed and live plague vaccines showed that the latter was more efficient when immunized animals were challenged with the fully virulent strain of Y. pestis by three routes of administration in three strains of mouse (5). The live plague vaccine has been adopted for human use for more than 70 years, and it is still the vaccine of choice in the former SU countries for those working around plague. The vaccine is considered to From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

be effective against all forms of plague; however, the safety of this vaccine in humans is questionable, because it retains some virulence, and in most countries (including the United States) live vaccines such as this are not licensed (6–8). Currently, the vaccine considered to present the best prospects for human use is a subunit vaccine consisting of either the mixture of LcrV and F1 antigens or their peptide fusion form F1-LcrV (3, 8). The LcrV protein (V antigen) of Y. pestis is a multifunctional protein involved in modification of innate immune responses to this pathogen, as well as in regulation of expression and translocation of Yop effectors of the type III secretion system (T3SS; refs. 9 and 10). The F1 antigen is a capsular subunit protein located on the surface of the Y. pestis cell, which is thought to have anti-phagocytic properties (11, 12). In this study, we have compared both humoral and cellular immune responses to the formaldehyde-killed and live plague vaccines elicited in BALB/c mice. Since it is not possible to test the safety and efficacy of these vaccines in human volunteers, it was important to identify immune correlates of protection. We had the rare opportunity to characterize the Y. pestis-specific antibody and T-cell-mediated immune responses of individuals previously immunized with the liveattenuated vaccine strain EV and compare these responses to those elicited in BALB/c mice.

49.2

Results and Discussion

49.2.1 Experimental Outline for Immunization in the Murine Model Y. pestis vaccine strain EV76 was used to make both killed and live plague vaccines for immunization of BALB/c mice and following in vitro assays. Killed vaccine was prepared by the same methods used in the past to produce a plague vaccine for human immunization (13). The procedure included the growth of Y. pestis on a complex solid medium for 72 hours at 37°C, followed by killing the cells with formaldehyde. The killed vaccine stocks were stored in bulk at 4°C with 0.5% of phenol 473

474

as a preservative. Live vaccines were prepared freshly prior to each immunization and consisted of the cells grown either at 26 or 37°C in the Heart Infusion Broth for 30 hours with aeration. Final cell concentration was measured by spectrophotometer at an optical density of 600 nm, followed by plating of the culture in 10-fold dilutions (colony-forming units). Groups of female BALB/c mice were immunized at the age of 6 to 8 weeks either with killed or live vaccines (Figure 49.1). Killed vaccine at a dose of 2 x 108 formaldehydetreated cells was administered either with or without Inject Alum adjuvant (Pierce, Rockford, IL) via the subcutaneous (s.c.) route. Live vaccines, grown at two temperatures, were inoculated at a dose of 2 ´ 105 by the same s.c. route. Mice were boosted twice at intervals of 14 days by killed vaccine and once with live vaccines on day 28 after primary immuni-

Figure 49.1. Immunization and bleed schedule.

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zation. Blood samples were collected at days 0, 29, 35 and 43, followed by a terminal bleed and spleen harvest on day 57.

49.2.2

Murine Humoral Responses

Several major antigens of Y. pestis were cloned and expressed in E. coli as N-terminal fusions with a polyhistidine Tag, followed by affinity purification using an Ni2+- charged resin. To test the antibody response in mice immunized with killed and live vaccine, we used the T3SS effectors YopM and YopE, known protective antigens LcrV and F1, and plague plasminogen activator Pla. The quality of purification of Y. pestis antigens and the results of Western blots using sera collected on day 43 are depicted in Figure 49.2. We found that the sera of mice immunized with live vaccines prepared from cells grown at 26 or 37°C reacted with LcrV and capsular antigen F1 (Figure 49.2B). Since the expression of both LcrV and F1 antigens is practically nonexistent in Y. pestis cultured at 26°C, the identical picture of Western blots from sera obtained after immunization with live vaccines prepared from cells grown at two different temperatures was the indicator that live vaccine organisms replicated in vivo. In contrast, antisera from mice immunized with killed vaccine, with or without adjuvant, contained antibodies to F1 but lacked immunoglobulin specific to LcrV (Figure 49.2C). This would be expected since F1 but not LcrV is a constituent of the input-killed Y. pestis prior to inactivation with formaldehyde. Since the killed vaccine could not replicate in the mouse, there was no LcrV production in vivo. These results were confirmed in enzyme-linked immunosorbent assays (ELISA; Table 49.1). Results for pooled bleeds showed that both live and killed vaccines elicited F1-specific antibodies. Antisera

Figure 49.2. Antigenic specificity of immune antisera. (A) The silver-stained PAGE gel of purified recombinant Y. pestis antigens: 1- YopM, 2- YopE, 3- LcrV, 4- F1, 5- Pla. (B, C) Results of representative Western blots using antisera from mice immunized with live and killed vaccines, respectively.

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Table 49.1. ELISA antibody titers to F1 and LcrV antigens in vaccinated mice

although anti-LcrV titers seemed to be higher when Y. pestis EV76 cells were cultured at 37°C (Figure 49.3B).

Anti-F1, IgG Vaccine

Week 4

Week 5

Week 6

Terminal

Mock

<1/100

<1/100

<1/100

<1/100

Killed

>1/12,500

>1/12,500

>1/12,500

>1/12,500

Killed+ Alum

>1/12,500

>1/12,500

>1/12,500

>1/12,500

Live, 26°C

>1/12,500

>1/12,500

>1/12,500

>1/12,500

Live, 37°C

>1/12,500

>1/12,500

>1/12,500

>1/12,500

Anti-LcrV, IgG Vaccine

Week 4

Week 5

Week 6

Terminal

Mock

<1/100

<1/100

<1/100

<1/100

Killed

<1/100

<1/100

<1/100

<1/100

Killed + Alum

<1/100

<1/100

<1/100

<1/100

Live, 26°C

<1/100

>1/1,000

>1/1,000

<1/1,000

Live, 37°C

<1/100

>1/1,000

>1/1,000

<1/1,000

from immune mice bled on days 29, 35, and 43 showed titers greater than 12,500. The titers to F1 were high at four weeks and remained high regardless of the immunization protocol. Mice immunized with killed vaccine, however, did not develop antibodies specific for V antigen. Mice receiving live-attenuated vaccine showed low titers to LcrV at 4 weeks; these levels peaked at 6 weeks and began to decline at terminal bleed. Thus, the kinetics of antibody development to F1 differed from that to V antigen. Determination of IgG isotype usage to F1 antigen did not reveal a principal difference between groups of mice immunized with live or killed vaccines (Figure 49.3A). Similarly, there was little difference in IgG subtype pattern to LcrV in mice immunized with live vaccines grown at two different temperatures,

49.2.3

Murine T-cell-mediated Responses

In order to examine the murine T-cell-mediated responses, spleens were removed at the time of terminal bleed and splenocytes harvested for in vitro T-cell studies. A portion of the splenocytes were frozen and stored in liquid nitrogen for future studies. Another portion was stimulated with heat-killed (HK) Y. pestis in vitro. Samples from these cultures were tested for 3 H-thymidine incorporation on days 3 and 7 of culture. The remaining cells from this in vitro stimulation were frozen and stored in liquid nitrogen for future studies. Following initial studies using spleens from individual mice from each group, the spleens were pooled for each experimental group. Preliminary data indicated that the immune splenocytes are stimulated to proliferate in the presence of HK Y. pestis (data not shown). As illustrated in Figure 49.4, immune splenocytes, but not naïve splenocytes, produced IFN-γ when stimulated with HK Y. pestis. Figure 49.5 shows the results of testing culture supernatants for IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ. IL-4, and IL-2 were below detection possibly due to consumption of these cytokines in the 3-day cultures. The most telling difference between the cytokines produced by splenocytes from mice given killed vs. live vaccine lay in the lack of IFN-γ, as was the case for the former, but not the latter. When the splenocytes were from mice vaccinated with live vaccine, and stimulated with HK versus live Y. pestis, differences were seen for the cytokines and chemokines produced. Stimulation with live cells of Y. pestis resulted in significantly higher production levels of KC, CM-CSF, IL-3 and IL-6, in comparison with the stimulation by HK cells (Figure 49.6). Also, stimulation of splenocytes with capsular protein F1 from mice immunized with killed Y. pestis resulted in an antigen-specific T-cell proliferative response (Figure 49.7).

A

B

Anti-LcrV IgG subtypes

0.350

anti-F1 IgG subtypes

IgG1

0.300

IgG2a

0.250

IgG2b

0.350 IgG1

0.300 0.250

IgG2a

0.200

IgG3

0.200 0.150

0.150

IgG2b

0.100

0.100

0.050

IgG3

0.000

killed killed+alum live 26

live 37

0.050 0.000

Live 26

Live 37

Figure 49.3. IgG isotypes determined on day 43. (A) Immunoglobulin subtypes to capsular antigen F1 for all groups of mice immunized either with killed or live vaccines; (B) Immunoglobulin subtypes to LcrV for the groups of mice immunized with live vaccine cells grown either at 26 or 37°C.

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49.2.4

Human Humoral Responses

Blood samples were obtained from two volunteers who had previously been immunized with live plague vaccine Y. pestis EV, line NIIEG. Donor A1 was immunized once approximately one month prior to the beginning of experiments.

IFNgamma (pg/ml)

6000

4000 d3 d6 2000

0 media

PHA

Y. pestis

naive

media

PHA

Y. pestis

live vaccine

Figure 49.4. IFN-γ production by murine splenocytes stimulated in vitro with HK Y. pestis. Splenocytes from naïve mice, or mice immunized with live Y. pestis were stimulated in vitro with medium, 10 µg/ml PHA, or killed Y. pestis. Culture supernatants were harvested on days 3 and 6 and tested for IFN-γ.

A pre-immune serum from Donor A1 was available. Then, Donor A1 was bled monthly for 8 months post-immunization. Testing of antisera Donor A1 in Western blots and ELISA for reactivity to purified Y. pestis antigens YopM, YopE, LcrV, F1, and Pla produced negative results. Donor A2 had multiple immunizations with the live plague vaccine with the last inoculation occurring at least 5 years prior to the current testing, therefore, pre-immune serum from this donor was not available. Despite a prolonged period of time that passed from the last immunization, Donor A2 had anti-F1 immunoglobulin in the blood, as detected via Western blot (Figure 49.8, lane 4). Also, we observed a weak positive signal of Donor A2’s antiserum with Pla antigen (Figure 49.8, lane 5). However, this reaction was likely non-specific, since Pla protein displayed a similar signal when the sera of three non-immune donors participating in this study was used as a control (data not shown). Determination of IgG isotype usage to F1 antigen revealed that there was no a dominant subtype in Donor A2’s antiserum (Figure 49.9). Thus, we observed a long-lasting circulation of anti-F1 antibodies in the blood of the person immunized with live vaccine, and this fact is in good agreement with previously published findings on human vaccination with Y. pestis EV, line NIIEG (14). Specific antibodies to LcrV were not detected in Donor A2’s serum. This was not a surprising outcome, taking into account our observation that antibody titers to LcrV in mice immunized with live plague vaccine reached their maximum at 5 to 6 weeks, but then started to decline by week eight post-immunization (Table 49.1).

Figure 49.5. Y. pestis elicited Th1/Th2 cytokines. Splenocytes from mice immunized with killed (top panel) or live (bottom panel) Y. pestis were stimulated in vitro with medium, 10 µg/ml PHA, or HK Y. pestis. Culture supernatants were harvested on days 3 and 6 and tested for IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ (See Color Plates).

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477

Figure 49.6. Cytokine and chemokine responses elicited by in vitro stimulation of immune T cells with live or HK Y. pestis. Splenocytes from mice immunized with live Y. pestis were stimulated in vitro with HK or live Y. pestis. Culture supernatants were harvested on days 5 or 6 and tested for RANTES, MIP-1α, KC, G-CSF, IL-17, IL-3, IL-6, GM-CSF, IL-10, IL-5, and IFN-γ.

16000 14000 12000 10000 8000 6000 4000 2000 0 killed immune killed immune killed immune killed immune + media + Y. pestis + ConA + F1

Figure 49.7. Capsular protein F1 stimulation of Y. pestis immune splenocytes. Splenocytes from mice immunized with killed Y. pestis were stimulated in vitro with medium, killed Y. pestis, 1 µg/ml ConA, or F1. Cultures were assayed on days 3 for 3H-thymidine incorporation.

49.2.5

Human T-cell-mediated Responses

Despite the lack of identifiable antibodies to several purified proteins of Y. pestis in the serum of Donor A1, we have found antigen-specific T-cell recall responses in this donor. The Tcell-mediated responses of Donor A2 have been tested as well. For examination of human T-cell-mediated responses, peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from Donors A1 and A2 by centrifugation

Figure 49.8. Antigenic specificity of immune antisera from Donor A2. The left panel shows the silver-stained electrophoretic gel of purified Y. pestis antigens (lanes 1.YopM, 2. YopE, 3. LcrV, 4. F1, 5. Pla and 6. whole bacterial lysate of Y. pestis EV76 grown at 37°C. The right panel shows results of Western blot using antisera from Donor A2, who had been vaccinated with live attenuated Y. pestis.

over Lymphocyte Separation medium. A portion of PBMCs were frozen and stored in liquid nitrogen for future studies. A portion of PBMCs were stimulated with HK Y. pestis grown at 37 or 26°C. These cultures were tested at days 3 and 7 for 3 H-thymidine incorporation. The results of the stimulation have shown that both cells grown at 37 and 26°C provided a strong proliferative response, particularly on day seven (Figure 49.10). The remaining stimulated cells were frozen

478

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0.25

0.20

0.15

0.10

0.05

0.00 anti-lgG

anti-lgG1

anti-lgG2

anti-lgG3

anti-lgG4

Figure 49.9. IgG isotypes to capsular antigen F1 in the serum of Donor A2.

3H-TcR-incorporation (cpm)

35000

d3 d7

30000 25000

Figure 49.11. Antigen-specific stimulation of human PBMC. PBMCs from non-vaccinated Donor B2, and from Donor A1, vaccinated once with live attenuated Y. pestis were stimulated with PHA or HK Y. pestis. 3H-TdR incorporation was determined in day 6 cultures.

20000 15000 10000 5000 0

medium

PHA

Y-pestis 26

Y-pestis 37

Figure 49.10. PBMC stimulation with killed Y. pestis. PBMCs of Donor A1 were stimulated with medium, 10 µg/ml PHA, or HK Y. pestis grown at 26 or 37°C. Quadruplicate cultures were pulsed with tritiated thymidine on days 3 and 7.

for future studies. T-cell-depleted PBMCs were transformed with EBV to generate autologous antigen-presenting cells for use in in vitro re-stimulation to propagate antigen-specific Tcell lines. PBMCs from non-immune (B2) and immune (A1 or A2) donors were stimulated with Y. pestis for proliferation assays, as well as for cytokine production. The PBMCs from B2 did not proliferate in the presence of Y. pestis, while PBMCs from A1 did (Figure 49.11). Figure 49.12 shows the results of testing culture supernatants of in vitro stimulation of human PBMC from immune Donor A2 with PHA, HK Y. pestis grown at 37°C and purified capsular antigen F1 for the number of cytokine and chemokine responses.

Figure 49.12. Cytokine and chemokine responses elicited by in vitro stimulation of human PBMC from immune Donor A2 with PHA, HK Y. pestis grown at 37°C and purified capsular antigen F1. Culture supernatants were harvested on day 3 and tested for IL-10, IFN-γ, IL-4, TNF-α, IL-5, IL-7, IL-12, IL-13 and IL-17 (See Color Plates).

49. Live Plague Vaccine

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6000 IL-8 5000 IL-10 4000

IFN-g

IL-1b

3000

G-CSF

2000

MCP-1 1000 MIP-1b 0 A2

A1

B5

B4

Figure 49.13. Cytokine and chemokine responses elicited by in vitro stimulation of human PBMC from immune Donors A1 and A2 as well as from naïve Donors B4 and B5 with heat-killed Y. pestis. Culture supernatants were harvested on day 3 and tested for IL-8, IL-10, IFN-γ, IL-1β, GM-CSF, MCP-1, MIP-1β (See Color Plates).

Finally, we have compared cytokine and chemokine responses of PBMCs from both immune donors A1 and A2 with those from two naïve donors (B4 and B5) after stimulation with HK Y. pestis cells (Figure 49.13). The results shown in Figures 49.11 to 49.13 clearly demonstrated plague cellular immunity in vaccinated individuals.

49.3

Conclusions

Thus far, our findings support the premise that killed plague vaccine does not elicit antibodies to LcrV, and we would expect to find a lack of T-cell-mediated response to this protein after immunization with this vaccine. Using the live vaccine in the mouse, we have been able to elicit antibodies to LcrV and expect to find LcrV-specific T cells derived from these mice. We have yet to find Y. pestis-specific antibodies to LcrV in the human sera; however, our experiments in mice suggested that the time of circulation of anti-LcrV antibody at detectable levels might be short. In contrast, anti-F1 antibodies could be detected in human serum for years post-vaccination. The F1 antigen-specific T-cell recall responses could be detected in both humans and mice that were followed after all types of immunizations with plague vaccines. Overall, T-cell stimulation studies of PBMCs from human donors immunized once or multiple times with live plague vaccine indicated that there were Y. pestis-specific memory T cells in the blood of the vaccinated individuals. Our data are in good

agreement with recently published observations on long-lasting T-cell responses in veterans of the Gulf conflict (1990–1991) immunized with killed plague vaccine (15).

Acknowledgements. We thank Dr. Valentina A. Feodorova for helpful discussions. This work was supported by grant U54 AI0577156 to NIH/NIAID Western Regional Center of Excellence for Biodefense and Emerging Infectious Diseases.

References 1. Inglesby TV, Dennis DT, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Friedlander AM, Hauer J, Koerner JF, Layton M, McDade J, Osterholm MT, O’Toole T, Parker G, Perl TM, Russell PK, Schoch-Spana M, Tonat K. (2000) Plague as a biological weapon: medical and public health management. Working Group on Civilian Biodefense. JAMA 283:2281–2290. 2. Perry RD, Fetherston JD (1997) Yersinia pestis—etiologic agent of plague. Clin Microbiol Rev 10:35–66. 3. Titball RW, Williamson ED (2001) Vaccination against bubonic and pneumonic plague. Vaccine 19:4175–4184. 4. Anisimov AP, Lindler LE, Pier GB (2004) Intraspecific diversity of Yersinia pestis. Clin Microbiol Rev 17:434–464. 5. Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, Titball RW (1995) A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model. Vaccine 13:1551–1556.

480 6. Meyer KF, Smith G, Foster L, Brookman M, Sung M (1974) Live, attenuated Yersinia pestis vaccine: virulent in nonhuman primates, harmless to guinea pigs. J Infect Dis 129:S85–S112. 7. Welkos S, Pitt ML, Martinez M, Friedlander A, Vogel P, Tammariello R (2002) Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague. Vaccine 20:2206–2214. 8. Titball RW, Williamson ED (2004) Yersinia pestis (plague) vaccines. Expert Opin Biol Ther 4:965–973. 9. Brubaker RR (2003) Interleukin-10 and inhibition of innate immunity to Yersiniae: roles of Yops and LcrV (V antigen). Infect Immun 71:3673–3681. 10. Cornelis GR (2000) Molecular and cell biology aspects of plague. Proc Natl Acad Sci USA 97:8778–8783. 11. Baker EE, Somer H, Foster LW, Meyer E, Meyer KF (1952) Studies on immunization against plague. I. The isolation and

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13.

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characterization of the soluble antigen of Pasteurella pestis. J Immunol 68:131–145. Du Y, Rosqvist R, Forsberg A (2002) Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis. Infect Immun 70:1453–1460. Marshall JD JrBartelloni PJ, Cavanaugh DC, Kadull PJ, Meyer KF (1974) Plague immunization. II. Relation of adverse clinical reactions to multiple immunizations with killed vaccine. J Infect Dis 129:S19–S25. Devdariani ZL, Fedorova VA, Gromova OV, Taranenko TM (1997) Comparative incidence of detection of specific antibodies to Yersinia pestis capsular antigen and lipopolysaccharide in humans immunized with pest vaccine. Klin Lab Diagn 4:39–41. Allen JS, Skowera A, Rubin GJ, Wessely S, Peakman M (2006) Long-lasting T cell responses to biological warfare vaccines in human vaccinees. Clin Infect Dis 43:1–7.

Chapter 44 Dendritic Cells: Biological and Pathological Aspects Jacques Banchereau, John Connolly, Tiziana Di Pucchio, Carson Harrod, Eynav Klechevsky, A. Karolina Palucka, Virginia Pascual, and Hideki Ueno

44.1

Introduction

Our skin and mucosa are covered by considerable numbers of microbes, yet we stay healthy. However, when the microbes break these barriers, the immune system faces several challenges. First, it needs to decide whether or not to respond. Second, if a response is made, it must be tailored to fight that particular microbe. Generating the right class of immune response can be a matter of life and death itself. For example, in leprosy, the tuberculoid form of the disease is characterized by a Type 1 response which keeps the disease in check, but the lepromatous form induces an often fatal Type 2 response (1, 2). Microbe-specific immunity must therefore limit the extension of the infection as well as remove the infected cells. This requires the participation of different cells of the innate immune system as well as cells of the adaptive immune system, specifically T and B lymphocytes that are educated and activated by dendritic cells (DCs; ref. (3). Studies to characterize DCs started in the 1970s (4), but the techniques to generate DCs in vitro were discovered in the 1990s (5–8), allowing the identification of many of their biological and molecular properties. The current vaccines, essentially designed by microbiologists, rely on the generation of antibody responses. However, the development of T cell-mediated immune responses is required for chronic diseases for which no effective vaccines exist. The initiation of T-cell immunity poses several challenges. First, the frequency of microbe-specific T cells is extremely low. Second, infected cells express very few peptide-MHC complexes recognized by the specific T cells (100 or less per cell). Third, infected cells lack the co-stimulatory molecules needed to drive T cell clonal expansion and the production of cytokines, thus lacking the capacity to generate cytotoxic/helper T cells. These challenges are overcome From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

by DCs, which capture microbes, present their antigens, and provide signals necessary for T-cell expansion and differentiation. There are two different paths for the uptake of microbial antigens by DCs: 1) the “classical” way where DCs in tissues capture the invading microbes and migrate into secondary lymphoid organs, and 2) the “novel” way where the microbial proteins are rapidly drained into secondary lymphoid organs and captured by lymphoid-resident DCs (Figure 44.1). Both migrating DCs and DCs resident in lymphoid organs undergo maturation and activate antigen-specific T cells. DCs are also important in launching humoral immunity partly through their capacity to directly activate B cells (9, 10). They also activate innate immune cells, such as natural killer (NK) cells (11, 12) and natural killer T (NKT) cells (13). In addition to the ability to recognize and eliminate what is foreign or aberrant, the immune system has built-in tolerance mechanisms to ignore components of “self” (14). DCs appear to be essential in the maintenance of immunological tolerance both in the thymus and in the periphery (14). Thus, their alteration might contribute to the break of tolerance and thereby to the pathogenesis of autoimmune diseases. Just like lymphocytes, DCs are composed of several subsets with distinct functions (15). This review will emphasize the role of DC subsets in disease pathogenesis and how we can exploit these subsets for therapy.

44.2

DC Biology

44.2.1 Activation of DCs and Launching of Protective Immunity DCs initiate adaptive immune responses and control their quality and magnitude. Immature DCs act as immunological sensors for potentially dangerous microbes, either by directly recognizing microbial components or by receiving signals delivered by the cells of the innate immune system that are exposed to microbes. 409

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Periphery 2 nd wave DC-migration

Antigen DCs in tissue

Afferent lymphatics

1st wave Antigen-draining

Lymphoid Organs

T

Afferent lymphatics

T Lymphoid-resident DCs

Figure 44.1. Two waves of T-cell stimulation by distinct DC subsets. Soluble antigens in the periphery are carried into the draining secondary lymphoid organs in two paths. First, soluble antigens rapidly diffuse into the draining LNs through lymphatics and are captured by lymphoidresident DCs, which stimulate antigen-specific T cells (first wave of stimulation). Second, DCs present in the tissues capture soluble antigens and migrate into the secondary lymphoid organs where they stimulate T cells (second wave of stimulation). These two waves of T-cell stimulation result in the induction of distinct types of T-cell responses (See Color Plates).

Elicitation of the anti-microbial immunity requires DCs to undergo a complex maturation process. Numerous agents activate DCs, including microbes, dying cells, cells of the innate immune system, and cells of the adaptive immune system (Figure 44.2).

Innate Immune System

44.2.1.1 Microbes Dendritic Cells

Adaptive Immune System CD4+ T cells

CD8+ T cells

B cells

Figure 44.2. DCs ferry information from the innate immune system to the adaptive immune system. Immature DCs act as immunological sensors for potentially dangerous microbes, either by directly recognizing microbial components or by receiving signals from cells of the innate immune system that also sense microbes. Immature DCs decode and integrate such signals, and ferry this information to cells of the adaptive immune system. Thus, the type of adaptive immune responses is highly dependent on the nature of the activating stimuli which DCs receive from the innate immune system (See Color Plates).

Activation of DCs by Microbial Components

Many cells recognize microbes through pattern-recognition receptors (PRRs; refs. 16 and 17). The microbes express a limited set of conserved molecular patterns, referred to as pathogenassociated molecular patterns (17), the combination of which is unique to a microbial type. Microbes can directly activate DCs as well as other cells such as stromal cells or neutrophils through at least three PRR families of molecules: Toll-like receptors (TLRs; ref. 16), cell surface C-type lectin receptors (CLRs; refs. 18–20), and intracytoplasmic NOD-like receptors (NLRs; refs. 21 and 22). TLRs have been given the most attention. It appears that DCs express large numbers of receptors and their expression is complex. First, distinct DC subsets express different TLRs. For instance, blood plasmacytoid DCs express TLR1, 6, 7, 9, and 10, but not TLR4, while blood myeloid DCs express TLR1, 2, 3, 4, 5, 6, 7, 8, and 10, but not TLR9 (23, 24). Epidermal Langerhans cells (LCs) isolated from skin lack the expression of TLR4 and TLR5, while dermal interstitial DCs (IntDCs) express many TLRs including TLR2, 4, and 5 (25). In contrast to human myeloid DCs, murine myeloid DCs express TLR9 (16, 26),

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the receptor for CpG/ODN. Thus, the information gathered from mouse studies with respect to the use of CpG/ODN as vaccine adjuvants is of limited interest in the design of vaccines for human use. Similarly, the mouse genome expresses more CLR genes than the human genome. In the human, CLRs allow for distinguishing DC subsets with BDCA2 specifically expressed on plasmacytoid DCs (27), Langerin expressed on LCs (28), and DC-SIGN expressed on IntDCs (29). Many other C-type lectins are more promiscuous and are, as is the case with TLRs, expressed on various cell types including endothelial cells and neutrophils. C-type lectins expressed on DCs act as anchors for a large number of microbes—including viruses, bacteria, parasites and fungi—and allow their internalization, but they also act as adhesion molecules between DCs and other cell types including endothelial cells, T cells, and neutrophils (30). The third family, NLRs, is composed of 22 family members, some of which compose inflammasomes involved in the secretion of interleukin (IL)-1 (21, 22). Very little is known about the expression of NLRs in DCs. Two factors that activate DCs via NALP3 and NOD1/2 have been identified: monosodium urate (31) and muramyl dipeptide (32, 33) respectively. The study of CLRs and NLRs represents a fertile area of DC research for the coming years.

Microbes

44.2.1.2 Activation of DCs by Products of Dying Cells Lysates of dying cells induce the maturation of DCs (34), and some components involved in dying cells enhance antigen presentation by DCs, leading to T-cell immunity (34, 35). These endogenous activating molecules are collectively called damage-associated molecular pattern molecules (36). They include heat shock proteins (37), high-mobility group box 1 protein (38), β-defensin (39), and uric acid (40).

44.2.1.3 System

DCs as Choreographers of the Immune

DCs have long been known to secrete a variety of chemokines (41–43). However, it is remarkable that both pDCs and mDCs secrete sets of “redundant” chemokines in three waves, which correspond to the three stages of the immune response to a microbe. (Figure 44.3; ref. 44). The first chemokines that are produced within 2 to 4 hours in vitro are CXCL1, CXCL2, CXCL3, (which attract innate effectors such as NK cells) and CXCL8 (which attracts neutrophils). This set of cells might limit the spread of infection. The next wave produced within 4 to 8 hours involves CXCL9-11 and CCL3-5, which attract activated memory T cells and monocytes that could replenish the pool of DCs or of tissue

Periphery

Immature DCs

EARLY CXCL1 CXCL8 CXCL2 CXCL3

neutrophils CTLs NK

LATE CCL3 CXCL9 CCL4 CXCL10 CCL5 CXCXL11

Effector memory T

Afferent lymphatics

Mo

Mature DCs

Lymphoid Organs

CCL19 CXCL13 CCL22 CCL21

Naïve T

B,Tfh

T reg

Figure 44.3. DCs are choreographers of the immune system. Both pDCs and mDCs secrete sets of “redundant” chemokines in three waves which correspond to three stages of the immune response to a microbe. The first chemokines that are produced within 2-4 h attract innate effectors such as NK cells and neutrophils. This set of cells might limit the spread of infection. The next wave produced within 4-8 h attracts activated memory T cells and monocytes which could replenish the pool of DCs or of tissue macrophages. At a late stage (>12 h) when mature DCs land in the draining secondary lymphoid organs, they attract B cells, Tfh cells for maturation of humoral responses, and naïve T cells to broaden the immune response. T regs are also attracted to control the extent of the immune response (See Color Plates).

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INNATE IMMUNE SYSTEM

IL-10

TSLP

IL-4

TNF

Activated monocytes

IFNg

IFN a

IL-15

immature mDC

Mo GM-CSF Flt3L

IL-15-DC

IL-10-DC IFN-DC

TSLP-DC IL-4-DC

TNF-DC

IFNg-DC

Figure 44.4. The innate immune system controls the adaptive immune system by modulating the type and function of mDCs. Innate immune cells secrete different sets of soluble factors in response to various stimuli. Granulocytes and macrophages secrete IL-1, IL-6, and TNF upon microbial recognition. NK cells secrete IFN-γ. Mast cells secrete GM-CSF, IL-4 and TNF. Keratinocytes secrete IL-15 and GM-CSF, as well as thymic stromal lymphopoietin (TSLP) in allergic lesions. Plasmacytoid DCs secrete large amounts of Type 1 IFN upon viral encounter. Immature DCs and/or monocytes activated by GM-CSF and/or Flt3L during extravasation are exposed to these factors, resulting in the differentiation into mature DCs with distinct phenotypes. These distinct DCs promote distinct types of T-cell immunity. Thus, mDCs are the key players to convey information from the innate immune cells to the adaptive immune cells (See Color Plates).

macrophages. Finally, at a late stage (>12 h) when mature DCs land in the draining secondary lymphoid organs, they secrete CXCL13 (which attracts B and T cells specialized for humoral responses - aka, follicular helper T cells: Tfh), CCL19 and CCL21 (which attract naïve T cells), and CCL22 (which attracts T regs and might finally permit the termination of the immune response).

44.2.1.4 Activation of DCs by Innate Immune Cells and Tissue Environment Pathogen invasion leads to activation of innate immune cells including neutrophils, basophils, mast cells, and pDCs. Neutrophils are dedicated to phagocytosis and killing of bacteria, while eosinophils, basophils, and mast cells are dedicated to killing parasites. pDCs may have evolved to control viral infection (45). Neutrophils, macrophages, mast cells, and pDCs secrete various proinflammatory cytokines that lead to DC activation (46–48). Epithelial cells also produce numerous cytokines (49, 50). Furthermore, neutrophils activate DCs through cell-to-cell contact between Mac-1/CEACAM-1 and DC-SIGN (51), or through the secretion of β-defensins (39).

DCs also have a reciprocal interaction with innate immune cells. The interaction of DCs with NK, NKT, and γ/δ T cells can occur in the periphery and the secondary lymphoid organs (reviewed in ref. 52). Activated NK cells enhance the ability of DCs to promote Type 1 responses (53). Mature DCs also activate NKT and γ/δ T cells (13, 54–58). In return, CD40L expressed on NKT cells induce the strong activation of DCs (52). The innate immune cell factors activate immature DCs and their precursors into mature cells with distinct phenotypes (Figure 44.4). Thus, Type I IFN, TSLP, TNF, IL-10, IFNγ, or IL-15 yield DC differentiation into IFN-DCs (59–62), TSLPDCs (63, 64a), TNF-DCs (65), IL-10-DCs (66, 67), IFNγ-DCs (53), or IL15-DCs (68, 69), respectively. These distinct DCs induce distinct types of T-cell immunity. For example, TSLPDCs skew T-cell development into inflammatory Type 2 cells, which secrete large amounts of TNF as well as Type 2 cytokines (63). IL-10-DCs promote IL-10-secreting regulatory T-cell (T reg) development (66, 67). IFNγ-DCs promote potent Type 1 T-cell responses through the upregulation of IL-12 secretion (53). IL-15-DCs, which express Langerin and share many characters with LCs (68, 69), are powerful activators of CTLs (68). Thus, the innate immune system, which responds to microbial

44. Dendritic Cells: Biological and Pathological Aspects

invasion, controls the adaptive immune responses by modulating the type and function of mDCs.

44.2.1.5

DC Interaction With Adaptive Immune Cells

Once loaded with the microbial antigens and activated, DCs migrate into the draining lymphoid organs, where they interact with lymphocytes. DCs stimulate CD4+ and CD8+ T cells by presenting antigens in the context of MHC-class II and class I molecules, respectively (70). Microbial lipid and lipopeptide antigens are presented in the context of CD1 family molecules, resulting in activation of αβT cells, γδT cells, and NKT cells (71). DCs also present antigens to B cells (10, 72). At least three families of molecules are involved in the lymphocyte activation: cytokines, B7 family members and TNF family members. Molecules of the IL-12 family, including IL-12 (73, 74), IL-23 (75–77), and IL-27 (78, 79), are secreted from DCs, but differentially regulate immune responses. While IL-12 promotes the differentiation of T cells into Type 1 (73, 74), IL23 promotes the differentiation of T cells into inflammatory Th17 cells (80, 81). IL-27, in contrast, appears to act as an anti-inflammatory agent in vivo, and inhibits the differentiation of Th17 cells (82, 83). Molecules of the B7 family, including CD80 (B7-1), CD86 (B7-2), ICOS-ligand, PD-L1 (B7-H1), and PD-L2 (B7-DC), are essential to the regulation of T cell-mediated immunity and tolerance (84). When compared to IntDCs, LCs express higher levels of CD80 and lower levels of CD86. The expression of these molecules, though typical of DC maturation, does not determine the specific DC function. For example, DCs infected by respiratory syncytial virus (RSV) express high levels of CD80 and CD86 (85) and are potent suppressors of immune responses in vitro (Connolly et al., unpublished observations). The expression of ICOSL, a ligand for ICOS (86), is differentially regulated from CD80 or CD86 (87). Although ICOSL is widely expressed on APCs including B cells, monocytes, and macrophages, high levels of ICOSL appear to be limited to DC subsets specialized in the induction of T regs (88, 89). Among TNF receptor/ligand family molecules, TNF (5) and CD40 ligand (90) were found early to act as activators of DCs. Importantly, CD40 ligation also induces DCs to express other TNF-family molecules such as CD70, 4-1BBL, and OX40L. OX40L on DCs polarizes T cell differentiation into Type 2 (91) and shuts down IL-10 secretion from T regs (92). CD70 is critical for the priming of naïve CD8+ T cells (93, 94) and for the differentiation into IFN-γ secreting cytotoxic T cells (95) or memory T cells (96). 4-1BBL expression is important in the priming of naïve CD8+ T cells and the survival of memory CD8+ T cells (97, 98). DCs also express TNF family molecules associated with B-cell priming and/or differentiation such as BAFF/Blys (99, 100) and its closely related APRIL (101).

44.2.2

Maintenance of Tolerance by DCs

A relatively new theme of research in DC biology is their role in the maintenance of tolerance towards tissue components. DCs

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are involved in the control of both central and peripheral tolerance (14). In the thymus, high affinity autoreactive thymocytes are eliminated upon encountering self-MHC peptide complex (central tolerance). There is evidence that both thymic epithelial cells as well as mature DCs in the thymus are involved in this process (102). However, this step is imperfect and autoreactive T cells are released in the periphery. Thus, mechanisms operate in the periphery to prevent the development of autoimmunity (peripheral tolerance). An important crossroad of central and peripheral tolerance is found in the human thymic Hassall’s corpuscles. There, resident mDCs stimulated by TSLP drive the positive selection of self reactive CD4+CD25+ T regs (64b), which are critical for the maintenance of self tolerance in the periphery. Peripheral DCs are also involved in the maintenance of peripheral tolerance. Non-activated immature DCs continuously present self antigens to autoreactive T-cells in the absence of costimulation, leading to their anergy or deletion (103–105). However, mature DCs also appear to be involved in the maintenance of peripheral tolerance. Mature mDCs can expand functional T regs both in vitro and in vivo (104, 106–108). The apparent contrast between the induction of immunity and tolerance by mature DCs may be best explained by the existence of various stages of DC maturation. Possibly, peripheral tolerance is actively maintained by “tolerogenic” DCs (109). In addition to deleting T cells, tolerogenic DCs induce the differentiation and proliferation of T cells with regulatory/suppressor functions (110, 111). Some pathogens have a capacity to actively render DCs tolerogenic (112). Although the specific markers of tolerogenic DCs are yet to be determined, expression of inhibitory immunoglobulin-like transcript (ILT) receptors might be their feature (113). In vitro-generated DCs exposed to IL-10 express ILT3, which is associated with their tolerogenic functions (114). RSV induces DCs to upregulate the expression of ILT4 and ILT5 as well as PDL1 and renders the DCs unable to activate allogeneic naïve CD4+ T cells. Furthermore, very few of these RSV-infected DCs potently suppress allogeneic CD4+ T-cell proliferation induced by activated DCs (Connolly et al., unpublished observations). This might explain the pathophysiology of RSV infections which are often recurrent due to the inefficient induction of specific adaptive immunity. A few studies indicate that plasmacytoid DCs (pDCs) might be involved in tolerance induction as well. pDCs stimulated via CD40 induce IL-10-secreting regulatory CD4+ T cells (89) as well as suppressor CD8+ T cells (115).

44.3

DC Subsets

There are two main pathways of DC ontogeny from hematopoietic progenitor cells (HPCs). One pathway generates myeloid DCs (mDCs) while another generates plasmacytoid DCs (pDCs), a subset capable of secreting large amounts of type I IFN in response to viral stimulation (48, 116). FLT3-L appears as a major factor governing DC homeostasis in the steady state in mouse and humans. FLT3-L enhances the

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generation of both mDCs and pDCs in vivo (117–119) and in vitro (120, 121). Conversely, FLT3-L deficient mice show a considerable decrease in numbers of DCs in both peripheral and lymphoid tissues (122).

44.3.1

Myeloid DC subsets

Myeloid DCs are found in three compartments: (1) peripheral tissue, (2) secondary lymphoid organs, and (3) blood. In the skin, two distinct types of mDCs are found in two distinct layers. LCs reside in the epidermis, while intDCs are present in the dermis (Figure 44.5; ref. 123). CD34+HPCs, when cultured with GM-CSF and TNF-α (5), give rise to both CD1a+CD14– LCs and CD1a-CD14+ intDCs (124), which display different phenotypes and biological functions. For example, intDCs, but not LCs, produce IL-10 in response to CD40L stimulation (125) and express non-specific esterases (126). IntDCs induce the differentiation of naïve B cells into IgM-secreting plasma cells through the secretion of IL-6 and IL-12 (126, 127) but are not very efficient at priming naïve CD8+ T cells. In contrast, LCs are particularly efficient at inducing cytotoxic high avidity CD8+ T cells (Klechevsky et al., submitted) and they are not able to activate naïve B cells into IgM-secreting plasma cells (126). Both in vitro-derived and epidermal LCs are also strong activators of naïve CD4+ T cells, inducing their polarization into T cells secreting IFN-γ (Th1) as well as cells secreting IL-4, IL5, and IL-13 (Th2). Both in vitro-derived and dermal intDCs can also expand IFN-γ-producing CD4+T cells, but they are particularly efficient in inducing a specific type of CD4+ T

cells—follicular helper T cells (Tfh)—which help immunoglobulin production from B cells (Figure 44.6; Klechevsky et al., submitted). LCs and IntDCs appear to be equally potent at activating the proliferation and differentiation of memory T and B lymphocytes. These recent findings have led us to propose that intDCs (dermal DCs) preferentially induce humoral immunity, while LCs preferentially induce cellular immunity. These in vitro findings with human DC subsets isolated from skin or generated from CD34+ HPC cultures are in accord with several in vivo data in the mouse. Mouse dermal DCs migrate into the outer paracortex, just beneath the B-cell follicles (128, 129), whereas LCs migrate into the T cell-rich inner paracortex (129). This concept may be particularly important in vaccines designed to activate humoral responses or cellular responses, or both (discussed later). The concept of differential regulation of T-cell immunity by distinct DC subsets also applies to the mouse (130, 131). Targeting CD8α+ mDCs with anti-DEC205 Ab-OVA conjugates preferentially induces CD8+ T-cell immunity, while targeting CD8α- mDCs with anti-DCIR2 Ab-OVA conjugates preferentially induces CD4+ T-cell immunity (132). Peripheral lymphoid organ-resident DCs are also involved in both immunity and tolerance. LN-resident DCs capture microbial antigens rapidly delivered through lymphatics and conduits, and upon stimulation through PRRs, these DCs induce the proliferation and IL-2 secretion of antigen-specific T cells (128). In the steady state, LN-resident DCs capture self antigens and induce tolerance. The germinal center of secondary lymphoid organs contains germinal center DCs (133), whose function remains to be established.

Skin Epidermal Langerhans cells

Blood Dermal Interstitial DCs

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C-type lectin

Langerin

DC-SIGN Mannose Receptor

(DC-SIGN) (Mannose Receptor)

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CD11c CD1a E-cadherin

CD11c CD1a /CD14 CD11b CD36 Factor XIIIa

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1, 2, 3, 4, 5, 6, (7), 8, 10

1, 6, 7, 9, 10

TLRs

1, 2, 3, 6, (7), (10)

Figure 44.5. Human DC subsets in vivo. In the skin, two mDC subsets, Langerhans cells (LCs) and interstitial DCs (intDCs), reside in two distinct layers. Blood contains two major DC subsets, mDCs and pDCs. These DC subsets express different sets of molecules, including C-type lectin receptors (CLRs), or Toll-like receptors (TLRs) (See Color Plates).

44. Dendritic Cells: Biological and Pathological Aspects Microbes

Epidermis

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Periphery Epidermal Langerhans cells

Dermis Dermal Interstitial DCs

Lymphoid Organs

B cell follicle

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T T

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Figure 44.6. Interstitial DCs preferentially induce humoral immunity while Langerhans cells induce cellular immunity. Upon recognition of microbes, epidermal LCs and dermal IntDCs migrate to the secondary lymphoid organs through afferent lymphatics. Dermal IntDCs migrate into the outer paracortex, just beneath the B-cell follicles, whereas LCs migrate into the T cell-rich area. LCs are particularly efficient at inducing high avidity cytotoxic CD8+ T cells and are also strong activators of naïve CD4+ T cells, inducing their polarization into T cells secreting IL-4, IL-5 and IL-13 (Th2). In contrast, IntDCs are particularly efficient at inducing the differentiation of naïve B cells into IgMsecreting plasma cells and CD4+ T cells, which help immunoglobulin production from B cells (follicular helper T cells: Tfh). Both LCs and IntDCs are equivalently efficient at inducing the differentiation of CD4+ T cells that secrete IFN-γ (Th1) (See Color Plates).

44.3.2

Blood DC Subsets

Myeloid DCs and plasmacytoid DCs circulate in the blood and can be found as linnegHLA-DR+ cells. mDCs express CD11c, while pDCs express IL-3Rα chain (CD123) as well as BDCA-2 (Figure 44.4; refs. 48, 116, 134–136). mDCs and pDCs differentially express TLRs, indicating a specialization of DC subsets for the recognition of microbes. pDCs appear to directly migrate into the inflamed secondary lymphoid tissues through the HEV (137). mDCs are thought to first migrate to the inflammatory site and then into the secondary lymphoid tissues through afferent lymphatics (45). Plasmacytoid DCs exposed to viruses secrete large amounts of Type I IFN (48) as well as IP-10, TNF, and IL-6 (138). pDCs also differentiate into cells with the typical morphology and functions of DCs. Autocrine TNF is involved in the maturation of pDCs into APCs, and in the downregulation of Type I IFN secretion (138). pDCs act as antigen-presenting cells in vitro, and perhaps in vivo as well. We recently found that pDCs display unique MHC class I compartments, which permit direct vesicular loading of MHC class I ligands and thereby allow

prompt activation of cytotoxic CD8+ T cells (DiPucchio et al., unpublished observation). pDCs activated with IL-3 and CD40-ligand (CD40L) have been shown to secrete negligible amounts of IL-12, as well as to prime Th2 responses (139) and CD8+ T cells with regulatory/suppressor function (115). However, pDCs also induce Th1 responses in vitro when stimulated with both viral antigens and CD40L (26, 140). In humans, CD2 distinguishes two pDCs subsets (Matsui et al., unpublished observation). Both CD2- and CD2+ pDC subsets are able to secrete Type I IFN in response to viral exposure; However, CD2+ pDCs, which represent 20% to 30% of blood pDCs, efficiently kill target cells in a TRAIL-dependent fashion. Furthermore, CD2+ pDCs are more potent than CD2pDCs at inducing the proliferation of allogeneic naïve CD4+T cells (Matsui et al., unpublished observation). Recently, interferon-producing killer DCs (IKDCs) have been identified as a novel mouse DC subset able to secrete large amounts of Type I and II IFNs as well as to kill target cells (141, 142). Whether CD2+ pDCs represent a counterpart of mouse IKDCs is yet to be established.

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DC Subsets Regulate B-cell Responses

Little is known about the ability of DCs to present antigens to B cells, although early studies showed that DCs loaded with proteins induce humoral responses when administered to animals (143). DCs can retain unprocessed antigens (144) and transfer them to B cells (145). Immune complexes captured by DCs through the inhibitory Fc receptor, FcγRIIB, are retained in a non-degradative intracellular vesicular compartment, and presented as a native antigen to B cells (72). Two-photon intravital imaging further revealed the direct interaction of DCs and B cells in the extrafollicular region in lymph nodes (10). Both LCs and IntDCs derived from CD34+ HPCs promote the proliferation of CD40-activated B cells as well as the differentiation of memory B cells into plasma cells secreting IgG and IgA (10, 146). However, only IntDCs induce the differentiation of CD40-activated naïve B cells into IgM-secreting plasma cells in an IL-12-dependent manner (127). DCs appear to switch isotypes of B cells through BAFF and/or APRIL, molecules of the TNF family (101, 147). Co-cultures of naïve B cells with DCs induce class switch towards IgA1 and IgA2 in response to IL-10 and TGFβ (148). pDCs stimulated with influenza virus also promote B-cell differentiation into Ig-secreting plasma cells in a Type I IFNand IL-6-dependent fashion (149).

44.4

DCs in Diseases

Dysregulation of the DC system leads to the development of diseases including autoimmunity and allergy. DCs are also targets that microbes use for their survival.

44.4.1

DCs in Autoimmunity

DCs bearing self antigens are able to induce autoimmunity in mouse models of autoimmune cardiomyopathy (150), and systemic lupus erythematosus (SLE), a systemic disease in which antibodies are formed against several self antigens, especially nucleoproteins (151). In our view, a pivotal step in a specific autoimmune disease is excessive production of a particular cytokine, which results in the activation of DCs along a unique path (152). For instance, TNF plays an essential role in rheumatoid arthritis (RA; ref. 153). Indeed the best demonstration of the role of TNF is the beneficial effects of TNF antagonists in RA patients (153), as well as several other diseases including psoriasis. Thus an excessive production of TNF might result in ectopic maturation of DCs that would otherwise control peripheral tolerance. DCs themselves might represent a major source of TNF, as observed in psoriasis where large amounts of TNF are secreted by mDCs infiltrating the inflamed skin lesions (154). SLE appears to be associated with an increased production of Type I IFNs (59). Monocytes from SLE patients’ blood act as DCs, inducing the robust proliferation of allogeneic naïve CD4+ T cells. A combination of Type I IFN and

GM-CSF results in the differentiation of monocytes into mature DCs. Such mature DCs can present antigens from dying cells in an immunogenic rather than tolerogenic manner (59). Genomic studies on blood cells indicated that most if not all SLE patients overexpress IFN-induced genes (155, 156). The clinical relevance of the IFN signature in SLE is indicated by its loss upon treatment of patients with high dose glucocorticoids (156), a standard treatment of disease flares which is associated with a total disappearance of pDCs from the circulation (157). In SLE patients, the secretion of Type I IFN might happen in the secondary lymphoid organs or in the skin lesions, which are infiltrated by pDCs (158). It is possible that immune complexes present in the serum of SLE and/or TLR activation also contribute to the DC maturation (159, 160). DCs generated in the presence of SLE sera also drive the differentiation of CD8+ T cells toward fully active cytotoxic effector T lymphocytes (59, 161), which might be actively involved in the generation of autoantigen fragments through the destruction of target tissues. These autoantigens could be captured and presented by mDCs, thus further broadening the autoimmune process. pDCs and Type I IFN are also proposed to be pathogenic in other autoimmune diseases, including psoriasis (162), insulin-dependent diabetes mellitus (163), dermatomyositis (164, 165), and Sjogren’s syndrome (166). Studies in mice suggest that DCs might be used in the treatment of autoimmunity through their ability to induce T regs. Repetitive injections of “semi-mature” DCs induce antigen-specific protection of mice from experimental autoimmune encephalomyelitis and thyroiditis (167, 168). In NOD mice, which spontaneously develop diabetes, DCs can induce the generation of T regs in vitro, which provides a therapeutic benefit even after onset of disease (169). Indeed, T regs appear to suppress DCs that induce autoimmunity by presenting autoantigens (107, 169). In keeping with this, animals depleted of T regs show autoimmunity that is associated with expansion of activated DCs (170, 171). Thus, tolerogenic DCs, such as those generated with IL-10 (66, 172) or those infected with RSV, might be considered for the treatment of autoimmunity or the induction of specific tolerance in organ transplants.

44.4.2

DCs and Allergy

In healthy individuals, mDCs at mucosal surfaces are thought to capture harmless environmental antigens such as pollens and dust mites and silence the corresponding T cells by inducing IL-10-producing T regs through interaction between ICOS/ICOSL (88). Plasmacytoid DCs may be involved in the induction and maintenance of tolerance (89, 173), as in vivo depletion of pDC in mouse results in the exacerbation of asthmatic symptoms (173). Furthermore, ICOSL is expressed on activated pDCs at higher levels than on activated mDCs in vitro, and promotes the differentiation towards IL-10-producing T regs (89).

44. Dendritic Cells: Biological and Pathological Aspects

In allergy, mDCs polarize T-cell differentiation towards Type 2. TSLP, which is secreted by epithelial cells at the allergic inflammatory sites, induces DCs to express high levels of OX40L without secreting IL-12 family molecules (174), or Type I IFNs. These DCs skew T cell response towards inflammatory Type 2, characterized by the secretion of not only IL-4, 5, and 13, but also high levels of TNF (63, 175). TSLP also allows the expansion of circulating memory Th2 cells (176). TSLP has thus been implicated in atopic dermatitis (63, 177), as well as asthma (178). Proinflammatory cytokines such as TNF and IL-1, and Type 2 cytokines such as IL-4 and IL-13, promote TSLP secretion from keratinocytes (179). Novel anti-allergy drugs are being designed to modulate DC functions. For instance, sphingosine 1-phosphate receptor agonist (FTY720) (180) or iloprost, a prostacyclin-2 analog (181) abrogates experimental asthma by affecting DC function. Activation of the D prostanoid (DP)1 receptor suppresses asthma by modulating lung function and inducing T regs (182).

44.4.3

DCs and Infection

Pathogens have developed multiple approaches to alter DCs (183). Certain bacteria such as Yersinia pestis deliver toxins into phagocytes including DCs (184). Many viruses such as measles virus and herpes simplex virus-2 induce apoptotic cell death in DCs (185, 186). Viruses have evolved to selectively block antigen presentation on MHC class I and II in virallyinfected cells (187). HCMV-infected DCs (188) and RSV infected DCs (Connolly et al., unpublished observations) show a partial downregulation of their MHC, leading to a reduced antigen presentation. HCMV-infected DCs express Fas-L and TRAIL, which allow them to delete activated T lymphocytes (188). Herpes simplex virus-1 inhibits DC migration from the periphery to lymphoid organs by blocking CCR7 expression (189). Many pathogens, including herpes simplex (190), HIV (191), and anthrax (192), actively block DC maturation. Another strategy for pathogens is to alter the T cell polarizing function of DCs (i.e., switching responses from protective Th1 to nonprotective Th2 in infections with Candida albicans; ref. 193). Exposure of DCs to Bordetella pertussis (112) and RSV (Connolly et al., unpublished observations) results in IL-10 production, leading to immunological tolerance. DCs exposed to RSV are, in our hands, the most potent inhibitors of mixed lymphocyte reaction compared with “tolerogenic” DCs prepared by exposing monocytes to cytokines such as IL-10 (67) and TGFβ, or pharmacological reagents such as VitD3 (194) and steroids (195). In addition to the alteration of mDC functions, microbes may also have mechanisms to evade pDCs. Interestingly, pDCs are diminished in the blood of patients with several infections including HIV (196, 197) and RSV (198). Some pathogens also use DCs for their own replication and spreading in the infected host. For example, HIV-1, CMV and ebola virus bind to the antigen uptake receptor DC-SIGN/

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CD209 and use it to enter into the endocytic system of DCs, which later transmit infectious virus to other targets like T cells (199–202). In stark contrast to DC-SIGN, which mediates HIV transmission, Langerin appears to be a natural barrier to HIV transmission (203), since it binds HIV and directs it to Birbeck granules where it is degraded.

44.4

DCs and Cancer

Tumors employ several approaches to subvert the immune system (reviewed in ref. 204) with three major consequences: (1) prevention of specific immunity, (2) induction of specific tolerance, and (3) triggering of suppressive pathways. Stat3, which is constitutively activated in diverse cancers of both hematopoietic and epithelial origin, acts as a critical regulator of inflammation (205). Constitutive Stat-3 activity in tumors inhibits the production of proinflammatory cytokines while promoting the release of soluble factors that suppress DC functions (206). Furthermore, these factors upregulate Stat-3 expression in DCs, resulting in the induction of anti-tumor tolerance rather than immunity (207). Several cytokines also have been implicated in suppression of DCs in the tumor beds, including vascular endothelial growth factor (208, 209), IL-10 (67, 210–212), and IL-6 (213). Tumor cells can interfere with the DC antigen capture and presenting pathways through molecules such as MUC1 (214). Furthermore, at the early stages of disease, the immune response can simply be misled and used to promote cancer development. In breast cancer, tumors attract immature DCs (215) and trigger their maturation leading to the skewing of CD4+ T-cell differentiation towards Type 2 (216). In particular, IL-13 secreted from such T cells is responsible for the tumor growth, since blocking of IL-13 inhibits the tumor growth in a humanized mouse model of breast cancer (216). However, DCs also possess anti-tumor functions. In particular, they might be involved early in the capture of cancer cells for the generation of “spontaneous” anti-tumor immunity. Furthermore, under certain circumstances DCs express cytotoxic molecules. Plasmacytoid DCs express granzymes but no perforin (Matsui et al., unpublished observations; ref. 217). Type I IFN enables mDCs to kill tumor cells by co-expressing TRAIL (218). Furthermore, immature DCs can induce tumor apoptosis (218–220).

44.5 Design of Vaccines Through DC Biology Given their capacity to modulate immune responses, DCs represent an attractive target for the development of both preventive and therapeutic vaccines. Two approaches to DC based vaccines are being developed: antigen loaded ex vivo generated DCs and in-vivo DC targeting.

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J. Banchereau et al.

Ex Vivo DC-based Vaccines

Ex vivo DC vaccines were initially tested in a few healthy volunteers (221, 222) and more extensively in patients with different forms of cancer (reviewed in refs. 223 and 224). Studies performed with DCs generated by culturing monocytes with GM-CSF and IL-4 (IL-4-DCs; refs. 221 and 222) led to the conclusion that, when matured with a cocktail of proinflammatory cytokines, DCs can induce broad T-cell immunity. Priming of KLH-specific CD4+ T cells, and boosting of TTspecific CD4+ T cells as well as influenza matrix-specific CD8+ T cells (222, 225) have been documented. Maturation is important since the injection of immature DCs results in inhibition of antigen-specific CD8+T cell effector function and the appearance of antigen-specific IL-10-producing cells (221). Most trials in cancer-bearing patients have utilized IL-4DCs (7, 8, 226–230). However, monocytes are not the only source of DC precursors/progenitors that have been used in clinical studies. Blood DCs loaded with specific idiotype proteins (231) and recombinant fusion proteins (232) have been used in a variety of cancers. We have vaccinated patients with metastatic melanoma with antigen-loaded DCs derived from CD34+ hematopoietic progenitor cells (CD34-DCs; ref. 3). CD34-DC vaccination elicited melanoma-specific immunity and patients who survived longer were those who mounted immunity to more than two melanoma antigens (233). These results justify the design of larger follow-up studies with a range of different DC vaccines to assess their immunological and clinical efficacy. An important question is the loading antigens of the loading of antigens by the DCs. We have shown in prostate cancer (234), melanoma (235), and breast cancer (236) that DCs loaded with a killed allogeneic tumor cell line can induce tumor-antigen-specific T-cell immunity. Thus, we have vaccinated 20 patients with stage IV melanoma with autologous IL-4-DCs loaded with a killed Colo829 allogeneic melanoma cell line followed by activation with TNF and CD40L (227). The estimated median overall survival is 22 months with a range of 2 to 53 months. In two patients who failed previous therapy, they induced durable objective clinical responses, one complete regression (CR) and one nearCR lasting 18 and 46 months, respectively. In one of these patients, vaccination led to elicitation of CD8 T-cell immunity specific to a novel peptide-derived from MART-1 antigen, suggesting that cross-priming/presentation of melanoma antigens by DC vaccine had occurred. These early phase I studies have concluded that DC vaccines are safe and can induce immune responses as well as some clinical responses.

44.5.2

Targeting DCs In Vivo

The ex-vivo generated DC vaccines discussed above will permit us to acquire useful knowledge about DC biology in vivo in humans and eventually permit us to treat patients. However, novel strategies have been proposed to directly target the antigens to DCs in vivo. Multiple DC surface molecules have been considered as targets. Such targets must allow internal-

ization of the antigen cargo and its processing for presentation on both MHC class I and class II molecules. This eventually may lead to the eradication of infectious agents such as HIV (237, 238) and malaria (239). Different targets are expressed on different murine DC subsets, which yield different functional outcomes, but much less is known regarding human DCs. We have recently observed that conjugates of influenza hemagglutinin or matrix protein with anti-Langerin, ASGPR, Dectin-1, Lox-1, can be cross-presented to peripheral blood CD4+ and CD8+ T cells (unpublished observations). We are expecting considerable activity in the field of DC targeting because of its potential for yielding a wealth of vaccines, possibly the first vaccines generated by immunologists.

44.6

Conclusion

Considerable progress has been made in the understanding of the basic biology of DCs, both in vitro and in vivo as well as in the context of diseases. Much remains to be done to translate this new knowledge into medicine. The complexity of the DC system requires their rational manipulation. We foresee that the improved vaccines targeting DCs will permit us to treat and prevent many chronic infectious diseases due to viruses (HIV, hepatitis C) bacteria (Mycobacteria) and parasites (malaria) as well as cancer. We also foresee that the manipulation of DCs will be used to dampen immune responses possibly by turning on T regs, therefore helping patients suffering from allergy, autoimmunity and those in need of organ grafts.

Acknowledgments. This manuscript is dedicated to all the patients and volunteers who participated in our studies and clinical trials. We thank former and current members of the Institute for their contributions to our progresses. We thank Cindy Samuelsen for continuous help. We thank Dr. Michael Ramsay and Dr. William Duncan for their continuous support. We thank the NIH (AI068842, AR054083, 5U19AI057234, CA84512, 2R01CA078846, & 5R01AR050770), the Alliance for Lupus Research, the Dana Foundation, the Baylor Health Care System, and the Baylor Health Care System Foundation for their support.

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Chapter 45 Immunomic and Bioinformatics Analysis of Host Immunity in the Vaccinia Virus and Influenza A Systems Magdalini Moutaftsi, Bjoern Peters, Valerie Pasquetto, Carla Oseroff, John Sidney, Huynh Hoa-Bui, Howard Grey, and Alessandro Sette

45.1

Introduction

In the following study we will present a review of our efforts aimed at the characterization of host immunity directed against the vaccinia and influenza A viruses. In the case of vaccinia virus (VACV), our investigations combined bioinformatics methods with biochemical and immunological experimentation; while in the case of influenza A we have exclusively undertaken a bioinformatics-based analysis of various databases and data present in the scientific literature. Over the last few years our laboratory has been interested in the study of the cellular responses directed against poxviruses in general, and in particular those induced in humans and mice by immunization with live VACV. Vaccination with VACV has been in use as a means to protect against smallpox and other poxviruses for over two centuries. Furthermore, experimental VACV infection has been widely utilized in various animal models, and indeed poxviruses are one of the most commonly used viral vectors. Despite its common use, until recently remarkably little was known regarding the specificity of antigens and epitopes that are recognized following VACV infection or immunization. Mapping cellular immune responses against VACV is of interest because exact knowledge of the epitopes recognized allows one to determine whether they are conserved in variola major virus, the causative agent of smallpox, and in other poxviruses of practical and medical significance, such as monkeypox virus, a poxvirus responsible for human outbreaks, or modified VACV Ankara (MVA), a poxvirus widely utilized as a viral vector for human vaccine studies. The information acquired from studies with VACV can be used to estimate the fraction of the immune response likely to cross-react with these heterologous viruses, and thereby is of significance in the con-

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

text of protective immunity. Furthermore, the knowledge of which epitopes are recognized allows for the establishment of accurate ELISPOT and intracellular cytokine staining assays, and the production of tetrameric-staining reagents, which can be utilized to evaluate new vaccines that are currently under development. Finally, detailing VACV immune responses offers a unique opportunity to understand and characterize immunodominance in a complex pathogen. In the case of influenza A, we have taken a purely bioinformatic approach to analyze the wealth of information existing in the scientific literature relating to the B- and T-cell epitopes recognized by a variety of different hosts. The purpose of this analysis was twofold. First, we wanted to compile and make easily accessible to the scientific community all data relating to influenza A-derived epitopes. This information can be of potential use in basic investigations, as well as for the evaluation of influenza diagnostics and vaccines. Second, we also wanted to examine whether any gaps exist in the collective knowledge available in the published literature, thereby highlighting areas for potential further investigations and inspiring future research directions. Herein, we present the results of these analyses.

45.2 Demonstrating the Success of Bioinformatics-based Epitope Predictions Using VACV as a Model Pathogen Our overall strategy to map responses against VACV entailed the combined use of bioinformatics-based predictions and experimental validation. Responses following VACV vaccination that are restricted by common human and murine class I molecules were investigated, allowing the generation of three different data sets. The first data set, comprised of the T-cell responses in C57BL/6 (H-2b) mice, led to the validation of the overall approach. The second and third sets characterized the responses in HLA transgenic mice and human vaccines, respectively. As a read-out, IFN-γ production in response to various peptides or infected cells was 429

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determined by using common assays such as ELISPOT and intracellular cytokine staining.

45.2.1 Validation of Bioinformatics-based Epitopeprediction in the H-2b Murine Model System In our first study, we utilized C57BL/6 mice as a murine model system to address whether the totality of CD8+ T-cell responses could be identified by the bioinformatics prediction approach (1). A specific concern with the bioinformatics-based approach was that a large fraction of epitopes might represent non-canonical sizes and/or motifs and thereby will preclude the accurate and comprehensive identification of the epitopes responsible for the overall anti-VACV response. This experiment was necessary to validate the bioinformatics approach, since our previous study utilizing an expression library approach (2) identified a set of five distinct epitopes accounting for only about 40% of the total VACV-specific CD8+ T-cell response, based on the level of IFN-γ production. The study by Moutaftsi et al. addressed the experimental question whether it was possible to identify the remaining 60% of the total response (1). As such, the study was designed to address whether bioinformatics methods might be superior or inferior to empirical methods, and more specifically, whether the issue of non-canonical motifs, post-translational modifications, and unusually long or short peptides represented a serious concern. For this purpose, the whole VACV genome was scanned for all possible 8-, 9- and 10-mer peptides, covering a total of approximately 175,000 different sequences. Utilizing our in-house Vaccinia ORF s 8-mer peptides 9-mer peptides 10-mer peptides

258 58744 58486 58228

Top 1%of MHC binding predicted

Kb 8-mers Kb 9-mers Db 9-mers Db 10-mers Total

564 564 564 564 2256

Test pools of 10 peptides

Number of pools Positive pools

227 68

Deconvolution of positive pools

Number of peptides Positive peptides

674 54

Distribution of positive peptides

Kb

8-mers Kb 9-mers Db 9-mers Db 10-mers Total

18 9 18 4 49

Figure 45.1. Distribution of Kb and Db VACV-specific epitopes.

developed algorithms, each peptide was scored for its predicted capacity to bind Kb and Db molecules. The top 1% scoring 8- and 9-mer peptides in the case of Kb, and 9- and 10-mers in the case of Db, were selected and synthesized (Figure 45.1). Splenocytes obtained from mice that were experimentally infected for 7 days with VACV intra-peritoneal (i.p.) were utilized to screen all 2,256 peptides for T-cell reactivity. Due to the large number of peptides, they were initially tested in pools of 10, followed by the deconvolution of the positive pools to identify the discrete epitopes responsible for the specific activity. From these experiments, a total of 27 different Kb and 22 Db VACV-specific epitopes were identified, thus revealing a much broader spectrum of responses than originally suspected. Importantly, we were able to show that a pool containing all 49 identified epitopes could account for 95% of the total antiVACV response, measured as the total number of CD8+ T cells secreting IFN-γ in response to VACV-infected target cells. Furthermore, it was found that 43 of the 258 possible VACV proteins could elicit CD8+ T-cell responses. Together, these data suggest that the cellular immune response against VACV is remarkably broad rather than directed against a few immunodominant epitopes/antigen(s). From these experiments we thus concluded that predictable epitopes account for the majority of CD8+ T-cells responses to a complex pathogen such as VACV. In addition, the results highlight an unsuspected complexity of responses directed against VACV and suggest that coupling of genomic information with accurate epitope predictions should allow drawing definite maps of the interaction between complex pathogens and their hosts.

45.2.2 Identification of HLA-restricted Class I VACV-specific Epitopes 45.2.2.1 VACV-specific CD8+ T-cell Epitope Identification in HLA-transgenic Mice In an independent series of experiments we undertook the identification of VACV-specific epitopes restricted by human class I molecules. In a first set of experiments, we targeted epitopes restricted by the common HLA molecules B∗0702, A∗0201 and A∗1101 using HLA-transgenic mice (3). Each of these molecules represents a prototype allele of the B7, A2 and A3 supertypes, respectively (4). In an approach similar to that described above, a set of epitopes predicted to bind with high affinity to the corresponding HLA molecules were tested in HLA-transgenic mice expressing the respective molecule. Altogether, 14 A∗0201, 4 A∗1101 and 3 B∗0702 putative epitopes were identified. HLA restriction of the 21 newly identified epitopes was demonstrated by stimulating purified CD8+ T cells derived from VACV-infected HLA-transgenic mice with HLA-matched and HLA-mismatched antigen-presenting cells (APCs) that were pulsed with each individual peptide. We were able to demonstrate that the epitope-induced T cells recognized human APCs

45. Immunomic and Bioinformatics Analysis Vaccinia and Influenza A Viruses

that were infected with VACV in vitro, suggesting that the epitopes are naturally processed by human cells, and thereby excluding differential processing in human and murine cells as a factor limiting the usefulness of epitope identification in transgenic mice. Additionally, in the HLA transgenic mouse studies, it was observed that co-expression of different major histocompatibility complex (MHC) molecules can dramatically influence the repertoire of epitopes recognized. It was also noted that the identified VACV epitopes were highly homologous with corresponding MVA and variola sequences, highlighting their potential relevance to anti-variola immunity, and also suggesting that these epitopes could be utilized to monitor responses of humans vaccinated with experimental constructs based on MVA as a viral vector delivery system.

45.2.2.2 VACV-specific CD8+ T-cell Epitope Identification in Human Vaccines We have also performed experiments utilizing predicted class I binding peptides, to characterize responses in PBMC from recently vaccinated human volunteers (5). These studies led to the identification of 48 new epitopes, derived from 35 different viral antigens. These responses were restricted by the HLA A1, A2, A3, A24, B7, and B44 supertype molecules. Some antigens (D1R, D5R, B8R, C10L, C19L, C7L, F12l, and O1L) appeared to be dominant, as multiple donors, multiple epitopes, and multiple HLA molecules will recognize them, further supporting the observation mentioned above that the response to VACV is broad. In general, however, it was observed that a large degree of diversity existed amongst different individuals at the level of specific epitopes recognized. In the case of six different individuals expressing the A∗0201 molecule, no single epitope was recognized in all individuals, and no individual recognized all different epitopes. The molecular basis of this phenomenon is unclear, but is consistent with the observation made in the case of HLA transgenic mice where the repertoire of co-expressed MHC molecules seemed to influence the repertoire epitope. Consistent with this observation, only a limited overlap was noted to exist between the repertoire of VACV specificities recognized in HLA transgenic mice and humans.

45.2.3 Structural Features of the Antigens Recognized by Cellular Immunity Based on our data, and on additional data from studies contained in the literature, a map of VACV antigens that are recognized by murine and human immune responses is starting to emerge. An epitope set has been defined covering murine and human MHC Class I, while the definition of class II epitopes is still in progress. There is a high degree of homology between the vaccinia epitopes identified and the corresponding variola, MVA, and monkeypox virus sequences. This epitope set could be of use in the evaluation of new vaccines and diagnostics, as well as for basic studies evaluating

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poxvirus-specific immunity in mice and humans. The definition of immunodominant epitopes and antigens following VACV immunization allows for some insights into the breadth of immune responses and into the mechanisms of immunodominance in a complex pathogen. In general, it appears that cellular immunity can recognize a large number of VACV antigens. The antigens recognized are dispersed fairly evenly throughout the genome, in both the more conserved central region and the more variable adjacent regions. Compilation of these data allowed a preliminary analysis of the structural features associated with cellular immune recognition. It was found, that in the case of class I responses, early antigens are recognized most frequently, but late antigens are also recognized. In fact, about a third of the antigens recognized by class I responses are late antigens. This is surprising because it was widely believed that only early antigens are recognized by class I-restricted responses. In terms of viral protein function, class I-restricted responses are directed against virulence factors, structural proteins and viral regulation molecules with frequencies approximately equal to their distribution in the viral genome. As mentioned above, the response to VACV is broad and diverse. This is of particular significance in the context of viral evolution, and with respect to concerns over bioterrorist threats involving the use of variola virus, the causative agent of smallpox. If the immune responses induced by VACV vaccination were focused on only one or few epitopes/antigens, then it would conceivably be possible for a mutated or engineered virus to escape protective immunity. The data gathered in the studies summarized above suggest that this is not the case. Indeed, the fact that immune responses against poxviruses are highly broad and diverse implies that it is difficult for these viruses to escape immune responses through epitope mutation, and might explain why these viruses have evolved a diverse array of virulence factors to interfere with the host immune responses.

45.3 Immune Epitope Database and Analysis Resource (IEDB) and Mapping the Known Immune Responses Against Influenza A Virus In the second part of this paper we will describe our analysis of immune epitope data relating to influenza A virus. This analysis relies on the IEDB, and for this reason the database structure and functionality will be briefly summarized in this and the following paragraphs. A more detailed account can be found in recent review articles (6, 7).

45.3.1

The IEDB

The purpose of the IEDB is to catalog and organize an evergrowing body of immunological information. Both B- and T-cell epitopes from infectious pathogens, and experimental

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and self-antigens are all considered within the scope of the database, with priority assigned on Category A-C pathogens and emerging diseases. Epitope information is captured from a variety of host species, including humans, non-human primates, rodents, and other species for which detailed epitope information is available. It is anticipated that the database could assist in the development of new methods to predict and model immune responses, and also to assist in the development of vaccines and diagnostics (www.immuneepitope.org). A clear and rigorous definition of what constitutes an epitope is obviously crucial to the effort of designing and populating the database. The IEDB defines an epitope as “the chemical structure recognized by specific receptors of the immune system (antibodies, MHC molecules, and/or T cell receptors).” A narrower definition of epitope would correspond to “the structure recognized by antibodies and TCRs.” However, this definition was broadened to allow inclusion of MHC binding and elution data, as this data is widely utilized to generate epitope predictions. It was further noted that although MHC binding does not prove immunogenicity, it implies, depending on the given experimental circumstances, potential immunogenicity. In this respect, epitope-related data inherently is very contextdependent. Without question, there are key features of epitope-associated data that are context-independent, such as those associated with the sequence or structure of the epitope, or with the specific (literature) citation. However, there are many context-dependent attributes, such as those related to the host recognizing the epitope and the natural or experimental environment. Representation of these context-dependent features of an epitope is a novel component of the IEDB. Based on these principles, the IEDB structure organizes more than 300 different data fields in various classes of concepts that are in turn organized in an informal ontology (8). Examples of classes include information related to the epitope source, chemical structure, the antigen-presenting cell, the antibody and T-cell receptor, the assay utilized to read the response, the antigen utilized in such an assay, and the immunogen. Obviously, in each specific context, and for any given type of epitope, the information relating to these classes might or might not be present. For example, an MHC binding assay context does not include any information relating to immunization, while antibody responses associated with a given epitope do not carry any information related to antigen presentation or MHC restriction.

45.3.2 Populating and Querying the Database and its Associated Analysis Resource As mentioned above, the database is currently being populated according to a priority list mutually agreed upon with Program Officers from the National Institute of Allergy and Infectious Diseases, the agency funding the project. Curation of A-C pathogens, including influenza A, and all emerging pathogens, is now virtually complete. Next, curation efforts will be focused on epitopes derived from other infectious diseases and allergens, followed by epitopes derived from

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self-antigens involved in autoimmunity, transplant rejection antigens and other allo-antigens. The data is curated following a formalized process, described in some detail elsewhere and still in evolution, that is based on a combination of automated text recognition and expert review (9). As a first step, automated queries are performed on all scientific literature abstracts accessible through PubMed. These queries are very general and inclusive, as they aim to extract as many of the papers potentially relevant for a given target or epitope source category as possible. The abstracts corresponding to the selected papers are then scanned for the presence of relevant information. Typically, only about a third of the abstracts are found to be potentially relevant. The full-length articles corresponding to those abstracts are then obtained and reviewed by the curation staff. The data contained in each paper is curated according to well-established procedures, described and formalized in the IEDB Curation Manual. Other curators and a council of senior immunologists review the curated records and edit; corrections are introduced when necessary. Finally, approved curated records are promoted to the IEDB system. Currently, the IEDB has processed more than 20,000 abstracts, and admitted to curation of a total of 6,275 potentially relevant papers, 2,894 of which have been finalized. The high level of complexity inherent in immunological data requires extensive human intervention, and substantial immunological domain-specific expertise. Likewise, the high complexity and large volume of data can become overwhelming for the end-user. To facilitate extracting and reviewing the data contained in the IEDB, we have designed three different levels of query, ranging from a Google-like quick search, to a simple, user-friendly search, and finally to a more advanced query that allows searching retrieval information from every single field of the database. We expect that the query and display interface of the database will continue to evolve as more surveys and usability studies are conducted to indicate the best strategies to serve the database users and display the epitope information. Linked to the IEDB is an analysis resource, hosting various bioinformatic tools and algorithms, useful for the generation of epitope predictions and the analysis of epitope data (10–17). The hosted prediction tools allow the prediction of MHC binding capacity for many different alleles and host species, as well as antibody prediction tools. Additionally, the analysis resource hosts tools allowing the visualization of epitope structures available from the Protein Data Base. Finally, other tools allow calculation of projected population coverage afforded by a given set of epitopes, or the conservancy of the epitope set within a given set of protein sequences.

45.3.3 An Analysis of the Influenza A Data Available in the Scientific Literature We recently undertook an analysis of all data related to influenza A published in the scientific literature (Bui et al. in press, PNAS; Figure 45.2). The purpose of this analysis was to provide researchers in the field with a compilation of

45. Immunomic and Bioinformatics Analysis Vaccinia and Influenza A Viruses

More than 16 million references available in Pubmed References related to influenza 2063 (~0.01%) References of potential relevance following abstract scan 743 (~36%)

References curated into IEDB 429 (~58%)

Figure 45.2. Analysis of influenza A data available in the scientific literature.

available epitopes, representing potentially useful resources for analyzing immune responses and assisting in the development and evaluation of flu vaccines and diagnostics. At the same time, we expected that this analysis would also allow identification of gaps in knowledge, thus ultimately highlighting areas for further research and investigation. A number of different features were considered in this analysis. We specifically investigated the number of well defined antibody versus T-cell epitopes, and the antibody epitopes were further classified in linear versus conformational. The distribution of the various classes of epitopes was compiled according to the viral protein of origin and the host species from which the epitopes were originally defined. Additional criteria considered were the influenza strain in which the epitope was originally defined, and the degree of conservancy of the structure of each particular epitope in different influenza strains. To maximize the biological relevance of the analysis, we only included epitopes for which native antigen or virus was utilized as either the immunogen, or the antigen utilized in the assay detecting the responses. Several general conclusions were drawn from this analysis. First, in terms of current knowledge, the total number of immune epitopes captured by the database is 412 for T cells and 190 for antibodies. Of the antibody epitopes, about 60% are linear sequences, and only about 6% have 3D antigen–antibody complex structures available. The relative preponderance of T cell versus antibody epitopes was somewhat unexpected, given the well appreciated importance of antibody responses in protection from influenza, and may reflect the relative ease by which T-cell epitopes can be identified in comparison to antibody epitopes. In terms of strain distribution, epitopes were identified for 13 different influenza A subtypes and 58 strains, but only two epitopes were reported for the H5N1 avian subtype. In terms

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of the host species distribution of the epitopes, antibody epitopes were defined mostly in mouse and rabbits, with very few epitopes being defined in humans and ferrets. This bias was again unexpected and likely to reflect technical ease of epitope definition, rather than biological relevance. Indeed, by comparison, T-cell epitopes were defined mostly in mouse and human hosts. In the case of both antibody and T-cell epitopes, few epitopes were defined in birds and non-human primates. About 50% of T-cell epitopes exhibit 80% or higher conservancy among influenza strains, including avian flu. By comparison, antibody epitopes are much more variable, likely reflecting the fact that these epitopes are mostly derived from the more variable HA and NA viral antigens. In terms of knowledge gaps, we identified five areas of potential interest or concern. First, few protective antibody and T-cell epitopes are reported in the literature. Second, there is a paucity of well-defined antibody epitopes. Third, the epitopes are derived from a limited number of host species, and few epitopes are defined in chicken, ferrets, nonhuman primates, and, in the case of antibody epitopes, humans. Fourth, a limited number of epitopes has been reported for avian influenza strains/subtypes. Finally, compared to the HA and NP proteins, there were relatively fewer epitopes reported for the other influenza proteins, suggesting that more broadly based epitope identification studies might be of interest.

45.4

Conclusion

In conclusion, utilizing bioinformatics and experimental approaches, we have identified a relatively large set of VACV epitopes. In conjunction with additional epitopes described by other groups (18–21), this epitope set defines immunodominant antigens following VACV immunization and should allow efficient coverage of the most frequent murine and human MHC Class I and II molecules. It is our hope that this epitope set might be of use for the evaluation of new vaccines and diagnostics. Furthermore, this data provides insight into the breadth of the immune response to VACV and has certain implications for immune escape and biodefense as discussed above. Finally, this dataset also provided important insights into mechanisms of immunodominance in a complex pathogen. In this paper, we also reviewed the establishment of the IEDB. In addition to its role as a repository of epitope-related information, the IEDB represents a tool to assist basic and applied investigators in the design and interpretation of experimental results, and in the evaluation and design of new diagnostic and therapeutic constructs. As an example of the functionality of the IEDB, we presented an analysis of available data pertaining to influenza. This analysis revealed a wealth of existing knowledge related to influenza A epitopes potentially of service to the broad scientific community. At the same time the analysis also revealed gaps in our current knowledge, thus highlighting opportunities for further research.

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Acknowledgments. We would like to acknowledge the SAIC and IEDB team, O. Lund and S. Buus. This work was supported by the National Institute of Health: Contract #HHSN26620040006C, contract #HHSN266200400124C, RO1 grant #AI-56268, and NO1 grant #AI30039. It is publication number #860 of LIAI.

10.

11.

References 1. Moutaftsi M, Peters B, Pasquetto V, Tscharke DC, Sidney J, Bui HH, Grey H, Sette A (2006) A consensus epitope prediction approach identifies the breadth of murine T(CD8+)-cell responses to vaccinia virus. Nat Biotechnol 24:817–819. 2. Tscharke DC, Karupiah G, Zhou J, Palmore T, Irvine KR, Haeryfar SM, Williams S, Sidney J, Sette A, Bennink JR, Yewdell JW (2005) Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines. J Exp Med 201:95–104. 3. Pasquetto V, Bui HH, Giannino R, Banh C, Mirza F, Sidney J, Oseroff C, Tscharke DC, Irvine K, Bennink JR, Peters B, Southwood S, Cerundolo V, Grey H, Yewdell JW, Sette A (2005) HLA-A∗0201, HLA-A∗1101, and HLA-B∗0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J Immunol 175:5504–5515. 4. Sette A, Sidney J (1998) HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr Opin Immunol 10:478–482. 5. Oseroff C, Kos F, Bui HH, Peters B, Pasquetto V, Glenn J, Palmore T, Sidney J, Tscharke DC, Bennink JR, Southwood S, Grey HM, Yewdell JW, Sette A (2005) HLA class I-restricted responses to vaccinia recognize a broad array of proteins mainly involved in virulence and viral gene regulation. Proc Natl Acad Sci USA 102:13,980–13,985. 6. Peters B, Sidney J, Bourne P, Bui HH, Buus S, Doh G, Fleri W, Kronenberg M, Kubo R, Lund O, Nemazee D, Ponomarenko JV, Sathiamurthy M, Schoenberger S, Stewart S, Surko P, Way S, Wilson S, Sette A (2005) The immune epitope database and analysis resource: from vision to blueprint. PLoS Biol 3:e91. 7. Peters B, Sidney J, Bourne P, Bui HH, Buus S, Doh G, Fleri W, Kronenberg M, Kubo R, Lund O, Nemazee D, Ponomarenko JV, Sathiamurthy M, Schoenberger SP, Stewart S, Surko P, Way S, Wilson S, Sette A (2005) The design and implementation of the immune epitope database and analysis resource. Immunogenetics 57:326–336. 8. Sathiamurthy M, Peters B, Bui HH, Sidney J, Mokili J, Wilson SS, Fleri W, McGuinness DL, Bourne PE, Sette A (2005) An ontology for immune epitopes: application to the design of a broad scope database of immune reactivities. Immunome Res 1:2. 9. Vita R, Vaughan K, Zarebski L, Salimi N, Fleri W, Grey H, Sathiamurthy M, Mokili J, Bui HH, Bourne PE, Ponomarenko J, de CastroR JrChan RK, Sidney J, Wilson SS, Stewart S, Way S,

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Peters B, Sette A (2006) Curation of complex, context-dependent immunological data. BMC Bioinformatics 7:341. Bui HH, Sidney J, Dinh K, Southwood S, Newman MJ, Sette A (2006) Predicting population coverage of T-cell epitope-based diagnostics and vaccines. BMC Bioinformatics 7:153. Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Mothe BR, Chisari FV, Watkins DI, Sette A (2005) Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications. Immunogenetics 57:304–314. Buus S, Lauemoller SL, Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J, Holm A, Brunak S (2003) Sensitive quantitative predictions of peptide-MHC binding by a ‘Query by Committee’ artificial neural network approach. Tissue Antigens 62:378–384. Nielsen M, Lundegaard C, Worning P, Lauemoller SL, Lamberth K, Buus S, Brunak S, Lund O (2003) Reliable prediction of Tcell epitopes using neural networks with novel sequence representations. Protein Sci 12:1007–1017. Peters B, Bui HH, Frankild S, Nielson M, Lundegaard C, Kostem E, Basch D, Lamberth K, Harndahl M, Fleri W, Wilson SS, Sidney J, Lund O, Buus S, Sette A (2006) A community resource benchmarking predictions of peptide binding to MHC-I molecules. PLoS Comput Biol 2:e65. Peters B, Bulik S, Tampe R, Van Endert PM, Holzhutter HG (2003) Identifying MHC class I epitopes by predicting the TAP transport efficiency of epitope precursors. J Immunol 171:1741–1749. Peters B, Sette A (2005) Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method. BMC Bioinformatics 6:132. Tenzer S, Peters B, Bulik S, Schoor O, Lemmel C, Schatz MM, Kloetzel PM, Rammensee HG, Schild H, Holzhutter HG (2005) Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding. Cell Mol Life Sci 62:1025–1037. Jing L, Chong TM, McClurkan CL, Huang J, Story BT, Koelle DM (2005) Diversity in the acute CD8 T cell response to vaccinia virus in humans. J Immunol 175:7550–7559. Mathew A, Terajima M, West K, Green S, Rothman AL, Ennis FA, Kennedy JS (2005) Identification of murine poxvirus-specific CD8+ CTL epitopes with distinct functional profiles. J Immunol 174:2212–2219. Terajima M, Cruz J, Raines G, Kilpatrick ED, Kennedy JS, Rothman AL, Ennis FA (2003) Quantitation of CD8+ T cell responses to newly identified HLA-A∗0201-restricted T cell epitopes conserved among vaccinia and variola (smallpox) viruses. J Exp Med 197:927–932. Tscharke DC, Woo WP, Sakala IG, Sidney J, Sette A, Moss DJ, Bennink JR, Karupiah G, Yewdell JW (2006) Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice. J Virol 80:6318–6323.

Chapter 46 Immunoreactions to Hantaviruses Alemka Markotić and Connie Schmaljohn

46.1

What Are Hantaviruses?

Hantaviruses, of the Bunyaviridae family, are tri-segmented negative-strand RNA viruses with large (L), medium (Μ), and small (S) genome segments that encode the viral RNA-dependent RNA polymerase (RdRp), two envelope glycoproteins (Gn and Gc), and the nucleocapsid protein (NP; ref. 1). They include a number of pathogens that cause in humans hemorrhagic fever with renal syndrome (HFRS) and the hantavirus pulmonary syndrome (HPS). Besides pathogenic hantaviruses, which cause HFRS (Hantaan [HTNV], Seoul [SEOV], Puumala [PUUV], and Dobrava [DOBV] viruses) or HPS (e.g., Sin Nombre [SNV], Andes [ANDV], New York [NYV], viruses, etc.), there are some non-pathogenic or conditionally pathogenic hantaviruses (e.g., Prospect Hill [PHV], Tula [TULV], Saarema [SAAV] viruses, etc.) that do not cause a manifest disease in humans (2, 3). The main reservoirs of hantaviruses are small rodents (e.g., Apodemus flavicollis, Apodemus agrarius, Clethrionomys glareolus, Peromyscus maniculatus, Oligoryzomis longicaudatus, etc.) and their co-evolution through millennia produced their distinctively congruent phylogenies (4–6). Hantaviruses persistently infect their reservoirs causing no evident pathology, whereas a broad spectrum of clinical conditions has been recognized in HFRS/HPS patients, from unapparent or mild illness to a fulminant hemorrhagic disease with severe renal or cardio-pulmonary failure and death (2, 3). Although there have been suggestions (7) that antigenic variations could explain differences in the HFRS/HPS severities, clinical symptoms, and the epidemiological characteristics, so far there is no firm evidence to support this (7). In spite of numerous genetic and serologic analyses of different hantaviral isolates worldwide, especially because HPS has been recognized in the United States, little is known about the From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

pathogenesis of HFRS/HPS (2, 8). To date, several instructive review articles on immunopathogenic mechanisms of hantaviruses have been published (9–18). To avoid redundancy, in this review, we concentrated mostly on cell receptors, innate immune responses originating from the main target cells for hantaviruses, and apoptosis mechanisms.

46.2

Cell Receptors

Gavrilovskaya et al. provided the first information about hantavirus receptors (19). They showed that HPS-associated hantaviruses used β3 integrins for cellular entry. Evidence for this included studies in which infection with NYV or SNV was inhibited by antibodies to β3 integrins and by the β3 integrin ligand, vitronectin. In contrast, infection with PHV, which does not cause manifest disease in humans, was inhibited by fibronectin and β1-specific antibodies (19). A year later, the importance of the same receptor (β3 integrin) was confirmed also for the HFRS-associated hantaviruses (20). These findings implicated integrins as cellular receptors for hantaviruses and suggested that hantavirus pathogenicity correlates with integrin usage and subsequent downstream cell signaling. The integrins are part of a large family of αβ heterodimeric cell-surface receptors that are expressed on a wide variety of cells. As adhesion molecules, they mediate cell–cell and cell– extracellular matrix interactions and are involved in the pathogenesis of many diseases and viral infection cycles (21). The binding of HFRS- and HPS-associated hantaviruses to human αvβ3 integrin maps to the plexin-semaphorin-integrin domain present at the apex of inactive, bent, αvβ3-integrin structures. Previously, it was found that both HFRS- and HPS-associated viruses dysregulate endothelial cell migration. Because cellular β3 integrins are critical adhesive receptors on platelets and endothelial cells and regulate both vascular permeability and platelet activation and adhesion, the use of these receptors by hantaviruses could disrupt these functions and might contribute to hantavirus pathogenesis (22, 23). 435

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The processes of hantaviral cell infection after attachment to the cell surface are not well understood. It appears that hantaviruses enter cells through the clathrin-coated pit pathway and use low-pH-dependent intracellular compartments for infectious entry (24). Although β3 receptors are important for cell infection with pathogenic hantaviruses, additional receptors for cellular entry are likely. A novel, 70-kDa protein was suggested as a candidate receptor or alternative cellular component for cell interaction with HTNV (25).

46.3 The Struggle Between Cells and Hantaviruses Various human cells are susceptible to infection with hantaviruses (26), but monocytes/macrophages and endothelial cells appear to be the main target cells (17, 18, 27).

46.3.1

Monocytes/macrophages

The first published evidence that macrophages are permissive for hantavirus replication came from a study in newborn rats infected with SEOV, in which virus could be isolated from macrophages for several months after infection (28). From this, the researchers suggested that monocytes/macrophages may be the way hantaviruses spread from the primary site of infection (28). Although monocytes/macrophages are important in innate immunity, there is still a big gap in the knowledge about their function and role during hantaviral infection. As there are no adequate animal models of infection for most hantaviruses, information has been primarily derived from rare clinical studies and from in vitro experiments. Histological and immunohistological studies of eight kidney biopsies in patients with HFRS caused by PUUV, showed mild to moderate interstitial infiltration of lymphocytes, plasma cells, monocytes/macrophages, and polymorphonuclear leukocytes (29). Another study implicated macrophages in the local host response in the lower respiratory tract (30). It is well known that hantaviruses are usually spread by contaminated aerosols from excreta of their reservoirs, small wild rodents. Compared to a reference group of 15 healthy individuals, bronchoalveolar lavage fluid of patients infected with PUUV contained significantly higher total numbers of cells and significantly higher numbers of lysozyme-positive macrophages, CD8+ T cells, and natural killer (NK) cells (30). There is no evidence of cytopathic effects in hantavirus-infected macrophages, thus mononuclear phagocytes might serve also as a long-term storage depot of viruses, and therefore be involved in hantavirus dissemination during HFRS (8, 17, 18, 27). The infection of macrophages with hantaviruses is also associated with intracellular metabolic changes. The contact of phagocytes with hantaviruses activates the oxygen-dependent metabolism and nitric oxide-synthase in the cells. In one study, the nitric oxide-synthase-dependent system of the infected macrophages was activated earlier than their oxygen-depen-

dent system and the intracellular contents of acid and alkaline phosphatases increased within the first hours after the infection (31). Activation of monocytes/macrophages, production of certain cytokines, and the role of their differentiation has been described in several studies (8, 15, 32, 33). In one study, human alveolar macrophages were found to produce low levels or no tumor necrosis factor (TNF)-α after infection with SNV. Also, supernatants from SNV-infected human alveolar macrophages did not cause a significant increase in endothelial monolayer permeability (32). In another study, infection of primary human monocyte/macrophages with PUUV showed a low production of interferon (IFN)-α, suggesting that PUUV is a poor IFN inducer. In addition, antiviral MxA protein was detected three days post-infection, but did not mediate resistance to PUUV infection (33). Our recent study with non-pathogenic TULV showed that infected macrophages produce chemokines (interleukin [IL]-8, monocyte chemotactic protein [MCP]-1 and macrophage inflammatory protein [MIP]1β), which are important for recruiting inflammatory cells, but no significant changes in cytokine levels were measured (34). Another interesting observation was that differentiation of monocyte/macrophages increases their susceptibility to PUUV infection and suggests that after differentiation to tissue macrophages, they might function in the spread of the virus (33). In addition, a virulent HTNV clone (cl-1) strongly induced macrophage-to-macrophage fusion in mice and suppressed cytotoxic T-cell activity (35). In our experiments with the human monomyelocytic THP-1 cell line and HTNV (MOI = 10), we observed cell differentiation toward macrophages (Figure 46.1B–F), migration, and cell-to-cell fusion (Figure 46.1C–F). Generally, human macrophages could be found in all tissues and had the capacity, in specific but rare instances, to undergo homotypic fusion and to differentiate into multinucleate osteoclasts or giant cells, which are usually found in bone or in chronic inflammatory reactions (36, 37). The molecular mechanisms used by macrophages to adhere to and to fuse with each other, and possibly with other cells, are an essential step that remains to be characterized. Cell-to-cell fusion and, in particular, macrophage fusion could become a therapeutic tool for delivering genes or drugs in a cell-specific targeted manner. The therapeutic applications of such a strategy encompass cancer, infectious diseases, and genetic disorders (36, 37).

46.3.2

Dendritic Cells (DCs)

DCs are the most potent antigen-presenting cells of the immune system and play a critical role in the regulation of the adaptive immune response. They also present key connections between innate and adaptive immune response (8). Immunohistochemistry in four HPS fatal cases showed the widespread presence of hantaviral antigens in endothelial cells of the microvasculature, particularly in the lung, but also in follicular DC, macrophages, and lymphocytes (38). An in vitro study by Raftery et al. (39) showed that HTNV, which causes HFRS, productively infects human DCs and activates immature

46. Immunoreactions to Hantaviruses

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Figure 46.1. Morphological changes in a human monomyelocytic THP-1 cell line infected with HTNV. Cell differentiation (B–F), migration and cell-to-cell fusion (C–F) are presented. Slides with infected or uninfected THP-1 cells, 11 days after infection, were stained with Wright Giemsa stain to provide better resolution of cellular details.

DCs, resulting in upregulation of major histocompatibility complex (MHC), co-stimulatory, and adhesion molecules. HTNV-infected DCs were able to stimulate T cells, but displayed reduced antigen uptake as the DC matured. Infection of DCs with HTNV induced the release of various proinflammatory cytokines such as TNF-α and IFN-α. So, it seems that at least HFRS-associated hantaviruses are not immunosuppressive and do not have mechanisms to disrupt DC maturation (39). In a recent study, polyoma virus-derived, virus-like particles carrying a 120-amino acid-long sequence of PUUV NP were able to activate antigen- presentation by murine spleen cell-derived DCs. Efficient uptake of the VLPs and activation of murine DCs were demonstrated, which not only suggests strong antigenicity of chimeric VLPs, but also may indirectly suggest a role for PUUV NP in the process of DC maturation (40). Of course, additional information is needed to confirm this hypothesis.

46.3.3

Endothelial Cells

Hemostasis, inflammatory reactions, and immunity involve close interactions between immunocompetent cells and the vascular endothelium. Hantaviruses demonstrate a high tropism for endothelial cells (ECs) throughout the body (41). The main pathologic finding in infected ECs is an increased vascular permeability, which is accompanied by endothelial activation, increased expression of adhesion molecules, and recruitment of inflammatory cells in the organs involved. Vascular cells are both a target for cytokines/chemokines and a source of their production (27). One of the main perpetrators of permeability is TNF-α. In several studies, TNF-α and other cytokines have been found to have a significant influence on ECs during hantaviral infections (Table 46.1; refs. 41–43). In a recent study, we examined cytokines and chemokines produced in human vein ECs (HUVEC)

(ATCC CRL 1730) infected with HTNV strain 76118 (44), ANDV strain Chile-9717869 (45), and SNV strain CC107 (46). Cells were maintained in MCDB 105 medium (SIGMA) supplemented with 10,000 units/L of heparin (SIGMA) and 30 mg/L of ECGS (Becton Dickinson), and the viral stocks were propagated in Vero E6 cells (Vero C1008, ATCC CRL 1586). Cells were infected at multiplicities of infection (MOI) of 10–1 pfu/cell (SNV), 100 pfu/cell (ANDV), or 101pfu/cell (HTNV). The different MOI were used due to our inability to achieve equivalent MOI, because of the low-titer seed stocks of some of the hantaviruses, like SNV. As controls for the infected cultures, uninfected cells were treated identically to infected cultures; that is, control cells were incubated in the same medium that we used for diluting the viruses before adsorption. The cells were then washed, re-fed, and maintained with appropriate medium. The cells were incubated for seven days at 37°C, 5% CO2 and the medium was not changed during the observation period. All infected and uninfected cells were tested for Mycoplasma contamination by enzyme-linked immunosorbent assay-polymerase chain reaction (ELISA-PCR; Roche Diagnostics Corporation, Roche Molecular Biochemicals, Indianapolis, IN, USA), and were found to be Mycoplasma free. The cytokines that we measured were IL-1β, IL-6, IL-10, IL-12p40, granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-γ, and TNF-α. The chemokines that we measured were IL-8/CXCL-8, regulated upon activation, normal T-cell expressed, and secreted (RANTES/CCL5), monocyte chemo-attractant protein (MCP-1/CCL2). The macrophage inflammatory protein (MIP-1α/CCL3 and MIP-1β/CCL4) levels were determined in the supernatants of infected (HTNV, SNV, ANDV) and uninfected cells by specific ELISA (Pharmingen or R&D Systems, Inc., Minneapolis, MN, USA)

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A. Markotić and C. Schmaljohn Table 46.1 Comparison of published data about cytokines/chemokines produced by human endothelial cells infected with hantavirusesi.

Authors

Chemokines

Cytokines

Viruses

*

IL-1 IL-6 IL-10 IL-12 GM-CSF TNF-α IL-8 MIP-1α MIP-1β RANTES MCP-1

Pensiero et al., 1992 (13) (HSVEC, mRNA)

Sundstrom et al., 2001 (18) (HMVEC-Ls, mRNA, protein)

HTNV

HTNV

SNV

Ø Ø↑ n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t.

Ø Ø n.t. n.t. n.t. Ø Ø Ø Ø ↑ Ø

Ø Ø n.t. n.t. n.t. Ø Ø n.t. n.t. ↑ Ø

Geimonen et al., 2002 (32) (HUVEC, mRNA)

Markotic´ & Schmaljohn, 2007 (HUVEC, protein)

HTNV NY-1V HTNV Ø ↑ n.t. n.t. ↑ n.t. ↑ n.t. n.t. ↑ n.t.

Ø Ø n.t. n.t. Ø n.t. Ø Ø Ø Ø n.t.

Ø ↑ ↑ ↑ Ø Ø Ø↑ Ø Ø ↑ Ø

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Ø ↑ ↑ ↑ Ø Ø ↓ Ø Ø ↑ ↑

Ø Ø Ø Ø Ø Ø ↓ Ø Ø ↑ Ø

n.t., not tested; Ø, no cytokines/chemokines; ↑, increased levels; ↓, decreased levels.

according to the instructions provided by the manufacturer. Samples were collected at various time points and stored at –70°C until assayed. HUVEC were cultivated for 3 days and then were infected with one HFRS- (HTNV) or two HPS-associated viruses (SNV, ANDV), and maintained for 7 days without medium change. ANDV induced continuous production of RANTES/CCL5 (Figure 46.2D). In addition to RANTES/CCL5, both HTNV and SNV induced production of IL-6, IL-10, and IL-12p40 (Figure 46.2A–D). However, although production of these cytokines and chemokines could be detected in HTNV-infected cells continuously for 6 to 7 days, they were observed in the SNV-infected cells only for the first three days post-infection (Figure 46.2A–D). In addition, only the SNV-infected cells continuously produced MCP-1/CCL2 (Figure 46.2E). Interestingly, both HPS-associated viruses (SNV and ANDV) suppressed production of IL-8/CXCL8, while the HFRSassociated HTNV induced a slight increase during the first two days of infection (Figure 46.2F). Our results are consistent with those of others with regard to the IL-1 and RANTES/CCL5 induction by HFRS- and HPS-associated hantaviruses in ECs, although differing results were obtained for IL-6 and IL-8 induction (Table 46.1; 41–43). To our knowledge, we are the first to observe production of IL-10 and IL-12p40 in HUVEC infected with hantaviruses. There are limited data about the role of IL-10 in ECs. It was reported that IL-10 inhibits antigen presentation by human dermal microvascular ECs and its effect on IL-8 production is still unclear (47). Recently, it was shown that IL-12 has potent in vivo anti-angiogenic activities (47). However, in HFRS/HPS its main role could be its influences on T cells. Chemokines are a key element in the cascade process of leukocyte recruitment (48) and are produced by ECs in response to different molecules involved in the inflammatory reactions (47).

In summary, our study showed that hantaviruses induced production of several cytokines (IL-6, IL-10, and IL-12) and chemokines (RANTES/CCL5, MCP-1/CCL2, and IL-8/CXCL8). IL-6 in conjunction with its soluble receptor may induce chemokine production and indirectly, plays an important role in leukocyte recruitment (47). Further, our results showed that hantaviruses activate ECs to produce various cytokines/chemokines, which are known to participate in inflammatory and immune reactions in HFRS/HPS. Our findings may point to new research directions. For example, additional studies are needed to identify specific intracellular signals that are responsible for the differences in the dynamics of cytokines/chemokines production between HFRS- and HPS-causing viruses, and to determine why ANDV did not induce cytokines/chemokines production in ECs (except RANTES/CCL5). There are very few studies of ANDV and ECs. Recently, in a study by Spiropoulou, et al. (49), ANDV was observed to be a poor inducer of IFN-β. In addition, phosphorylated Stat-1/2 levels were found to be significantly lower after infection of primary lung ECs with ANDV than with non-pathogenic Prospect Hill virus (PHV). Other investigators have found that ANDV infection upregulates transcription of MxA RNA and expression of MxA protein in human ECs in vitro (50). In an earlier study, the late decrease in hantavirus production in HUVEC was reported to be the result of the induction of IFN-β, which could be reversed by adding anti- IFN-β serum to the culture medium (41). In another study, HFRS-associated HTNV clearly induced the production of IFN-β, whereas expression of this cytokine was barely detectable in the supernatants or in extracts from non-pathogenic TULV-infected HUVEC (51). However, the upregulation of HLA class I on both TULV- and HTNVinfected HUVEC could be blocked by neutralizing anti-IFN-β antibodies. A delay in the induction of antiviral MxA in ECs after infection with HTNV was also observed. The researchers suggested, therefore, that this could allow viral dissemination and contribute to the pathogenesis leading to HFRS (51).

46. Immunoreactions to Hantaviruses

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Figure 46.2. Detection of cytokines and chemokines in the supernatants of human vein endothelial cells (HUVEC) cells by ELISA. In HUVEC, HTNV induced continuous production of (A) IL-6, (B) IL-10, and (C) IL-12p40 while SNV induced production for only 3 days and ANDV showed no induction. All three hantaviruses induced (D) RANTES/CCL5 production but only SNV induced (E) MCP-1/CCL2 production. HPS-causing viruses (SNV and ANDV) decreased production of (F) IL-8/CXCL8.

440

A. Markotić and C. Schmaljohn

Figure 46.3. (A) Mock- or (B) virus-infected HEK293 cells were fixed in methanol and stained. Indirect fluorescent antibody staining using hyperimmune mouse ascites fluid to HTNV and fluorescence microscopy were used to demonstrate the presence of viral antigens. Cytopathic effect in cells at 11 days after infection with SNV (B).

In one study on induction of the innate immune response by the HPS-causing hantavirus SNV, a variety of IFN-stimulated genes (ISG) were found to be induced between four and 24 hours after exposure to either live or UV-inactivated SNV. The levels of induction at early time points were generally higher in ECs treated with inactivated SNV particles, but SNV replication was required for continued ISG expression. These results suggest that hantavirus particles may themselves be capable of early induction of ISG but that ongoing production of viral particles during infection also contributes to the maintenance of the innate immune response (52). There are also obvious differences in IFN-specific transcriptional responses between pathogenic and nonpathogenic hantaviruses. One study showed that nonpathogenic PHV elicits early IFN responses after infection of human ECs and its replication is blocked in ECs. Co-infection of ECs with pathogenic and non-pathogenic hantaviruses decreased the induction of IFN-responsive MxA transcripts by non-pathogenic PHV by 60%, and further suggests the potential of pathogenic hantaviruses to regulate the early IFN response (53). The Gn protein of a pathogenic hantavirus regulated cellular IFN responses upstream of IRF-3 phosphorylation at the level of the TBK-1 complex. Further, the Gn cytoplasmic tail permitted the hantavirus to bypass innate cellular immune responses and provided a mechanism for pathogenic hantaviruses to successfully replicate within ECs (53). The researchers, therefore, suggest that the cytoplasmic tail of Gn is a virulence factor for pathogenic hantaviruses (53). In addition to cytokines, the increased capillary permeability observed during HFRS and HPS could be a result of other immunoreactions. By enzyme immunoassay, using HUVEC as the substrate, IgG class anti-EC antibodies (AECA) were detected in sera obtained from HFRS patients infected with PUUV. These AECA may be related to the capillary leak in HFRS (54). Hantavirus-specific CD8+ and CD4+ CTL might also contribute to the immunopathology and capillary leak syndrome observed in the HPS (55). A human CD8+ hantavi-

rus-specific cytotoxic T lymphocyte cell clone was found to recognize and lyse ECs infected with SNV, and, in a transwell permeability assay, was able to increase the permeability of ECs infected with SNV or with a recombinant adenovirus expressing the SNV Gc protein (56). The 70-kD heat shock protein (Hsp70), which is an important part of the cell’s machinery for protein folding and protecting cells from stress, is induced by HTNV infection in HUVEC and may play a role in the inhibition of viral replication and the protection of cells from viral infection (57).

46.4

Apoptosis

Apoptosis, or programmed cell death, is important for homeostasis in multicellular organisms and also plays a role in innate immunity to infectious agents. Apoptosis is an essential component of the cellular response to injury caused by pathogens, including viruses. After viral infection, cells may undergo apoptosis as a mechanism of self-defense with a result of aborting the production and release of progeny virus. Viruses may interact with different apoptotic pathways and their components to interfere with mechanisms of apoptosis or to develop regulatory mechanisms and take advantage of the process of programmed cell death (58). Research on apoptosis caused by hantaviruses began less than a decade ago (59). The first report showed that live, but not UV-inactivated HTNV or PHV induced apoptosis in Vero E6 cells with a subsequent significant reduction in the level of the proto-oncogenic Bcl-2 protein. However, the mRNA level remained unchanged in HTNV-infected cells, suggesting possible involvement and post-transcriptional regulation of this anti-apoptotic protein in the process (59). The interaction between death-associated protein 6 (Daxx) and PUUV-N protein suggests a possible role for Fas in apoptosis, in that Daxx is a protein identified originally as a Fas-mediated apoptosis enhancer (60). Furthermore, functional analyses have

46. Immunoreactions to Hantaviruses

demonstrated that Daxx binds to the Fas death domain and enhances Fas-mediated apoptosis (61). Additional evidence for a possible Fas/Fas ligand (L) role in apoptosis is that they show increased expression in peripheral blood mononuclear cells (PBMC) during acute and convalescent phases of the hantaviral infection. In addition, activation of the initializing (caspase-2, -8 and -9) and the effector (caspase-3, -7 and -10) caspases was also detected (62). FasL (CD178) was found to be expressed in CD4+ and CD8+ T-cell subsets (but mainly in CD8+ T cell subsets) in HFRS patients both in the early and later stages of disease (63). In a recent study, investigators have shown that the expression of membrane-bound FasL and TNF-related apoptosis-inducing ligand (TRAIL) was upregulated on the surface of PBMC (particularly on CD8+ T lymphocytes) isolated from the HFRS patients, as compared to healthy controls. The levels of TNF-α, sFasL, and sTRAIL in plasma from the HFRS patients in the acute phase also increased in comparison to levels in healthy donors. In addition, the percentage of Th1, Tc1, and Tc2 subsets in PBMC from the patients increased significantly compared with those from healthy donors. All these results indicate that dynamic changes occurred in both the membrane bound and soluble forms of these factors in HFRS patients. Both factors (apoptosis-inducing ligands and some Th1 and cytotoxic T lymphocytes) may play an important role in the etiology of hantaviral infection in humans (64). The apoptosis of lymphocytes is necessary for removing the excess of activated antigen-reactive T cells and downregulation of the immune response. So far, there are no data on the mechanisms of immune regulation during hantavirus infections in humans. A study with non-pathogenic TULV showed apoptosis in infected Vero E6 cells. TULV replication was found to be required for activating caspase-3 and the cleavage of poly (ADP-ribose) polymerase (PARP). At the same time, activation of caspase-8 was noted. TNF receptor 1 was induced during a late stage of TULV infection. Additionally, it seems that TNF-α may contribute significantly to apoptosis in a synergistic manner with TULV propagation. The important role of caspases in apoptosis induced in TULV-infected Vero E6 cells was indicated by the efficient inhibition of apoptosis during pretreatment with a broad-spectrum caspase inhibitor, z-VAD-fmk (65). Furthermore, it was demonstrated that the progressive replication of TULV in Vero E6 cells initiates several death programs that are intimately associated with endoplasmic reticulum (ER) stress: (i) early activation of ERresident caspase-12; (ii) phosphorylation of Jun NH2-terminal kinase (JNK), and its downstream target transcriptional factor, c-jun; (iii) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (iv) changes in the ER-membrane protein BAP31, implying cross-talk with the mitochondrial apoptosis pathway. An increased expression of an ER chaperone Grp78/BiP was also noted (66). In contrast, our study on human embryonic kidney cell line (HEK293) infected with pathogenic HFRS (HTNV) and HPS-

441

(ANDV, SNV)-associated hantaviruses showed no obvious differences in the expression of mRNA levels of FasL, Fas, Fasassociated death domain protein (FADD), DR3, Fas-associated phosphatase (FAP), Fas-associated factor (FAF), TRAIL, caspase-8, and TNF-receptor 1–associated death domain (TRADD) in comparison to non-infected (mock) cells, although apoptosis was confirmed in infected cells (67). Only a slight increase in TNFRp55 was seen. A central dogma of hantavirus infections is that they are maintained in nature in persistently-infected rodents and can also persistently infect cultured mammalian cells, causing little or no cytopathology (68). Subtle signs of cytopathology, however, have been reported. For example, two reports described low pH-dependent cell fusion that occurred in cultured cells infected with hantaviruses (69, 70). In another study, results indicated that HFRS-associated hantaviruses may induce a very low level of apoptosis in dividing Vero E6 and human alveolar basal epithelial A-549 cells, but not at all in confluent cells. No difference was found in the percentage of adherent cells, or of cells with condensed nuclei, between non-infected and virus-infected cells as far as 12 days after infection. TUNEL assays also did not show significant differences between infected and non-infected cells. The researchers suggested that non-pathogenic hantaviruses may differ from HFRS-associated hantaviruses with regard to ability to induce apoptosis (71). Another study by this same research group showed that elevated levels of extracellular perforin, granzyme B, and epithelial cell apoptosis are induced during acute PUUV infection, while the high levels of caspase-cleaved CK18 during the convalescent phase indicate that epithelial cell apoptosis may be increased for a prolonged time after infection (72). An unexpected outcome of one of our studies was the observation of cytopathic effects in infected HEK293 cells (67). To our knowledge, it is the first report of frank cytopathogenicity due to infection by a hantavirus in any cell line (Figure 46.3). Furthermore, in preliminary studies, we found that both at the gene expression level, and also at the protein level, there were differences in the apoptotic pathways used by pathogenic and non-pathogenic hantaviruses (unpublished results). It is obvious that current research has only begun to address the complex mechanisms of apoptosis caused by hantaviral infection and further intensive research with other in vitro models, as well as in clinical studies, is necessary. Such studies might also uncover better means for treatment of hantaviral infections.

46.5

Conclusion

HFRS/HPS are life-threatening diseases of importance for public health. So far, there is no commonly-accepted, adequate prophylactic or therapeutics for these diseases. The immunopathogenic mechanisms induced by hantaviruses are complex and are not well understood. Lack of animal models for studying HFRS is still a significant problem, although a promising hamster model for HPS caused by ANDV infection has provided a new means

442

to study immunopathogenesis (73). Despite the large gaps in knowledge, the recent intensive research on ECs has provided a starting point for additional studies such as their possible interaction with other important immune cells like macrophages and T cells. It is certain that macrophages have a distinctive role, especially in the early phase of innate immune responses to hantaviruses, but more remains to be learned. A better understanding of the mechanisms by which pathogenic and non-pathogenic hantaviruses interact with apoptotic pathways may also provide useful information for future designs of more effective therapeutics and vaccines.

Acknowledgements. Some original data presented here on monocytes/macrophages, endothelial cells (Figures 46.1, 46.2, and 46.3) were prepared while Dr. A. Markotić held The National Academies, National Research Council postdoctoral position at U.S. Army Medical Research Institutes in Infectious Diseases, Frederick, MD, USA. The text is also prepared in the scope of the project Immunoreactions to hantaviruses and leptospires (P.I. Dr. A. Markotić) funded by the Croatian Ministry of Sciences, Education and Sports.

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A. Markotić and C. Schmaljohn 69. Arikawa J, Takashima I, Hashimoto N (1985) Cell fusion by haemorrhagic fever with renal syndrome (HFRS) viruses and its application for titration of virus infectivity and neutralizing antibody. Arch Virol 86:303–313. 70. McCaughey C, Shi X, Elliot RM, Wyatt DE, O’Neill HJ, Coyle PV (1999) Low pH-induced cytopathic effect–a survey of seven hantavirus strains. J Virol Methods 81:193–197. 71. Hardestam J, Klingstrom J, Mattsson K, Lundkvist A (2005) HFRS causing hantaviruses do not induce apoptosis in confluent Vero E6 and A-549 cells. J Med Virol 76:234–240. 72. Klingstrom J, Hardestam J, Stoltz Μ, Zuber B, Lundkvist A, Linder S, Ahlm C (2006) Loss of cell membrane integrity in puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin. J Virol 80:8279–8282. 73. Hooper JW, Larsen T, Custer DM, Schmaljohn CS (2001) A lethal disease model for hantavirus pulmonary syndrome. Virology 289:6–14.

Chapter 47 Innate Immunity to Mouse Cytomegalovirus Djurdjica Cekinović, Irena Slavuljica, Tihana Lenac, Astrid Krmpotić, Bojan Polić, and Stipan Jonjić

47.1

Introduction

Innate immunity, the first line of defense against pathogens, plays an essential role in the initial phase of viral infection, before expansion, clonal selection, and differentiation of antigen-specific T- and B-cells occur. The innate immune system comprises cellular and humoral components. Among cellular components, major players in the innate immune response are macrophages, dendritic cells (DCs), and natural killer (NK) cells (1). Macrophages express an enormous phagocytic capacity, which makes them important for the clearance of infected cells as well as for the initiation of the innate immune response (2). DCs, major antigen presenting cells, are essential for the activation of NK cells as well as for T- and B-lymphocytes that lead to the final clearance of viral antigens (3). The rapid activation of NK cells and their recruitment to the sites of infection define their central position in the innate immunity. The antiviral functions of NK cells are mediated either through direct lysis of infected cells or the secretion of antiviral cytokines like IFN-γ and TNF-α (4). NK cells also play a role in the regulation of specific immune response thus linking innate and adaptive immunity (5). The cytomegaloviruses (CMVs) are members of the β-herpesvirinae subfamily of the herpesviridae. Primary human CMV (HCMV) infection usually passes asymptomatically due to the effective host’s immune response. Yet, in spite of the fully primed immune response, the virus is able to establish a lifelong latency from which the reactivation may occur whenever the immune response is compromised. In contrast to immunocompetent hosts, HCMV infection of immunodeficient individuals may induce severe illness and high mortality (6). Moreover, one of the most common viral congenital infections in humans is caused by HCMV, frequently resulting in developmental abnormalities of the central From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

nervous system and severe neurological impairments (6, 7). The species specificity of HCMV precludes the studies of the pathogenesis of HCMV infection in animal hosts. Therefore, animal CMVs, particularly mouse CMV (MCMV), have been the most commonly used models to study the CMV infection and pathogenesis (8). Cellular immunity is indispensable for the control of primary MCMV infection and for the establishment and maintenance of latency (9), whereas specific antibodies prevent the spread of the virus after reactivation (10). During the co-evolution with their hosts CMVs have developed multiple strategies to compromise or evade the immune response (11). However, as pointed above, mechanisms of innate and acquired immunity can overcome these viral immunomodulations and successfully control the infection. This game of hide-and-seek prompts further research, which could lead to a better understanding of the importance of NK cells and other components of innate immunity to CMV infection as well as the role of viral immunoevasins in their modulation.

47.2 Macrophages and DCs as Components of the Innate Immunity to MCMV Monocytes are bone marrow derived cells that circulate in the blood. In order to perform their function, they migrate into tissues and differentiate into macrophages that have great phagocytic potential (2). Macrophages can recognize, ingest and destroy microorganisms and other foreign antigenic material. Their phagocytosis is mediated by the expression of opsonic and non-opsonic receptors, which include Fc receptors, complement receptors, integrins, scavenger receptors, mannose receptors, and Toll-like receptors (TLRs; ref. 12). Besides phagocytosis, macrophages are able to produce a variety of proinflammatory cytokines and chemokines, and to present peptides in complex with MHC class II molecules to CD4+ T lymphocytes (Figure 47.1; ref. 13). 445

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Figure 47.1. Components of the innate immune response to MCMV. Following the MCMV infection, macrophages and dendritic cells secrete cytokines and chemokines that activate NK cells. Apart from cytokines, NK cells can be activated by engagement of activating receptors and their ligands on the infected cells (e.g. MCMV m157) or due to the lack of inhibition. Activated NK cells exert antiviral activity through the direct cytolysis of infected cells and the secretion of various antiviral cytokines. Cytokines secreted by NK cells can upregulate DCs’ and macrophages’ function in generating adaptive immune response.

Both monocytes and macrophages are permissive to CMV infection. After being infected, these cells are vehicles for the viral spread (14), and, most likely, macrophages also represent a cellular site of CMV latency (15). MCMV dissemination by infected monocytes is further amplified by the viral proinflammatory chemokine MCK (MCMV chemokine homolog), which recruits other cells to the sites of infection and by doing so facilitates the viral spread (16). Although the virus lacking the genes m131-m129 encoding the MCK is attenuated in vivo, the mechanisms of attenuation of this virus remain undefined (17). Members of the MCMV’s US22 gene family, m142 and m143, are essential for viral replication in macrophages. A virus lacking these genes replicates poorly in

cultured macrophages (18), but the importance of these genes for the viral biology in vivo still needs to be defined. Another member of the same gene family, M36, encodes protein with the antiapoptotic function in macrophages (18). MCMV also affects immune response by impairing the macrophage’s capacity to present antigens to CD4+ T cells (19). It is, in fact, able to reduce the IFN-γ-mediated MHC class II upregulation. This effect is possibly mediated through the MCMV induction of IFNα/β, since macrophages isolated from mice deficient for IFN-α/β receptor (IFN-α/βR-/-mice) show no decrease in MHC class II expression following MCMV infection (20). However, the work by Muller and colleagues showed IFN-β-mediated control of MCMV

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replication in macrophages (21). The experiments performed on macrophages isolated from mice lacking IFN-β, IFN-α/β receptor chain 1 (IFNAR) or tyrosine kinase 2 (Tyk2), emphasized the importance of IFN-α/β autocrine function and Tyk2 signaling in the macrophage-mediated control of MCMV infection. Tyk2-, IFN-β- and IFNAR-deficient macrophages are highly permissive to MCMV, while Tyk2-/-mice show increased viral titer in the liver, lungs and salivary glands compared to the wild type (wt) mice (21). The product of the MCMV gene m27 has been shown to negatively interfere with the IFN-γ signaling pathway (22) and the virus lacking this gene was shown to be heavily attenuated in vivo (22). Downmodulation of MHC class II molecules by MCMV may also be linked to the viral induction of IL-10, because IL-10-/-mice, following MCMV infection, develop a robust MHC class II induction in macrophages as compared to the wt mice (23). Altogether, although the functionality of MCMV infected macrophages is affected (24), they are still capable to induce NK cells to secrete proinflammatory cytokines (24). DCs are major antigen presenting cells that play a central role in translating innate into adaptive immune response. Through cytokine secretion (IFN-α/β, IL-2, IL-12, IL-15, IL18) and interaction with NK cells, they contribute to innate immunity (Figure 47.1). Immature DCs reside on peripheral tissues and sample the antigenic material they encounter. After contact with antigen or in a milieu rich with pro-inflammatory cytokines, DCs undergo maturation and migrate to the secondary lymphatic organs. When mature, DCs show lower capacity of antigen capturing but express high levels of MHC class I and II molecules as well as co-stimulatory molecules, which makes them exceptionally successful in priming of naive T cells (3). In addition, DCs are able to prime NK cells resulting in enhanced target cell lysis and IFN-γ production (25). NK cell activity can be augmented by DCs secreted cytokines. IFN-α/β and IL-15 stimulate NK cell proliferation and survival (26), whereas IL-12 (26) and IL-18 (27) promote IFN-γ secretion. In turn, activated NK cells enhance DCs maturation and IL12 production (28), a process dependent upon engagement of Nkp30 activating NK cell receptor and subsequent secretion of TNF-α and IFN-γ by NK cells (29). In mice, splenic DCs are divided into three major subpopulations: conventional or CD11b+ DCs (cDCs), CD8α+ DCs, and plasmacytoid DCs (pDCs) differing in cytokine production and antigen presentation during the infection (30). cDCs, in contrast to pDCs, represent a site of productive MCMV infection (31, 32). Infection disrupts cDCs’ function, thus resulting in impaired NK cell activation, lower capacity to prime T cells and general immunosuppression in vivo (31). The mechanisms by which MCMV affects cDCs’ function include reduced capacity of antigen capturing, selective downregulation of the surface MHC class I and II, CD40, CD54, CD86, CD88 (31), and CD80 (33) molecules and the loss of secretion function.

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pDCs rapidly produce a high amount of IFN-α/β in response to MCMV infection and induce a strong NK cell activation (34). However, recent work revealed that cDCs are also able to contribute to NK cell activation during MCMV infection (35). One of the specific features of MCMV infection is a dramatic loss of CD8α+ DCs from the spleen of MCMV sensitive BALB/c mice (36). The loss is not observed in MCMV resistant C57BL/6 mice, but can be induced by the depletion of Ly49H+ NK cells, indicating that these cells are essential for protecting CD8α+ DCs in the spleen. In turn, depletion of CD8α+ DCs results in the impaired proliferation of Ly49H+ NK cells during MCMV infection (36). The most important innate receptors by which DCs and macrophages recognize pathogens are TLRs, a major class of the pattern-recognition receptors. The activation of TLRs results in the production of reactive oxygen and nitrogen intermediates, the secretion of several cytokines (IFN-α/β and IL-12) and the upregulation of co-stimulatory molecules thus orchestrating both the innate and adaptive immunity (37). Initially, only molecules of bacterial and fungal origin were considered as TLR-activating ligands. However, it has become clear that viral components are also recognized by TLRs. Viral DNA, RNA and unmethylated CpG motifs are detected by intracellularly localized TLRs (38). At least three TLRs have a role in the early recognition and initiation of the immune response towards MCMV. Mice deficient in TLR9 or myeloid differentiation primary response gene 88 (MyD88), an adaptor molecule that mediates TLR9 signaling, show decreased serum concentration of IFN-α/β secreted by DCs, low IFN-γ production by NK cells and consequently elevated MCMV titers in the spleen and increased mortality (39, 40). The TLR3-Lps2 (TRIF) pathway is also activated during MCMV infection. TLR3-deficient mice show a lower secretion of IFN-α/β and IL-12 by DCs followed by impaired NK cell activity and higher susceptibility to MCMV (39). Recently, TLR2 has been identified to contribute to the MCMV control in the NK cell dependent manner. TLR2-/mice have increased viral titers in the spleen and in the liver, due to impaired NK cell activation by DCs (41).

47.3

NK cells and Their Receptors

NK cells are a separate subset of lymphocytes and essential effector cells of the innate immune system (Figure 47.1). They play a crucial role in the recognition and elimination of infected cells. The recognition of target cells relies on the integration of signals coming from both activating and inhibitory receptors expressed on the surface of NK cells (42, 43). Upon activation, NK cells kill infected cells through the granzyme- and perforin-dependent mechanisms. NK cells also release antiviral cytokines (e.g., IFN-γ and TNF-α) and chemokines (MIP-1 family and RANTES), which regulate the activity of other innate and adaptive immune effector cells

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(44, 45). Unlike T and B lymphocytes, NK cell receptors are encoded by separate genes that do not undergo rearrangements and that are mainly located within the NK complex of genes (NKC) situated on the mouse chromosome 6. Other major differences in comparison to T and B cells include the ability to respond early after infection as well as the lack of the immune memory, although some recent studies suggest that NK cells may acquire some sort of memory following antigen recognition (46). NK cell activity is tightly regulated by the inhibitory and activating signals received from the cell surface recep-

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tors. Normally, signal balance is shifted toward inhibition, thus preventing NK cell auto-activation (Figure 47.2). When activating signals overcome inhibitory signals due to decreased engagement of inhibitory receptors or increased engagement of activating ones, the balance shifts toward the NK cell activation. The key for understanding the recognition of infected cells by NK cells was the finding that NK cell responses were inhibited by the engagement of their surface receptors with MHC class I molecules as their cognate ligands (47, 48). According to the “missing self” hypothesis, the reduced levels of surface MHC class I molecules on target

Figure 47.2. Major NK cell receptors and their ligands in mouse. NK cell activity is strictly regulated by the signals from activating and inhibitory receptors. In contact with a healthy cell, engagement of receptors for MHC class I molecules provides inhibition that prevents autoimmune response by NK cells.

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cells, which is a frequent consequence of virus infection, leads to a decreased engagement of inhibitory NK cell receptors (49). Consequently, a downregulation of inhibitory signaling can lead to NK cell activation and cytolysis of target cells (Figure 47.3A). The surface level of classical MHC class I molecules is principally monitored by the polymorphic C-type lectin Ly49 family of NK cell receptors in rodents and the killer cell immunoglobulin-like (KIR) family of receptors in humans (42). The expression of non classical MHC class Ib molecules (human HLA-E and mouse Qa1) is monitored by the CD94/NKG2 receptors, which are expressed in both rodents and humans ( 50 ). A single CD94 gene product is linked to three (mouse) or four (human) NKG2 gene products. CD94 and NKG2 proteins are covalently linked and organized as type II membrane glycoproteins. Similar to Ly49 and KIR receptors, CD94/NKG2 receptors are expressed on most NK cells, but also on γδ T cells and on some effector/memory αβ T cells (51). Inhibitory receptors in their cytoplasmatic tails possess an immunoreceptor tyrosine-based inhibitory motif (ITIM; ref. 52). The ligand binding to the receptor results in the tyrosine phosphorylation of the ITIM and the recruitment and activation of protein tyrosine phosphatase SHP-1, resulting in the inhibition of NK cell activation (52). All three classes of NK cell receptors mentioned above also include the members of the activating receptors. However, while most of the inhibitory receptors recognize MHC class I molecules, activating receptors also bind various ligands which are predominantly expressed on stressed, transformed or virus-infected cells (44, 53). Altogether, it is the integration of

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signals from both inhibitory and activating receptors that ultimately dictates NK cell responses (Figure 47.2). Furthermore, the activating NK cell receptors associate with adaptor molecules containing either the immunoreceptor tyrosine-based activating motif (ITAM) or an YxxM motif (52). Most of the activating receptors associate with DAP12 adaptor molecule, while some associate with DAP10, FcεRIγ and CD3ζ adaptor molecules (54). The formation of the complex between the activating receptor and its ligand results in phosphorylation of tyrosine in the adaptor molecules which recruits and activates protein tyrosine kinases Syk/ZAP70 or PI3 resulting in the NK cell activation (52). The NKG2D is an activating receptor and an important regulator of immune responses mediated by both NK and T cells. Although belonging to the CD94/NKG2 family of C-type lectin receptor, NKG2D has a different structure and does not pair to CD94 (55). It is constitutively expressed on all NK cells but also on some T cells. While in humans it appears to be expressed on all CD8+ T cells, in mice it is expressed only on activated CD8+ T cells (53, 56, 57). Unlike on NK cells, NKG2D functions as a co-stimulatory receptor molecule on T cells. As a result of an alternative splicing, NKG2D exists in at least two isoforms differing in the length of its cytoplasmatic tail (58). Depending on its isoform, NKG2D associates either with both DAP10 and DAP12 (short isoform NKG2DS) or exclusively with DAP10 (long isoform NKG2D-L; refs. 59 and 60). However, recent work by Cosman and colleagues revealed that murine NKG2D-L can associate with both DAP10 and DAP12 adaptor molecules. NKG2D ligands in humans represent MHC class I chain-related molecules

Figure 47.3. Potential mechanisms of NK cell activation by MCMV infected cells. NK cells can be activated through (A) decreased engagement of inhibitory receptors or (B) increased engagement of activating receptors. Viral down-modulation of MHC class I molecules disables the engagement of inhibitory receptors and shifts the signal balance in favor of the NK cell activation. Interaction of the activating receptors and their ligands provides a signal that overcomes the inhibition and results in the NK cell activation.

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(MICA and MICB; refs. 53 and 61) and UL-16 binding proteins (ULBP-1, 2, 3 and 4 also called RAET; refs. 62 and 63). Three NKG2D ligands in mouse have been described so far: retinoic acid early transcript 1 (RAE-1) molecule consisting of five isoforms RAE-1α, β, γ, δ, and Î (64), a minor histocompatibility antigen H60 (65, 66), and a murine UL-16 binding protein-like transcript (MULT)-1 (Figure 47.2; refs. 67 and 68). Several other receptors expressed on the surface of NK cells also play an important role in the outcome of NK cell antiviral response. The NK cell receptor protein-1 (Nkrp1) family (69, 70) expressed in both humans and rodents, includes the Nkrp-1c (NK1.1) activating receptor, which serves as a NK cell marker in some mouse strains. Receptor for the Fc portion of immunoglobulins, FcγRIII/CD16, is an activating NK cell receptor (71), which is also expressed on macrophages and DCs and which functionally connects innate and acquired immune response (72). The binding of the antibodies bound to antigens on infected cells through the FcγRIII/Fc IgG fragment triggers the killing of these cells via a process known as antibody dependent cellular cytotoxicity (ADCC).

47.4 NK Cells in MCMV Infection: Resistant and Sensitive Mouse Strains NK cells play an important role in the control of herpesviral infections. Patients suffering from NK cell deficiency are prone to severe herpesvirus infections including infections with HCMV (73). Mice genetically deficient in NK cells or mice depleted from NK cells show an increased susceptibility to MCMV infection (74, 75). Adoptively transferred NK cells protect both immunosuppressed adults and newborn mice form MCMV disease. Furthermore, one of the most intensively studied systems of virus control by NK cells is the infection of mice with MCMV. NK cells use at least two distinct effector mechanisms to control the MCMV infection: (a) direct cytolysis of infected cells by exocytosis of granules containing perforin and granzyme, and (b) non-cytolytic mechanisms based on the secretion of antiviral cytokines such as IFN-γ and TNF-α, which also activate other components of the immune response (4). Some researchers reported that these two types of virus control by NK cells may in fact have some organ-specific characteristics: perforin-mediated NK cell antiviral control in the spleen, and IFN-γ mediated in the liver (76). However, subsequent studies by other researchers were not able to completely confirm these findings, showing that mice deficient either in perforin and granzyme or IFN-γ show an increased viral titers in both spleen and liver (76, 77). In our own study, we noticed that the role of cytolytic versus noncytolytic mechanisms in different tissues may also depend on the virus virulence. For instance, while mice lacking perforin and/or granzyme can control the infection with tissue-culture grown MCMV, this is not the case

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with the more virulent salivary gland-derived MCMV (Jonjic et al., unpublished). In their pioneering study, Scalzo and colleagues demonstrated that the murine NK gene complex (NKC) encoding for the Ly49 family of NK cell receptors controls susceptibility/resistance of mice to MCMV infection (78–80). Resistant mice, like those of C57Bl/6 (B6) strain, mount robust antiviral NK responses and effectively control virus replication. Later on it was demonstrated that in B6 mice resistance to MCMV maps to the activating Ly49H receptor that remarkably uses MCMV protein m157 as its ligand (81, 82) and no other cellular or viral ligands could be demonstrated (83). More recently, additional genetic loci have also been implicated in NK cell-dependent resistance (84–87). Vidal and colleagues have recently shown that genetic resistance maps to Ly49P in MCMV-resistant MA/My mice and that NK cell-mediated resistance to infection in these mice involves recognition of a viral protein in the context of MHC I H-2Dk (85). An additional locus, cmv4, is also suggested to encode for an NK cell activating receptor and to mediate resistance in PWK mice (Figure 47.3B; ref. 84). Loci outside of the NKC complex may also regulate NK cell-resistance in other mouse strains (87). In contrast, MCMV susceptible strains lacking Ly49H or any other resistant locus mentioned earlier, fail to mount an effective NK cell response, leading to increased viral loads and disease burden during the early days after infection. These findings underscore the fact that NK cell responses can determine whether viral infections are effectively controlled by host immunity or whether infections progress to cause severe or life-threatening disease.

47.5 CMV Strategies to Evade NK Responses As mentioned above, MHC class I molecules represent the dominant ligands for inhibitory NK cell receptors (42). MCMV down-regulates MHC class I surface expression to avoid recognition and destruction by CD8+ T cells, whose antigen-specific receptor activation relies on the recognition of peptide-loaded MHC class I molecule on target cell. To that aim, MCMV uses three immunoevasive genes named m04, m06 and m152 (88–90). gp34, encoded by m04, forms a complex with MHC class I molecules in the endoplasmatic reticulum (ER), which is eventually transported to the cell surface (88, 91). gp48, a product of m06, prevents the surface expression of the MHC class I by redirecting them to lysosomes for degradation. Finally, m152 encodes gp40, a glycoprotein that retains MHC class I in the ER cis-Golgi intermediate compartment (90), and therefore strongly inhibits CTL activity in vitro (92) and in vivo (93). Through its ability to down-regulate the NKG2D ligand RAE-1, m152 also evades the NK cell function (see later; ref. 94). However, it appears that there is a complex interaction of these three immunoevasins. More recent work by Reddehase and

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colleagues indicate that m04 functions as a counter-evasion protein able to restore antigen presentation by antagonizing the inhibitory function of m152 (95). Although the downregulation of MHC class I molecules compromises the viral control by CD8+ CTLs, it simultaneously exposes the virally infected cells to NK cell-mediated killing due to the lack of ligands for inhibitory receptors. To overcome this situation, like many other viruses, MCMV has evolved mechanisms to escape from NK cell recognition as well (11). One of the possibilities is encoding the surrogate MHC class I like molecules able to serve as ligands for inhibitory NK cell receptors (96). For instance, the m144 gene encodes the viral MHC class I homologue, whose immunomodulatory function was confirmed in vivo (97), but the receptor involved in the binding of m144 remains unknown. Another distantly related structural homologue of MHC class I is the above-mentioned product of m157 gene, a ligand for the acti-

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vating receptor Ly49H (82). The m157 belongs to the m145 gene family, several members of which have already been described to play a role in immunomodulation (Figure 47.4). In fact, apart from serving as a ligand for the activating NK cell receptor, the m157 protein may serve as a ligand for the Ly49I inhibitory receptor (81). This finding also indicated that the m157 gene product primarily served as a ligand for NK cell inhibitory receptors and became an activating ligand only by co-evolution with Ly49H NK cell receptor. Although it is still puzzling that the virus had preserved the gene encoding the ligand for activating NK cell receptor, it is remarkable that the exposure of the virus to Ly49H+ NK cells in vivo resulted in the mutation and deletion of m157 gene to the extent that the protein could not be recognized by Ly49H any longer (98–101). This way Ly49H+ NK cells might steer the evolution of the virus capable of avoiding innate immune control.

Figure 47.4. Down-modulation of NKG2D ligands by MCMV. NKG2D is an activating receptor whose engagement with inducible cellular ligands (RAE-1, H60, MULT-1) results in the NK cell activation. In order to evade NKG2D recognition and consequent NK cell activation, MCMV possesses genes (m138, m145, m152, m155) whose protein products are able to downregulate all known NKG2D ligands. Viral mutants lacking each of these genes are attenuated in vivo in NKG2D- and NK-cell dependent manner.

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47.5.1 MCMV Downregulation of NKG2D Ligands It is puzzling that most of laboratory mouse strains fail to generate an efficient NK cell response toward MCMV, although their NK cells do express various activating receptors that engage ligands inducible by infection. Among other questions, it was unclear why Ly49H-negative mice do not respond via NKG2D, a powerful NK cell activating receptor, whose engagement triggers cell lysis even when the cell expresses normal levels of MHC class I molecules (102, 103). We have solved this puzzle by showing that MCMV actively down-modulates cellular ligands for NKG2D receptor and therefore prevents NK cell activation (104). So far, four MCMV proteins have been characterized for their ability to down-regulate NKG2D ligands: H60, MULT-1 and RAE-1 family of proteins (Figure 47.4; refs. 94, 105–108). MCMV m152 was initially described as a gene whose product, gp40, induces the downregulation of MHC class I molecules and prevents the recognition of infected cells by CTLs (90). Due to the observation that a recombinant virus lacking m152 gene is attenuated in vivo at early days post infection, and far before CD8+ T cells initiate their immune control, it was assumed that m152/gp40 must also be involved in the regulation of NK cell function. This was confirmed by an experiment in which early attenuation of ∆m152 virus was abolished by the NK cell depletion (104). In vitro studies revealed that wt MCMV, in contrast to the ∆m152 mutant virus, downregulates the expression of NKG2D ligands from the surface of infected cells. Other researchers subsequently showed that the NKG2D ligands affected by m152/gp40 are RAE-1 proteins (94). Downregulation of only one of the NKG2D ligands could not be sufficient to prevent NK cell activation, since the remaining ligands would trigger the NK cell response. Therefore, we assumed that in addition to m152/gp40, there must be other MCMV proteins that regulate cell surface expression of H60 and MULT-1 as well. Using MCMV deletion mutants we managed to characterize two other genes that encode proteins involved in the down-modulation of NKG2D ligands. We showed that m145 gene and m155 gene products are involved in the down-modulation of NKG2D ligands MULT-1 and H60, respectively (105, 106). It is important to point out that these proteins as well as m152, belong to the m145 gene family, previously characterized as distantly related to MHC I molecules (109). In our attempts to characterize molecular mechanisms involved in the down-modulation of NKG2D ligands by the three viral proteins, we observed that there must be an additional viral inhibitor of the NKG2D ligands. Recently, we have shown that the product of m138 gene, previously identified as viral Fc receptor, is able to down-modulate surface resident MULT-1 as well as H60 proteins (107). Furthermore, we have shown that the m138 protein interferes

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with clathrin-dependent endocytosis of MULT-1 and causes its degradation in lysosomes (107). An important hint for this discovery was previous evidence that the mutant virus lacking m138 gene is attenuated in vivo in IgG-independent manner, suggesting that there must be another explanation for the early attenuation of the mutant virus (110). Indeed, we have shown that similarly to the mutant viruses lacking m152 and m155 genes, the virus lacking m138 gene is attenuated on day three post infection and that this attenuation can be abolished by NK cell depletion or blocking of NKG2D receptor (107). Although so far four viral inhibitors of NKG2D have been identified, the cellular mechanisms exploited by the viral proteins, apart from role of m138 mentioned above, still remain largely elusive. Further studies are needed to understand the expression level of different viral proteins involved in the regulation of NKG2D ligands during the course of the virus infection. Since NKG2D receptor is also expressed on T cells, additional studies are needed in order to answer the question whether the viral inhibition of NKG2D ligands has a consequence on the viral control by CD8+ T cells during the primary, but also during the chronic infection.

47.6

Conclusion

Although the MCMV model of herpesvirus infection resulted in numerous important discoveries, many fundamental questions related to the immunobiology and lifelong nature of this infection remain to be answered. The emerging new topic is the evolution and the existence of activating NK cell receptors specific for viral proteins. Here, we have reviewed our own work, as well as the work of other researchers on the role of viral regulators of NK cell response with the emphasis on inhibitors of NKG2D ligands. Even though several viral NK cell immunoevasins have been well characterized and their relevance for NK cell-mediated surveillance of virus infection has been proven both in vitro and in vivo , many aspects still remain elusive. The immediate question would be why the virus, which has evolved so many immunosubversive genes, is still not a significant pathogen after infection of the immunocompetent host. What could be the evolutional selective pressure for the virus to generate such an immunoregulatory potential? Many additional studies are needed to find out whether immunoregulatory proteins are important for the virus to spread from host to host or to maintain itself in the state of latency and reactivate in spite of fully primed immune response. Studies aimed at answering these questions should not only lead to a better understanding of the immune response during chronic and persistent virus infection, but could also stimulate the development of novel therapy and immunotherapy approaches to manage human diseases associated with altered NK or T cell responses.

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Acknowledgements. This work was supported by Croatian Ministry of Science Grants and FP6 Marie Curie Research Training grant 019248. A. Krmpotic is supported by the Howard Hughes Medical Institute Scholars grant.

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Chapter 53 Identifying Research Resources and Funding Opportunities Eugene Baizman, Hortencia Hornbeak, Peter R. Jackson, and Priti Mehrotra

53.1

Introduction

To develop and maintain a sustainable research portfolio, investigators must be aware of resources available to support their proposed research. This includes a thorough knowledge of the many databases, biomedical research-focused websites, and funding and training opportunities available both domestically and internationally. It is hoped that this information will assist the new researcher in finding and leveraging these resources to better navigate the “research enterprise” and enter at an appropriate level with the required background knowledge to achieve successful funding of a research project. Sections 1–3 of Part V provide information on the type and variety of National Institutes of Health (NIH) funding mechanisms available to members of the scientific community; the preparation and writing of a successful grant application; important federal policies governing the funding of applications and contract proposals; the NIH electronic grant application submission process and timelines; and peer review. This section highlights websites of selected resources that support many types of biomedical research activities, focusing principally on National Institute of Allergy and Infectious Diseases (NIAID) resources. Other NIH resources and additional national and international sites are listed if their resources, funding opportunities, or training programs are especially relevant for assisting individual and/or collaborative research activities. The following websites support individual or group research efforts. They include descriptions and links to more detailed information in three principal categories: (1) searchable literature and research resources databases; (2) NIAID and other NIH research networks and programs that support basic, clinical, behavioral, and translational research; and (3) national and international resources, including potential funding opportunities. From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

53.2

Glossary of Terms

To help the novice researcher in the initial navigation process, it is always helpful first to define some of the language used that may be unfamiliar. Helpful databases of terms and acronyms used within NIH sites and Part V of this book can be found at the following websites: • NIAID Glossary of Funding and Policy Terms and Acronyms (http://www.niaid.nih.gov/ncn/glossary/default5. htm): This site focuses on funding and policy terms, but contains an extensive list of other terms as well. • NIAID “FIND-IT” (http://www.niaid.nih.gov/ncn/find/): More general in nature, this site functions as an A to Z dictionary of terms, many with external links to NIH, NIAID, and DHHS policies, NIAID standard operating procedures, tools, and forms.

53.3 Literature Searching and Other Database Resources A key resource for researchers traditionally includes the library, where many hours are spent delving into the scientific literature to explore a seminal idea or trail of inquiry to support a research project. Electronic tools available to today’s investigator are unsurpassed for searchability, integration of related links, meshing of titles and subject headings, and the indexing of millions of articles from thousands of different journals covering every scientific specialty and subspecialty. New journals are appearing regularly, and it has become increasingly difficult to maintain currency in one’s chosen field without such tools to hone in on specific research topics, special problems, current issues, and novel techniques. This section lists resources for searching the scientific journal and book literature, and describes a variety of searchable biomedical databases available at the NIH and elsewhere. 507

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• The National Library of Medicine (NLM; http://www.nlm. nih.gov): The NLM has unique resources for the public, healthcare professionals, researchers, and others. The NLM gateway (http://gateway.nlm.nih.gov/gw/Cmd) includes links to search principal bibliographic, consumer healthrelated, and other information resources. Some of these information services include a Directory of Health Organizations, listing hospitals, medical centers, specialized clinics, etc.; TOXNET (databases specifically for literature citations from toxicology, hazardous chemicals, environmental health, and toxic releases) and other subsets of Medline; a Household Products database; and Profiles in Science, with biographical information of prominent researchers in the biomedical research, health, and medicine. • PubMed Central (http://www.pubmedcentral.nih.gov/): A key information resource, this site is a place to start any search of the scientific literature. PubMed Central is a free database of archived biomedical research journals, with open access content. It includes abstracts and full articles in more than 300 clinical, behavioral, and basic scientific research journals that are electronically indexed, linked, and fully searchable. • MedlinePlus (http://www.nlm.nih.gov/medlineplus/aboutmedlineplus.html): MedlinePlus is a database of information from NLM, NIH, and other government agencies and health-related organizations. Journals in the fields of medicine, nursing, dentistry, veterinary medicine, the healthcare system, and preclinical sciences are indexed and searchable. These include bibliographic and author information from more than 5,000 biomedical journals published within the United States and in numerous countries. Most records have abstracts in English. MEDLINE searches are included in MedlinePlus and give access to medical journal articles. MedlinePlus also has prescription and over-thecounter drug information, a medical encyclopedia, interactive patient tutorials, and current health news. The site also includes extensive information on many diseases and medical conditions. Also included on this site are lists of hospitals and physicians, health information in Spanish, and links to clinical trials. Standardized MEDLINE searches are included in MedlinePlus and help the researcher focus access to specific medical journal articles. • Entrez PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez): Accessible through the NLM gateway (see earlier), this database of research articles contains more than 17 million biomedical journal citation abstracts—some dating back to the 1950s—searchable by keyword, title, author, date, and many other techniques. Developed by the National Center for Biotechnology Information (NCBI) at the NLM, Entrez databases include PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, Medical Genetics resources (see later), and others (see http://www.ncbi.nlm.nih.gov/Database/index.html for a complete listing of databases). Entrez PubMed provides access to full-text articles at journal websites and other

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related web resources, some of which are free to the public. This site also provides links to other molecular biology resources. • The National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/About/sitemap.html): The NCBI, an NIH center whose mission is to develop new information technologies to help understand fundamental molecular and genetic processes controlling health and disease, is a public resource for molecular biology information. The NCBI creates “automated systems for storing and analyzing knowledge about molecular biology, biochemistry, and genetics; facilitating the use of such databases and software by the research and medical community; coordinating efforts to gather biotechnology information both nationally and internationally; and performing research into advanced methods of computer-based information processing for analyzing the structure and function of biologically important molecules.” NCBI bioinformatics-related resources may be accessed through its home page at: www.ncbi.nlm.nih.gov. The NCBI has three principal branches: 1. Computational Biology Branch (http://www.ncbi.nlm. nih.gov/CBBresearch/) 2. Information Engineering Branch (http://www.ncbi.nlm. nih.gov/IEB/) 3. Information Resources Branch, which plans, directs, and manages the NCBI technical operations A linked index of resources available to the public is listed at: http://www.ncbi.nlm.nih.gov/Sitemap/ResourceGuide.html. • Bookshelf ( http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=Books): Another resource of NCBI (http://www. ncbi.nlm.gov), Bookshelf is a collection of biomedical books that can be searched and queried directly for background information or research on new topics. Note that books are linked to PubMed abstracts, and book links can be selected when viewing an abstract.

53.3.1

Medical Genetics Resources:

• dbGaP: the NIH Genome-Wide Database (http://www.ncbi. nlm.nih.gov/sites/entrez?db=gap), abbreviated dbGaP for the database of Genotype and Phenotype, is a newer NCBI database of studies and results reporting interactions of genotype and phenotype. These include genome-wide association studies, medical sequencing, molecular diagnostic assays, and analyses of association between genotype and non-clinical traits. dbGaP provides open access to some non-sensitive data that can be viewed online and downloaded without prior permission in order to allow broad release. For sensitive data sets involving personal health information, there is controlled access; prior authorization provides oversight and investigator accountability. Study summaries and full text of study

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•

•

•

•

reports are generally available to the public, while access to individual-level data including phenotypic data tables and genotypes require varying levels of authorization. Data in dbGaP will not be protected by intellectual property patents, and the use of primary data from dbGaP to develop commercial products and tests to meet public health needs is encouraged. The Gene Reviews database (http://www.ncbi.nlm.nih. gov/books/bv.fcgi?rid=gene.TOC), funded by the NIH and developed at the University of Washington, Seattle, has peer-reviewed articles on genetic testing, and includes information on diagnosis and management of inherited disorders. GeMCRIS, the NIH Genetic Modification and Clinical Research Information System (http://www.gemcris.od.nih. gov/), is a public information resource for anyone with an interest in human gene transfer research. The GeMCRIS database has information about human gene transfer trials registered with the NIH, including medical conditions under study, involved institutions, investigators leading the trials, gene products being tested and their delivery route, and study protocol summaries. GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) is the NIH genetic sequence database, an annotated collection of publicly available DNA sequences (see Nucleic Acids Research 2006 Jan 1;34[Database issue]:D16-20; open access link to article: http://www.ncbi.nlm.nih.gov/sites/ entrez?cmd=Retrieve&db=pubmed&dopt=Abstract&list_ uids=16381837). Because many journals require sequence information to be submitted to a database prior to publication to provide readers with an accession number, GenBank includes BankIt, a web-based tool to aid rapid submission of such data. In addition, the NCBI (see earlier) provides submission software available for download at its website (MAC/PC/UNIX). Batch submissions of sequences are also possible; see the website for details. ClinicalTrials.gov (http://clinicaltrials.gov/): ClinicalTrials. gov is a database that provides updated information about federally and privately supported clinical research in human volunteers. Information about the purpose of the trial, who may be eligible, locations of the study, and phone numbers are some of the details listed at this site. Before searching, learn more about clinical trials by visiting a particularly helpful basic introductory page with Frequently Asked Questions, located at: http://clinicaltrials.gov/ct/info/resources;jsessioni d=812C903E5E4BB0DE53E303DDE7E2CCE6. The information provided on ClinicalTrials.gov should be used in conjunction with advice from health care professionals. NOTE: Investigators wishing to register trials should refer to http://prsinfo.clinicaltrials.gov.

• CRISP (http://crisp.cit.nih.gov/): The Computer Retrieval of Scientific Information on Scientific Projects (CRISP) searchable database contains information on federally funded biomedical research projects and programs. The

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research included in CRISP falls mostly within the broad category of extramural projects, grants, contracts, and cooperative agreements conducted primarily by universities, hospitals, and other research institutions and funded by the NIH (http://www.nih.gov) and other government agencies. A relatively smaller number of research grants are funded by the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Health Resources and Services Administration (HRSA), and the Agency for Health Care Research and Quality (AHRQ) and the Office of the Assistant Secretary of Health (OASH). CRISP also contains information on the intramural programs of the NIH and the FDA. The database is maintained by the Office of Extramural Research (OER; http://grants. nih.gov/grants/intro2oer.htm) at the NIH. Through the CRISP interface (http://crisp.cit.nih.gov/ crisp/crisp_help.help) public users can search for scientific concepts, emerging trends and techniques, or identify specific projects and/or investigators. This site also gives access to additional general information about the CRISP database, and answers frequently asked questions about CRISP. The home page also is a gateway to searching award information. The CRISP interface permits basic queries of the database that allow the user to access CRISP records dating back to fiscal year 1972. Results of such queries are returned in a “hit list” that includes the title of the project. Titles are linked to Abstracts containing additional information about the project, including the Abstract text.

53.4 Additional NIH/NIAID Research Resources and Networks A thorough scientific education, an advanced degree, and laboratory skills are necessary steps on the path to becoming a successful research scientist. However, building a successful research career typically also requires training from a more experienced scientific mentor to help further one’s research skills, assess appropriateness of proposed methods, and nurture one’s potential to become an independent scientist. Training opportunities are discussed in Selecting the Appropriate Funding Mechanism of Part V. Thus, a collaborative effort with a more experienced investigator or team, who may have access to resources or networks that may not be available to the individual, can be most helpful in the early stages of furthering one’s research career. Following is a list of sample resources that have been developed over the years by NIAID, specifically to encourage collaborations in a broad variety of areas with members of the scientific community having diverse training and expertise. NOTE: Website content and links may change, as programs advance, merge, separate, or are concluded successfully. • NIAID Resources (http://www3.niaid.nih.gov/research/ resources/): This website contains numerous links to

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database and network resources, covering all areas of interest to the Institute, to assist the extramural community and individual investigator in focusing attention on his or her own research efforts. Most sites list contact information as well. Research resources are listed under the Program Division that they support. These Program areas are the Division of AIDS (DAIDS), the Division of Allergy, Immunology and Transplantation (DAIT), and the Division of Microbiology and Infectious Diseases (DMID); two Interdivisional Programs are also listed. Note that in addition, several non-NIAID resources are included because they are supported by NIAID. The names and links to each resource are included. Particularly interesting secondary links also may be included for some resources. • Division of Acquired Immunodeficiency Syndrome (http:// www.niaid.nih.gov/daids/daidsover.htm): a. DAIDS Clinical Research Policies and Standard Procedure Documents: http://www3.niaid.nih.gov/research/ resources/DAIDSClinRsrch/Default.htm b. NIH AIDS Research and Reference Reagent Program: https://www.aidsreagent.org/Index.cfm c. HIV/AIDS Specimen Repository: http://www3.niaid.nih. gov/research/resources/reposit/default.htm d. HIV Immunology Database: http://www.hiv.lanl.gov/content/immunology/index.html/index.html e. HIV Sequence Database: http://www.hiv.lanl.gov/content/ hiv-db/mainpage.html; http://www.hiv.lanl.gov/content/ hiv-db/HTML/links.html f. HIV Drug Resistance Database: http://resdb.lanl.gov/ Resist_DB/default.htm g. Nonhuman Primate HIV/SIV Vaccine Trials Database: http://www.hiv.lanl.gov/content/vaccine/home.html h. Vaccine Reagent Resource: http://www3.niaid.nih.gov/ research/topics/HIV/vaccines/resources/reagent i. Primate Research Resources: http://www.ncrr.nih.gov/ comparative_medicine/resource_directory/primates.asp j. Simian Vaccine Evaluation Units: http://www3.niaid.nih. gov/research/topics/HIV/vaccines/resources/simian/ k. Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF):http://www.taacf.org/ l. HIV/OI/TB Therapeutics Database: http://chemdb2.niaid. nih.gov/struct_search/default.asp m. Resources for the Development of AIDS Therapies and Topical Microbicides:http://www.niaid.nih.gov/daids/ pdatguide/overview.htm •

Division of Allergy, Immunology, and Transplantation (http://www3.niaid.nih.gov/about/organization/dait)

a. Gene Knockout/Transgenic Mice NIAID Exchange Program: http://www.niaid.nih.gov/reposit/taconic.htm; http:/ /www.taconic.com/emerging/listing.htm b. NIAID Bioinformatics Integration Support Contract (BISC):http://www3.niaid.nih.gov/about/organization/ dait/bisc.htm

c. Database of HLA Genotypes and HLA Matching in Hematopoietic Cell Transplantation: http://www.ncbi.nlm.nih. gov/projects/mhc/MHC.fcgi?cmd=init d. International Bone Marrow Transplant Registry and Autologous Blood and Marrow Transplant Registry— North America: http://www.ibmtr.org/ e. Multiple Autoimmune Disease Genetics Consortium (MADGC): http://www.madgc.org/ f. Immune Tolerance Network (ITN): http://www.immunetolerance.org/index.shtml g. Non-Obese Diabetic (NOD) Mouse BAC Library: http:// www.niaid.nih.gov/dait/NODmice.htm h. NIH Nonhuman Primate Reagent Resources: http:// nhpreagents.bidmc.harvard.edu/NHP/default.aspx; http://nhpreagents.bidmc.harvard.edu/NHP/Available Reagents.aspx •

Division of Microbiology and Infectious Diseases (http:// www3.niaid.nih.gov/about/organization/dmid/)

a. Antimicrobial Acquisition and Coordinating Facility (AACF): http://niaid-aacf.org/ b. Antiviral Testing Program: http://www3.niaid.nih.gov/ research/topics/viral/resources.htm c. Bacterial Respiratory Pathogen Reference Laboratory: http://www.vaccine.uab.edu/ d. Bacteriology and Mycology Study Group (BAMSG): http://www.rhofed.com/bambu-bamsg/bambu-bamsg. html e. Biodefense and Emerging Infections Research Resources Repository (BEI Resources): http://www.beiresources.org/ f. Biodefense/Public Health DataBase (BioHealthBase): http://www.biohealthbase.org/GSearch/ g. Bioinformatics Resource Centers (BRC) for Biodefense and Emerging or Re-Emerging Infectious Diseases: http:// www.niaid.nih.gov/dmid/genomes/brc/ h. Collaborative Antiviral Study Group (CASG): http://www. casg.uab.edu/ i. Centers of Excellence for Influenza Research and Surveillance (CEIRS): http://www3.niaid.nih.gov/research/ resources/ceirs/ j. Filariasis Research Reagent Resource Center: http://www. filariasiscenter.org/ k. Food and Waterborne Diseases Integrated Research Network: https://web.emmes.com/study/fwd/ l. Hepatitis Resources for Researchers: http://www3.niaid. nih.gov/research/topics/hepatitis/resources.htm m. HCV Sequence Database: http://hcv.lanl.gov/content/hcvdb/index n. HPV Sequence Database: http://www.stdgen.lanl.gov/ o. In Vitro and Animal Models for Emerging Infectious Diseases and Biodefense: http://www3.niaid.nih.gov/biodefense/Research/invitro.htm p. In Vitro and Antiviral Screening Program: http://www3. niaid.nih.gov/research/topics/viral/resources.htm

53. Identifying Research Resources and Funding Opportunities

q. Influenza Genome Sequencing Project (NIAID): http:// www.niaid.nih.gov/dmid/genomes/mscs/influenza.htm r. Influenza Virus Genome Project (TIGR): http://msc.tigr. org/influenza/index.shtml s. Influenza Virus Resource (NCBI): http://www.ncbi.nlm. nih.gov/genomes/FLU/FLU.html t. Invasive Aspergillosis Animal Models Resources: http:// www.sacmm.org/iaam.html u. Leprosy Research Support:http://www.cvmbs.colostate. edu/mip/leprosy/globalleprosy3.html v. Malaria Research and Reference Reagent Resource Center (MR4):http://www.malaria.mr4.org/ w. Microbial Sequencing Centers: http://www.niaid.nih.gov/ dmid/genomes/mscs/default.htm x. Fungal Infections, Resources for Researchers: http:// www3.niaid.nih.gov/research/topics/fungal/resources. htm;http://www.sacmm.org/iaam.html; http://www. rhofed.com/bambu-bamsg/bambu-bamsg.html y. National and Regional Biocontainment Laboratories (NBL, RBL): http://www3.niaid.nih.gov/biodefense/ Research/RBL.htm z. Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA):http://ww.narsa.net/content/default.jsp aa. Pathogen Functional Genomics Resource Center (PFGRC): http://www.niaid.nih.gov/dmid/genomes/pfgrc/default. htm bb. The Plasmodium falciparum Genome Database (PFDB), TIGR: http://www.tigr.org/tdb/edb2/pfa1/htmls/ cc. Proteomics Research Centers (PRC): http://www.niaid. nih.gov/dmid/genomes/prc/ dd. PRC Administrative Resource Center: http://www. proteomicsresource.org/ ee. The Rabbit in Immunology and Infectious Disease Research: http://www3.niaid.nih.gov/research/ resources/ri/ ff. Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases (RCE): http://www3.niaid. nih.gov/research/resources/rce/default.htm; http://www3. niaid.nih.gov/research/resources/rce/sites.htm gg. Reservoirs of Antibiotic Resistance Network (ROAR): http://www.roarproject.org/ hh. Enteric Diseases Resources for Researchers: http://www3. niaid.nih.gov/research/topics/enteric/resources.htm https://web.emmes.com/study/fwd/ (Food and Waterborne Diseases Network)http://www.shigatox.net/cgi-bin/stec/ index (Shigatoxin Reference Center) ii. Schistosomiasis Resource Center: http://www.schistoresource.org/ jj. Tuberculosis Animal Research and Gene Evaluation Taskforce (TARGET): http://webhost.nts.jhu.edu/target/ kk. Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF):http://webhost.nts.jhu.edu/target/ ll. Tuberculosis Research Resources (NIAID):http://www3. niaid.nih.gov/healthscience/healthtopics/tuberculosis/ Research/Resources.htm

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mm. Tuberculosis Research Unit (Case Western Reserve University):http://www.case.edu/affil/tbru/index.htm nn. Tuberculosis Vaccine Testing and Research Materials (Colorado State University):http://www.cvmbs.colostate.edu/microbiology/tb/top.htm • Interdivision Programs: a. Clinical Research Toolkit:http://www3.niaid.nih.gov/ research/resources/toolkit/default.htm b. Tetramer Facility Guidehttp://www.niaid.nih.gov/reposit/ tetramer/index.html • NIAID-Funded Research Networks and Other Programs ( http://www.niaid.nih.gov/daids/fundedresearch.htm): Researchers wishing to join a network or program to pursue collaborative research will find this site has links to a variety of existing clinical research networks involving AIDS clinical trials in different patient populations, including vaccine and epidemiological studies. Many sites have instructions for researcher participation. NOTE: Establishing collaborations with these networks is at the discretion of the Principal Investigator. If there are difficulties identifying a contact person for the network listed, contact the NIAID Program Officer for that network. Links to the list of sites are included below. See HIV/AIDS Specimen Repository (earlier) for access to samples collected from trials conducted through these Networks/Programs. The six principal HIV/AIDS network resources are in bold (see: http://www3.niaid.nih.gov/about/organization/daids/ Networks/daidsnetworks.htm). Other NIAID programs and ongoing cohort studies make up the remainder of these resources: a. Acute HIV Infection and Early Disease Research Program (AIEDRP): http://www.aiedrp.org/ b. AIDS Clinical Trials Group (ACTG): http://www.aactg.org/ c. Centers for AIDS Research (CFARs): http://www3.niaid. nih.gov/research/cfar/ d. Comprehensive International Program for Research on AIDS (CIPRA):http://www3.niaid.nih.gov/about/organization/daids/CIPRA/(Note: Currently not accepting new applications) e. Evaluation of Subcutaneous Proleukin in a Randomized International Trial (ESPRIT): http://www.espritstudy.org/ f. HIV Prevention Trials Network (HPTN): http://www. hptn.org/index.htm g. HIV Vaccine Trials Network (HVTN): http://www.hvtn. org/ h. International Network for Strategic Initiatives in Global HIV Trials (INSIGHT): http://insight.ccbr.umn. edu/index.php i. Microbicide Trials Network (MTN): http://www.mtnstopshiv.org/ j. Pediatric AIDS Clinical Trials Group (PACTG):Renamed: IMPAACT: International Maternal Pediatric Adolescent AIDS Clinical Trials Group: http://www.hvtn.org/

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k. The Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA):http://www.cpcra.org/ l. The Women and Infants Transmission Study:http://www. niaid.nih.gov/daids/wits.htm m. Women’s Interagency HIV Study (WIHS): https://statepiaps.jhsph.edu/wihs/ n. Multicenter AIDS Cohort Study (MACS): http://statepi. jhsph.edu/macs/macs.html • Biodefense for Researchers (http://www3.niaid.nih.gov/ biodefense/default.htm): This site describes the role of NIAID and biodefense-related information for biomedical researchers, the public, and the media. Links are provided to a list of current and past Fiscal Year Biodefense Research Funding opportunities (e.g., http://www3.niaid.nih.gov/ Biodefense/Research/funding.htm). • NIAID International Grants and Contracts (http://www.niaid. nih.gov/ncn/grants/int/default.htm): With its focus on the international research community, this site provides a wealth of information in links to resources for non-US investigators. The webpage information is wide ranging and comprehensive, involving all aspects of application and qualification information; multilingual resources (Español, Francais, Portugués); ethics information; extramural research standard operating procedures; human subjects compliance and other policy information; international insights and some country-specific information; NIH post-award reporting requirements; contact information for NIH staff in the Office of International Extramural Activities; and, a list of NIAID research funding opportunity links, as well as payline, budget, and peer review information. A glossary of funding terms and an A to Z “Find It” list are also included to assist the non-U.S. investigator in navigating this comprehensive website. • Other NIH Resources: Each of the NIH Institutes and Centers includes on their website one or more resource pages. Below are two websites chosen by the authors because they do not have a specific disease focus and because of their cross-disciplinary nature and potential usefulness in helping the new investigator evaluate the available research and funding options at the NIH. a. The National Center for Research Resources (NCRR; http:// www.ncrr.nih.gov), one of the 27 Institutes and Centers one of the 27 Institutes and Centers of the NIH, provides clinical and translational researchers with training and tools to understand, detect, treat, and prevent a wide range of diseases. This support enables discoveries that span the molecular and cellular level, to animal-based studies, then to clinical research. It is anticipated that such research will result in cures and treatments for both common and rare diseases. NCRR links researchers with one another and with patients to share resources and research. NCRR programs are designed to accelerate and enhance research along the entire continuum of biomedical science. NCRR Funding Opportunities are published in the NIH Guide for Grants and Contracts (http://grants.nih.gov/

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grants/guide/), organized by date of issue, with more recent items appearing at the top of each section. Included are Program Announcements and Notices of Opportunities in Biomedical Technology (http://www.ncrr.nih.gov/biomedical_technology/funding.asp), Clinical Research (http:// www.ncrr.nih.gov/clinical_research_resources/funding. asp), Comparative Medicine (http://www.ncrr.nih.gov/ comparative_medicine/funding.asp), Research Infrastructure (http://www.ncrr.nih.gov/research_infrastructure/funding.asp), and Clinical/Translational Science (http://www. ncrr.nih.gov/research_funding/funding_opportunities/). A tabular summary of NCRR grant programs is also available (http://www.ncrr.nih.gov/research%5Ffunding/fundingtab.asp)—including brief descriptions, and staff contacts. NCRR also participates in the NIH Blueprint for Neuroscience Research (http://neuroscienceblueprint.nih.gov/) and the NIH Roadmap for Medical Research (http://nihroadmap.nih.gov/). Both programs are crosscutting and interdisciplinary, involving many NIH Institutes and Centers. The NIH Roadmap site in particular lists a large number of NIH resources available to investigators to stimulate translational—bench to bedside—research. NCRR participates in two federal grant programs that provide funding to small businesses—the Small Business Innovation Research (SBIR) program and the Small Business Technology Transfer (STTR) program (http://grants1.nih.gov/grants/funding/ sbir.htm). b. National Institute of Biomedical Imaging and Bioenginerring (NIBIB; http://www.nibib.nih.gov/HomePage) of the NIH succeeds in merging the physical and biological sciences to develop new technologies that improve health, accelerate discovery, and speed development of biomedical technologies that prevent or treat illnesses. Newer sophisticated imaging tools and techniques allow visualization of the human body not only with improved macro techniques, but also with microscale and nanoscale precision. Recent developments in bioengineering promise to enhance the body’s natural ability to recover from injury and disease. Unlike many other NIH institutes, the NIBIB is not limited to a single disease or group of illnesses but spans the entire spectrum. Bringing clinicians and researchers from every field of medicine, NIBIB joins them together with teams of scientists and engineers from many different backgrounds to develop innovative approaches to health care. Resources for researchers (http://www.nibib.nih.gov/ Research/Resources) include Scientific Resources listing sources of literature, biological reagents, and genetic and genomics tools (http://www.nibib.nih.gov/Research/ Resources/Scientific); a Resource Library with links to bioengineering and bioimaging sites, agencies, societies, and foundations (http://www.nibib.nih.gov/HealthEdu/ ResourceLibrary); and Image and Clinical Data References, with links to public clinical trial data supported by NIH such as Alzheimer’s disease, osteoarthritis, virtual colonos-

53. Identifying Research Resources and Funding Opportunities

copy, lung imaging, and brain mapping (http://www.nibib. nih.gov/Research/Resources/ImageClinData). c. Funding Opportunities: The NIBIB site searchable database of funding opportunities can be found at: http://www.nibib. nih.gov/FundingMain.

53.5 National/International Funding Resources Complementing the research mission and award mechanisms of the NIH are numerous private and public organizations worldwide dedicated to improving the health and welfare of mankind. It is especially important to note the research areas encompassed within the mission of these organizations and institutions, because most are also sources of potential funding for the investigators whose research efforts are focused in one or more of their areas of interest. Many organizations also award funds to an investigator’s institution. Some award funds only to the institution. These are noted as applicable. This section is designed to acquaint researchers with an overview of some of the principal domestic and non-US organizations that could support, through their private or public funding sources, individual researchers and projects to be performed within or outside the US. • National Resources—Government: a. The NIH Fogarty International Center (http://www.fic. nih.gov/): The John E. Fogarty International Center for Advanced Study in the Health Sciences (FIC) was created at the NIH in 1968. It is the principal international arm of the NIH, “addressing global health challenges through innovative and collaborative research and training programs” and fostering international partnerships. Training: The center website has numerous sources of support for clinical and basic scientists. One principal FIC program is the Clinical Research Scholars Resource and Support Center (http://www.fic.nih.gov/programs/ research_support/index.htm), which provides early training and career opportunities for U.S. graduate students in the health professions to participate in mentored clinical research in developing countries. The purpose of this Fogarty International Scholars program is to encourage the next generation of clinical investigators, to provide handson experience for potential new US and non-US investigators in low- and middle-income countries, and to strengthen relationships and collaborations between US and non-US institutions, researchers, and trainees. In addition, the program builds research capacity in the developing countries by providing financial and training support for students and newly graduated researchers in those countries. Regional health issues in foreign countries are a priority for the FIC. Thus, FIC has programs that address activities and opportunities in biomedical and behavioral sciences in

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Sub-Saharan Africa; South Asia; Russia, Eurasia, and the Arctic; the Middle East and North Africa; Europe; Asia and the Pacific; and the Americas and the Caribbean. Countryspecific information and instructions are also available for some countries. (For additional details, see: http://www.fic. nih.gov/programs/regional/index.htm.) Funding Opportunities (http://www.fic.nih.gov/programs/research_grants/index.htm): The Center lists on its website a number of current competitive research grant funding opportunities with research focused on the developing world. These include research on brain disorders relevant to low- and middle-income nations; the ecology of infectious diseases including transmission dynamics in animals and man; and fellowship and collaborative research grants to foster international participation between NIHsupported US scientists and non-US collaborators. Other programs include international studies on biodiversity; health and economic development; research and capacity building programs on tobacco and health; and research programs on the role of stigma in global health. A particularly interesting FIC initiative is the Global Research Initiative Program for New Foreign Investigators, which is designed to promote the productive return of NIH-trained researchers into their home countries to enhance that country’s scientific research infrastructure and stimulate locally relevant high priority research. This program also provides partial salaries to the non-US researcher returning home and support for research projects. (see: http://www.fic.nih.gov/programs/research_grants/grip/index.htm) b. The Centers for Disease Control and Prevention (http:// www.cdc.gov): The Centers for Disease Control and Prevention (CDC) was founded 60 years ago (July 1, 1946) as the Communicable Disease Center in Atlanta, Georgia, its present headquarters. The CDC’s mission is to protect and improve the public health, given 21st century challenges such as emerging infectious diseases, potential terrorism, environmental threats, an aging population, and lifestyle choices that are not always beneficial to an individual’s health or welfare. Funding and Training Opportunities: The CDC issues research initiatives using both grant and contract mechanisms. The CDC awards approximately $7 billion per year in more than 14,000 separate grant and contract actions, including simplified acquisitions. These initiatives help the CDC to accomplish its mission to promote health and quality of life by preventing and controlling disease, injury, and disability. CDC grants provide help to other healthrelated and research organizations that contribute to the CDC’s mission through health information dissemination, preparedness, prevention, research, and surveillance. (see: http://www.cdc.gov/about/business/funding.htm for Grants, funding and procurement). These websites have general information on CDC-sponsored grants (http://www.cdc. gov/od/pgo/funding/grants/grantmain.shtm) and contracts

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(http://www.cdc.gov/od/pgo/funding/contracts/contractmain.shtm), which are typically listed on FedBizOps (http://fedbizopps.gov/; Quick search: CDC). Many CDC programs are limited to the US. Training: The CDC offers a variety of training programs as well, encompassing epidemiology; public health management training; policy research and development; prevention practice; health communication; and applied and laboratory research, as well as other public health related training. These are listed at: http://www.cdc.gov/phtrain/training_programs.html.The CDC Foundation (http://www.cdcfoundation.org/) is an independent, private, nonprofit 501(c)(3) Georgia corporation, authorized by section 399F of the Public Health Service Act to support the mission of CDC in partnership with the private sector, including organizations, foundations, businesses, educational groups, and individuals. The Foundation helps the CDC to fight threats to health and safety, furthering the CDC mission. CDC scientists obtain additional resources through CDC Foundation partnerships that allow them to build programs that can substantially enhance CDC’s impact. The Foundation’s Applied Epidemiology Fellowships provide 10–12 months’ living expenses for second or third-year medical students to engage in an applied, hands-on training experience in epidemiology and public health at the CDC. Application information is found at: http://www. cdcfoundation.org/fellowships/cdcexperience/index.aspx. c. National Science Foundation (http://www.nsf.gov/): The National Science Foundation (NSF) is an independent federal agency created by Congress “to promote the progress of science; to advance the national health, prosperity, and welfare; to secure the national defense…” The NSF is the funding source for approximately 20 percent of all federally supported basic research conducted by America’s colleges and universities. In many fields such as mathematics, computer science, and the social sciences, NSF is the major source of federal backing. NSF Funding Opportunities (http://www.nsf.gov/funding/): Most NSF funding opportunities are divided into broad program areas, such as Biology, Computer and Information Sciences, Education, Engineering, Geosciences, International, Mathematics/Physical Sciences, Polar Research, Social, Behavioral Sciences, Science Statistics, and Cross-cutting Programs (see website for details). Program deadline and target date information can be found on the upcoming due dates list at: http://www. nsf.gov/funding/pgm_list.jsp?org=NSF&ord=date. It also appears in individual program announcements and solicitations. These publications can be found through the funding page http://www.nsf.gov/funding/. To receive rapid notification of new program information, by e-mail or via a custom webpage, subscribe at: http://www.nsf.gov/mynsf/. The NSF funds research and education in most fields of science and engineering, accounting for about one-fourth of federal support to academic institutions for basic research. NSF receives approximately 40,000 proposals each year for research, education, and training projects, of which approxi-

mately 11,000 are funded. In addition, the Foundation receives several thousand applications for graduate and postdoctoral fellowships. Training opportunities may be found in these program areas: • • • •

For undergraduate students (http://www.nsf.gov/div/index. jsp?div=DUE) For graduate students (http://www.nsf.gov/div/index. jsp?div=DGE) For postdoctoral fellows (http://www.nsf.gov/funding/ pgm_summ.jsp?pims_id=6201&org=DGE&from=home) For K–12 educators (http://www.nsf.gov/div/index. jsp?div=ESIE)

d. The US Civilian Research and Development Foundation (http://www.crdf.org/about/): The US Civilian Research and Development Foundation (CRDF) is a nonprofit organization authorized by the US Congress and established in 1995 by the NSF. A public-private partnership, the CRDF promotes international scientific and technical collaboration, primarily between the United States and Eurasia, through grants, technical resources, and training. The CRDF mission supports research that offers scientists and engineers alternatives to emigration and strengthens the scientific and technological infrastructure of their home countries; advance transition of non-US weapons scientists to civilian work; help move applied research to the marketplace and bring economic benefits both to the United States and the countries with which CRDF works; and strengthen research and education in universities abroad. CRDF Funding Opportunities: The CRDF staff, in-country offices, and support contractors, and proven track record in the region ensure that every project is administered in the safest and most reliable manner possible. The CRDF Grant Assistance Program (GAP) explores opportunities for collaboration in regions beyond Eurasia, including the Middle East and the Baltic Regions. For more information, see the CRDF GAP. See: Apply to the GAP (http://www.crdf.org/ gap/); More Information on GAP Services (http://www.crdf. org/gap/gap_list.htm?doc_id=290432); and, Information for Grantees (http://www.crdf.org/granteeinfo/; this page has administrative and financial project resources including Project Agreement Terms amp; Conditions, Request for Payment forms, Frequently Asked Questions and more). National Non-Governmental Resources a. The Bill & Melinda Gates Foundation (http://www.gatesfoundation.org/default.htm): The Bill & Melinda Gates Foundation was created to help reduce inequities in the United States and around the world. The Gates Foundation has been developing a process that helps decide how to spend time, effort, and money so it can accomplish that goal for as many people as possible. This process helps choose which issues to address and the groups to which grants are made. Foundation priorities, such as improving health, reducing extreme poverty in the developing world, and improving high school education in the US, establish high-level

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goals for its grant-making programs. Three program teams devise a strategy for meeting these goals, adhering to a specific process described at the following site: http://www. gatesfoundation.org/AboutUs/OurWork/OurApproach/#1. Funding Opportunities: The foundation awards the majority of its grants to US 501 (c)(3) organizations and other tax-exempt organizations identified by its staff. It does not award grants to individuals. In deciding how to invest its resources most responsibly, the Foundation looks for projects that: • Help reduce inequities in neglected areas • Produce measurable results • Catalyze increased momentum, scale, and sustainability of change • Collaborate with government, philanthropic, private sector, and not-for-profit partners • Favor preventative approaches • Leverage support from other sources • Advance our current strategies, accelerating the work we are already supporting The links below have additional details about grants and grantmaking priorities in areas of interest to the Gates Foundation: • Global Development Program—Grants in this program strive to reduce hunger and poverty in the developing world (http://www.gatesfoundation.org/ForGrantSeekers/GlobalDevelopment/) • Global Health Program—Grants in this program encourage the development of lifesaving medical advances and help ensure they reach the people who need them most(http:// www.gatesfoundation.org/ForGrantSeekers/GlobalHealth/) • United States Program—Grants in this program are designed to reduce inequities by improving access to educational and other opportunities for all residents (http://www.gatesfoundation.org/ForGrantSeekers/UnitedStates/) • Charitable Sector Support—In addition to the main program areas, the Foundation also gives grants to non-profit organizations that are serving and strengthening the overall charitable sector (http://www.gatesfoundation.org/ ForGrantSeekers/CharitableSector/) • Frequently Asked Questions—Includes answers to common questions about the Foundation’s grantmaking process and programs (http://www.gatesfoundation.org/ForGrantSeekers/FrequentlyAskedQuestions/) • Open requests for proposals (RFPs)—On occasion, the Foundation program teams issue RFPs. Notification of current RFPs is made at this site: http://www.gatesfoundation. org/ForGrantSeekers/OpenRFPs/. b. Howard Hughes Medical Institute (http://www.hhmi.org/): The Howard Hughes Medical Institute (HHMI) is a nonprofit medical research organization that helps to enhance science education at all levels and biomedical science worldwide through its grants program and other activities. The Institute conducts medical research with its own scientific

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teams. HHMI investigators work in Institute laboratories while also serving as faculty members at host institutions with which HHMI has long-term collaborations. These scientists are supported by research associates, technicians, and other personnel employed by the Institute, as well as by a headquarters staff. HHMI investigators come from university faculties and academic health centers. The Institute solicits nominations from these institutions to identify promising researchers with the potential to make significant contributions to science. Those selected as investigators are appointed for 5- or 7-year terms, which may be renewed after a rigorous review process. Funding and Training Opportunities: HHMI science education grants are administered through four programs: 1) a Graduate Science Education Program, 2) an International Program, 3) an Undergraduate Biological Sciences Education Program, and 4) a Precollege Science Education Program. HHMI grants for individuals are awarded through a competitive process having specific objectives and eligibility criteria. However, HHMI does not encourage and rarely funds unsolicited grant proposals for investigator-initiated research in the United States. Current Scientific Competitions for training opportunities can be found at: http://www. hhmi.org/research/competitions/. HHMI competitive grants do support promising individual biomedical research scientists working outside the US (http:// www.hhmi.org/grants/individuals/). Awards are made to scientists in Canada and Latin America (http://www.hhmi.org/ grants/individuals/canlatam.html), and Baltics and Central European States (http://www.hhmi.org/grants/individuals/ bceeru.html), as well as medical and dental students enrolled in a US institution (not the NIH) seeking research training (http://www.hhmi.org/grants/individuals/medfellows.html). Further HHMI resources can be found at the Institute’s Resource Center (http://www.hhmi.org/resources/). c. The Institute for Genomic Research (http://www.tigr.org/ index.shtml): The Institute for Genomic Research (TIGR) and The Center for the Advancement of Genomics (TCAG) merged in 2006 to form the J. Craig Venter Science Institute (JCVI; http://www.jcvi.org/). These have now become a large, multidisciplinary, genomic-focused organization. With scientists, staff, and laboratory space in Rockville, Maryland, and La Jolla, California, the new JCVI performs world-class genomic research. Comprehensive databases include microbial gene, fungal, parasite, and plan genomic databases represent a large repository of public research resources, most freely available to investigators. • International Resources: a. The Wellcome Trust (http://www.wellcome.ac.uk/): The Wellcome Trust is an independent privately endowed, United Kingdom (UK)-based charity funding research to improve human and animal health. The Trust’s mission is to foster and promote research with the aim of improving human and animal health. This broadly defined mission

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allows it to respond flexibly to medical needs and scientific opportunities. Major successes achieved through Wellcome Trust funding include sequencing of the human genome, development of the antimalarial drug artemisinin, pioneering cognitive behavioral therapies for psychological disorders, and establishing the UK Biobank. Welcome Trust Funding Opportunities: Grant applications are made through “funding streams”. See http:// www.wellcome.ac.uk/doc_wtd004064.html to choose one matching an area of interest. Alternatively, a complete list of grant schemes is located at: http://www.wellcome.ac.uk/ node2120.html. The websites below offer advice and guidance across all funding schemes. • Immunology and Infectious Disease: http://www.wellcome. ac.uk/funding/biomedicalscience/iid/ • Populations and Public Healthhttp://www.wellcome.ac.uk/ funding/biomedicalscience/pph/ • Neuroscience and Mental Health:http://www.wellcome. ac.uk/funding/biomedicalscience/nmh/• Physiological Sciences: http://www.wellcome.ac.uk/funding/biomedicalscience/ps/ • Molecules, Genes and Cellshttp://www.wellcome.ac.uk/ funding/biomedicalscience/mgc/ The Wellcome Trust funds a wide range of activities for health-related research conducted outside the UK as well, including the following: • Developing and Restructuring Countries: Global health research (http://www.wellcome.ac.uk/node2292.html)— biomedical research in developing and restructuring countries, with a particular emphasis on: research in infectious diseases, including tropical and neglected infectious disease, animal health, zoonoses and emerging infections public health, including communicable and non-communicable diseases. • Countries with Developed Market Economies: Major international scientific partnerships between countries with developed market economies, such as collaborations in genetics and genomics—for example, the International HapMap Project (http://www.wellcome.ac.uk/doc_WTD003500. html) and Structural Genomics Consortium (http://www. wellcome.ac.uk/doc_WTD003502.html). • There are also opportunities to apply for project grants ( http://www.wellcome.ac.uk/doc_WTD004411.html) and program grants (http://www.wellcome.ac.uk/doc_ WTD004407.html). b. The UK Medical Research Council (http://www.mrc. ac.uk/AboutUs/index.htm): The Medical Research Council (MRC) is a publicly funded organization whose mission is to improve human health through world-class medical research. The MRC supports research across the biomedical spectrum—from basic laboratory science to clinical trials—in all major disease areas. Research is supported within universities and hospitals, in its own units and insti-

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tutes in the UK, and in its units in Africa. An overview of the scientific areas covered in the MRC research portfolio is available on the site http://www.mrc.ac.uk/OurResearch/ ResearchPortfolios/index.htm. Currently, the MRC is partly funding the UK Biobank, a project to recruit half a million people aged 40–69 across Britain. It will track their health over the next 30 years to find out more about the causes and possible methods of treatment and prevention for many life threatening and debilitating diseases. MRC Funding Opportunities: In 2005, the MRC supported approximately 3,000 researchers working in universities and hospitals through research grants, and funded research training for more than 1,800 fellows and postgraduate students through MRC career awards. It also funded 32 of its own research establishments in the UK and Africa, which employ more than 3,500 scientists. The link to “Applying for a Grant” (http://www.mrc.ac.uk/ApplyingforaGrant/index.htm) explains the options available to researchers and the application and assessment process. It also provides useful background information for applicants, including MRC research priorities and recent award rates. MRC also funds research through a variety of studentship and fellowship schemes. To find out more, see the Careers section (http://www.mrc.ac.uk/Careers/index.htm). c. Europa: The European Union (http://europa.eu/abc/index_ en.htm)The European Union funds a variety of grants and programs listed at its website (http://ec.europa.eu/grants/ index_en.htm). A principal category that may be of interest to biomedical researchers is Public Health, with special topics such as bioterrorism, communicable diseases, mental health, nutrition, rare diseases, and many more (see: http:// ec.europa.eu/health/horiz_financing_en.htm). Research and Innovation, Agriculture, Environment, and perhaps other special topic areas also may interest the biomedical researcher. d. The Soros Foundations Network (http://www.soros.org/) Founded and chaired by George Soros, this network of privately endowed foundations makes grants to encourage changes in public policy to promote democratic governance, human rights, health, and socio-economic reform. The network, through their worldwide foundations, builds international alliances to accomplish their mission. Soros Networks publications and other linked resources that describe some of their accomplishments and work in progress may be found at: http://www.soros.org/resources. The network components listed below should be viewed in the context of giving the interested researcher the names of individuals or organizations providing potential collaborative opportunities in foreign countries. In addition, underserved and/or difficult to access populations could be reached through collaborative undertakings with Soros foundation staff in the host country. The resources page listed above identifies Soros grantees, conference speakers, authors, and Soros

53. Identifying Research Resources and Funding Opportunities

foundation staff, which therefore may serve as networking tools for identifying individuals with in-country knowledge of social, cultural, economic, and health situations overseas. Thus, this resource, like many others described in this section, can extend one’s search capabilities to find people with useful country-specific knowledge. The Open Society Institute (http://www.soros.org/about): The Open Society Institute (OSI) is one of the privately endowed foundations that awards grants to non-governmental organizations (NGOs), and scholarships and fellowships to individuals under a competitive process. The list of research initiatives for NGOs is country- and topic-dependent, and available at: http://www.soros.org/grants. Grants, Fellowships, and Scholarships to individuals focus on one of the following: Children and Youth; Economic Development; Education; Governance; Health; Human Rights; Law and Justice; Media, Arts and Culture, and; Women. Soros Foundations (http://www.soros.org/about/foundations): Soros foundations, located in 29 countries and two regions—Southern Africa and West Africa—consist of autonomous institutions that initiate and support Open Society activities. Priorities and specific activities of each Soros foundation are governed by a local board of directors and regional staff in consultation with George Soros and OSI boards and advisors. The regional African foundations make grants in 27 African countries. In addition support from the Open Society Institute, many of the foundations receive funding from other sources. The above site includes links to these foundations alphabetically by country.

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make available on the WHO website. The WHO Statistical Information system (WHOSIS; http://www.who.int/whosis/en/index.html) will help in such a search. These statistical databases include: burden of disease statistics, mortality data, statistical annexes of The World Health Report, statistics by disease or condition, global atlas of the health workforce, WHO Global Infobase Online, and external sources for health-related statistical information. • The WHO family of international classifications: i) The international statistical classification of diseases (http://www.who.int/classifications/icd/en/) ii) International classification of functioning, disability and health (http://www.who.int/classifications/icf/site/icftemplate.cfm) iii) Disability assessment schedule II (WHODAS II) (http:// www.who.int/icidh/whodas/index.html) • Geographical information tools: i) Public health mapping and geographic information systems (GIS) Program (http://www.who.int/health_mapping/ en/index.html) ii) Global health atlas (http://globalatlas.who.int/) iii) PAHO/AMRO SIG-Epi (http://www.paho.org/English/ DD/AIS/sigepi_web2003en.htm) • •

e. World Health Organization (http://www.who.int/about/en/) The World Health Organization (WHO) is the directing and coordinating authority for health within the United Nations. WHO tools available to aid researchers include the following: • Library database (WHOLIS), which indexes WHO publications and articles from WHO-produced journals and technical documents from 1985 to the present (http://dosei.who.int/uhtbin/cgisirsi/Mon+Jan+10+13:45:18+MET+2005/0/49). • Health statistics and health information systems (http:// www.who.int/healthinfo/en/index.html) is a WHO guide to epidemiological and statistical information includes mortality profiles, life tables, and vital registration data to name a few. Health information systems by country are also profiled. Most WHO technical programs develop healthrelated epidemiological and statistical information that they

•

WHO collaborating centers database (http://www.who. int/whocc/) Programs and Projects List (http://www.who.int/entity/ en/). WHO websites include information on a variety of training opportunities. Selected Information Resources sites:

iv) HIV/AIDS (http://www.who.int/topics/hiv_infections/ en/) v) Pandemic Influenza Preparedness (http://www.who.int/ csr/disease/influenza/pandemic/en/) vi) Avian Influenza (http://www.who.int/csr/disease/avian_ influenza/en/index.html) vii) Tropical Diseases Research (http://www.who.int/topics/ tropical_diseases/en/) viii) Infectious Diseases Resources list (http://www.who.int/ csr/disease/en/) ix) Overcoming Antimicrobial Resistance page (http://www. who.int/infectious-disease-report/2000/other_versions/ index-rpt2000_text.html)

Chapter 52 Preparing and Submitting a Competitive Grant Application Peter R. Jackson and Hortencia Hornbeak

52.1

Introduction

Preparing a competitive grant application can be a challenging endeavor. Grantsmanship encompasses the many time-intensive, complex tasks involved in planning, writing, and submitting a competitive grant application to a funding organizations. Due to limited funds and strong competition, successful biomedical researchers submit concurrent grants to several funding organizations throughout their careers. The goals and policies of the different funding organizations complicate grantsmanship for researchers and for research administrators at their institutions. However, a mastery of grantsmanship is critical for research success. Note: The National Institute of Allergy and Infectious Diseases (NIAID) maintains an extensive grantsmanship resource entitled “All About Grants Tutorials” (http://www.niaid.nih. gov/ncn/grants/) that may be of use as a general resource for all grant application processes. This section addresses several aspects of grantsmanship including why applications succeed or fail, tips for preparing a competitive grant application, the review criteria by which applications are evaluated, submission of an application, an overview of the peer-review system and what happens during a review meeting. In addition, relevant policy and resource guidance is provided concerning the submission of applications and post award management. Although this section focuses on the processes for applying for NIAID/National Institutes of Health (NIH) research funds, the information also has potential value to investigators seeking funds from other federal agencies or private organizations.

52.2 Why Applications Succeed or Fail in the Peer-Review Process All applications submitted to the NIH undergo rigorous peer review. In 2006, the success rate for NIH-supported investigaFrom: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

tor-initiated research awards was about 12%. However, NIH permits up to two resubmissions for most investigator-initiated grant applications, and success rates increase for applications revised according to the evaluative comments of the peer-review group. Experience demonstrates that successful NIAID applicants are persistent, aware of NIAID/NIH goals and policies, and knowledgeable about procedures for application submission, evaluation and revision. Based on years of review experience, the key elements of successful applications include: • • • • • • • •

new, original ideas focused research, administrative plans and/approaches appropriate resources and facilities knowledge of relevant published work experience with the essential methodology appropriate plans for data analysis clear future direction and contingency plans adequate staff with experience and training in essential methodology

In addition, application preparation should be undertaken with an understanding of the review criteria and all appropriate NIH/NIAID specific requirements of the funding opportunity announcement (FOA) and regulatory policies. On the other hand, unsuccessful applications often contain the following types of mistakes and flaws: • superficial, unfocused research or organizational/administrative plan(s) • insufficient knowledge of relevant literature and research area(s) • the selection of a principal investigator (PI) and/or key staff deficient in effort, experience, or training • excessive work for the requested period of support • insufficient detail about the significance of the work and potential future directions

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• inadequate attention to approaches and methods, alternative plans, and the statistical analysis of data • inadequate attention to preparing the application with respect to the review criteria and critical submission requirements

52.3 Developing a Competitive and Successful Application 52.3.1

Strategies for Success

Successful applicants include all relevant information called for in the application submission instructions of the FOA. Applicants must read the FOA thoroughly and verify that the all submission requirements are properly addressed and this includes the deadlines for submission. Scientific peer reviewers only evaluate submitted applications and support documents (if any). They do not compare applications. Successful applicants also understand the need to prepare their applications with attention to the review criteria and to verify that the application contains everything needed for the reviewers to make their evaluations. The five review criteria used to evaluate every NIH grant application are significance, approach, innovation, investigator and environment. However, each FOA can have specific additional review criteria and additional regulatory requirements that must be addressed by the applicants. See section 52.4 for detailed information. Successful applicants prepare a concise, clear, factual synopsis of the entire application. This description (i.e., abstract) section appears in the beginning of the application and is the first section read by the reviewers. Thus, the description can heavily influence reviewer enthusiasm for the entire submission. Therefore, to make the description comprehensive and an excellent orientation tool for the reviewers, applicants are strongly urged to write it after the application is completed. Successful applicants prepare submissions that are easy to evaluate. Reviewers are impressed by applications that are concise, well-written, well-organized and use charts, tables, diagrams, pictures, etc., in an appropriate manner. It is important to cross-reference, label, and number relevant items and to use appendices judiciously. Reviewers find it easier to evaluate applications that are prepared and organized according to the review criteria. Successful applicants prepare applications that are straightforward. Reviewers are experts in their fields and understand the strengths and weaknesses of various approaches and techniques. Reviewers expect applications to be honest about the strengths and weaknesses of the research plans and approaches. Thus it is critical to describe potential limitations or problems and how they will be addressed (or circumvented). Applications should be comprehensive and self-contained with all relevant information required for a full peer review and should never be prepared with the assumption that reviewers will know what the applicant means to say. Additionally, it is important not to over- or under-estimate the budget, indulge

in self-promotion, ignore some review criteria, or add irrelevant information to biosketches. Successful applicants rarely work alone. They seek collaborations and network widely. Some funded investigators report that reading successful applications improved their submissions and they benefited from receiving advice from department colleagues about draft applications. However, if a colleague is asked to read a draft application, it is important to allow enough time for honest and detailed feedback. Successful applicants are aware of changes in science and policies. Funding agency policies often evolve rapidly and there can be changes to submission rules. Applicants should keep abreast of such changes by reading relevant NIH, NIAID, and CSR Web pages, and by speaking with program officers (PO), scientific review officers (SRO) and grants management (GM) officers at the NIH, NIAID, and Center of Scientific Review (CSR), in addition to appropriate colleagues at your institution. Successful applicants do not give up! Applicants should read the evaluations in the Summary Statement and decide if the problems can be repaired. If so, then applicants should diligently address each criticism and develop a comprehensive, respectful reply that has a positive tone and attitude. Grantsmanship is a difficult process. Applicants should learn from the evaluative feedback and reapply. They are likely to succeed because the majority of applicants eventually are successful!

52.3.2

Checklists for the Application Process

Scientists who focus on the complexities of their research are often frustrated by the complex application submission requirements of funding organizations. The NIAID provides a comprehensive series of helpful checklists to simplify the planning, development, and submission of an NIH grant application. The NIAID checklists at http://www.niaid.nih.gov/ncn/ grants/charts/checklists.htm are revised to reflect changes in policy and procedure. They identify points critical to the development of a well integrated, comprehensive, convincing, and successful grant application. Checklists can help applicants submit competitive initial or revised applications, and to manage funded programs. They also help avoid the common mistakes in application preparation. Some checklist topics include the beginning, hypothesis, research plan, approach, background and significance, preliminary data, specific aims, design and methods, results, resources, budget, editing and proofreading, revising an application, notice of grant award, and managing the research. From the peer-review perspective, these points are critical in facilitating the evaluation of the application. For illustration, the details of the hypothesis checklist are. • What specifically am I setting out to prove? • Is the central research question important to the field? • Is the hypothesis logical, strong and testable by current methods? • Did I state my hypothesis in the abstract and specific aims sections of the application?

52. Competitive Grant Application

For a full appreciation of the scope and comprehensive nature of this resource, applicants are referred to the NIAID checklist site.

52.3.3 Advice for New Investigators The NIH is aware that new investigators traditionally have many questions and concerns about grant application development, submission and evaluation. The NIH, Office of Extramural Research (OCR), maintains a New Investigator Program at http://grants.nih.gov/grants/new_investigators/index.htm that provides information about specific grant programs and a compendium of policies pertaining to new investigators and useful resources http://grants.nih.gov/grants/new_investigators/resources.htm. The NIAID also maintains a link to useful Tips for New Investigators at http://www.niaid.nih.gov/ncn/ grants/plan/plan_i3.htm

52.4

The Review Criteria

Those who are successful in grantsmanship understand that every application should be developed with close attention to all relevant review criteria. The five standard NIH review criteria are significance, approach, innovation, investigator and environment. More information can be found at http://grants. nih.gov/grants/guide/notice-files/not97-010.html. Applicants must also remember that each FOA can have additional review criteria that need to be addressed in the application. The five evaluation criteria form the basis for the written evaluations of reviewers, their discussions of the scientific and technical merits of the application, and the assignment of Priority Scores. The five evaluation criteria are applied as explained below. While some FOAs may have additional evaluative elements and some reviews require different criteria (e.g., construction grants), most NIH research grant applications are evaluated in a similar manner. Successful applicants prepare their applications with these review criteria in mind in order for reviewers to undertake the evaluative process efficiently.

52.4.1

Significance

With respect to significance, the reviewers will be interested in determining if the proposed research is important and if the work has a clear rationale with focused aims and goals. They will also evaluate what scientific knowledge will be advanced, what benefits the work will provide, and how the work will affect the concepts or methods that drive the field.

52.4.2

Approach

With respect to the approach, reviewers will determine if the conceptual framework, design, methods, and analyses are adequately developed, well-integrated, and appropriate

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to the aims. It will be important for the applicant to identify potential problem areas and suggest alternative tactics. Depending on the grant mechanism, reviewers also will be interested in any preliminary data and/or relevant past research experience. The strategies for meeting milestones and setting priorities also will be assessed.

52.4.3

Innovation

With respect to innovation, reviewers will seek information about what is new and/or innovative about the research question(s) or method(s), and will determine if the project employs novel concepts, approaches or methods and if the aims are original and innovative. They also will seek to determine if the project challenges existing paradigms or develops new methodologies or technologies. The SRO will remind the review panel that innovative science is not always possible, and the SRO will advise the review panel to calibrate the weight of the innovation review criteria according to the research proposed.

52.4.4 Investigator With respect to the investigator, the reviewers will determine if the PI and key personnel have the training and experience necessary, to conduct the proposed research; if there are specific responsibilities and appropriate effort levels for the PI and key personnel; and if there are specific, relevant past accomplishments by the investigators. If multiple PIs are included in the application there are additional format and review considerations, see http://grants. nih.gov/grants/multi_pi/index.htm. A section of the research plan, entitled “Multiple PD/PI Leadership Plan” must be included in the research plan. A rationale for choosing a multiple Project Director/Principal Investigator (PD/PI) approach should be described. The governance and organizational structure of the leadership team and the research project also should be described and should include communication plans, processes for making decisions on scientific direction, and procedures for resolving conflicts. The roles and administrative, technical, and scientific responsibilities for the project or program should be delineated for the PD/PIs and other collaborators. If budget allocation is planned, the distribution of resources to specific components of the project or the individual PD/PIs must be delineated in the leadership plan. In the event of an award, the requested allocation may be reflected in a footnote on the Notice of Grant Award (NOGA). New investigators are critical to the continuation of the biomedical research enterprise. The NIH has developed a series of initiatives and programs to increase the number of new investigator awards. These programs and policies are found at http://grants1.nih.gov/grants/new_investigators/ index.htm.

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Environment

With respect to the environment, the reviewers will determine if the scientific environment in which the work will be conducted contributes to the probability of success and if the experiments take advantage of unique features of the scientific environment or employ useful collaborative arrangements. The applicants should include evidence of institutional support and should discuss the organizational framework and how it contributes to success. Plans for coordination and communication plans among staff and organizations will be assessed as will any special resources or facilities available and dedicated to the project. In addition to the previous criteria and in accordance with NIH policy, all applications also will be reviewed with respect to: • plans to include both genders, minorities, and their subgroups in the research • subject recruitment and retention plans • biohazards • plans for the use of select agents • model organism sharing plans • data sharing plans • foreign applications See the following webpages for more information: http:// grants.nih.gov/grants/policy/policy.htm#gps, http://grants.nih. gov/grants/peer/peer.htm#documents, and http://cms.csr.nih. gov/ResourcesforApplicants/PolicyProcedureReview+Guidel ines/Guidelines+for+Review+of+Specific+Applications/.

52.5

Submission of the Application

Successful applicants are aware that they can, for various appropriate reasons, request assignment of their application to a particular initial review group. The assignment of an application to the proper review group often is critical to its successful evaluation. Appropriate guidance is found at http://cms.csr.nih.gov/ResourcesforApplicants/Submission +And+Assignment+Process.htm and http://cms.csr.nih.gov/ ResourcesforApplicants/PolicyProcedureReview+Guidelines /OverviewofPeerReviewProcess/. Relevant information on the titles, scientific scope and rosters of the CSR Integrated review groups and study sections, is at http://cms.csr.nih.gov/PeerReviewMeetings/CSRIRGDescription/. Successful applicants are aware of the proper dates for the submission of their applications. There are rather strict policies for adhering to these dates; however, serious events such as deaths, illness, natural disasters etc can result in obtaining an approval from the CSR to submit an application late. The NIH Policy on Late Submissions is located at http://grants1.nih.gov/grants/guide/notice-files/NOT-OD06-086.html.

Note: See the “Additional Resources” in this section for more detailed instructions on submitting an application using the NIH systems including a segment designed to assist the potential grantee in navigating the electronic submission process.

52.6 Overview of the NIH Peer-Review System This section provides an overview of NIH and NIAID scientific peer review that covers conflict of interest (COI) and confidentiality, the involved personnel and their roles, the review process, and the outcome(s) of the review process. The NIH supports medical and behavioral scientific research in the pursuit of fundamental biological knowledge and the application of that knowledge to improve public health. Approximately 80% of the NIH budget is awarded as grants or contracts to extramural (non-NIH) institutions worldwide for basic and applied research. The NIH uses a robust scientific peer-review system to evaluate the scientific and technical merits of all submitted grant applications and contract proposals. The scientific peer-review system followed by the NIH is a competitive process that is managed by federal officials called SRO. Scientific peer review is conducted according to NIH policies and procedures that are based in law and overseen by the NIH Office of Extramural Research (OER) for Peer Review Policy, http://grants1.nih.gov/grants/peer/ peer.htm. Most NIH grant applications are of the type called Investigator Initiated Research Applications (or R01s) that contain the original research ideas of investigators in many biomedical and associated disciplines. The review of these applications is conducted by expert peers in scientifically aligned panels whose composition and activities are managed by personnel in the NIH Center for Scientific Review (CSR; http://cms.csr.nih.gov/). The CSR manages the peer review of most of the 80,000 applications submitted each year to the NIH. In the NIAID, scientific peer review is conducted according to NIH-NIAID policies and procedures overseen by the NIAID Division of Extramural Activities (DEA), http:// www3.niaid.nih.gov/about/organization/dea/default.htm, and the NIAID Scientific Review Program (SRP), http://www3. niaid.nih.gov/about/organization/dea/srp.htm. The SRP is responsible for the evaluation of grant applications and contract proposals submitted in response to NIAID-specific FOAs. NIAID-specific FOAs are located at http://www.niaid.nih.gov/ncn/budget/opps.htm. All NIH FOAs are published in the NIH Guide to Grants and Contracts http://grants.nih.gov/grants/guide/. Finally, all Federal government grant opportunities (including those of the NIH and NIAID), can be accessed at http://www.grants.gov/.

52. Competitive Grant Application

52.6.1

COI and Confidentiality

The NIH has a longstanding interest in research objectivity, in avoiding real and/or apparent financial, scientific, and personal COI and in maintaining confidentiality. NIH COI policies exist for NIH-supported institutions (http://grants2.nih.gov/grants/guide/notice-files/NOT-OD-05013.html) and grantees (http://grants2.nih.gov/grants/policy/ coi/coi_grantees.htm). There also is extensive, current webbased information on COI issues for NIH staff (http://grants2. nih.gov/grants/policy/coi/index.htm) and for reviewers (http:// grants2.nih.gov/grants/peer/COI_Information.doc). In the peer-review process, NIH staff and reviewers follow COI guidelines and must complete and sign pre-review (http:// grants2.nih.gov/grants/peer/Pre-Cert-Form.doc), and post-review (http://grants2.nih.gov/grants/peer/Post-Cert-Form.doc) COI/ Confidentiality certification documents. The NIAID adheres to the NIH COI guidelines (http://www. niaid.nih.gov/ncn/sop/coi.htm.) listed below: A review meeting participant (NIH staff or reviewer) must leave the review proceedings when: • his or her close relative (e.g., spouse, minor child, sibling, or parent) or partner (e.g., close professional associates or other colleagues) has a financial interest in the outcome of an activity such as peer review • he or she serves as an officer, director, member, owner, trustee, expert, advisor, consultant (with or without compensation), or employee of an applicant or other organization that would be affected by his or her decision • he or she is negotiating or has an arrangement for prospective employment with an applicant or other organization that would be affected by his or her decision Review meeting participants are urged to avoid any actions that might give the appearance of COI, even if they believe there may not be an actual conflict. The NIH has established a conflict of interest threshold of $10,000 for extramural researchers serving on NIH scientific peer-review panels, or “study sections,” that are used to evaluate research proposals. This financial threshold includes all sources of financial benefit, including honoraria, fees and stock holdings, both currently held and accruing over a 12month period. Interests held by immediate family members of the reviewer are included. Reviewers with a financial or other interest worth $10,000 or more in an application are disqualified from the review. Exceptions may be made by the NIH director under certain circumstances. This rule was published in the January 5, 2004, Federal Register. By completing the pre-review and post-review certification forms, NIH staff and reviewers certify that they will: • maintain the confidential nature of all grant application and review materials and of all review meeting proceedings • destroy or return all review-related material • not discuss review proceedings with anyone except the SRO • refer all questions concerning review proceedings to the SRO

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50.6.2

NIAID Scientific Review Staff Roles

By deliberate design, the NIH maintains a strict separation of responsibilities for federal personnel involved in the development and management of research programs, the peer review of grant applications, and the oversight of the award of federal funds. All aspects of the grant evaluation and award processes proceed according to policy and legal regulations. The evaluation of the scientific and technical merit of applications is determined by peers who are scientific experts, recruited by the SRO based on their relevant scientific expertise, research background, review experience and lack of COI with the applications. NIH staff do not take part in the evaluation of applications. The PO is involved in the development of research initiatives, some aspects of application submission before peer review, and interactions with applicants after peer review. SRO manage the thorough and fair evaluation of applications for scientific and technical merit according to peer review policies and procedures. GM officers are responsible for the business and budget parameters of awarded funds according to federal policy, http://grants.nih.gov/grants/policy/policy. htm#gps.

52.7 What Happens During a Review Meeting Successful applicants become familiar with the events that take place during a review meeting by reading guidance documents and, if possible, through participation in the peer review process of other applications as a member of an initial review group. Practical knowledge about the peer-review meeting process strengthens ones understanding of features that may increase, or decrease, reviewer enthusiasm for a submission. This knowledge often helps in the preparation of competitive applications. The following Web sites provide advice about, and even an actual video of, a peer review meeting. The CSR maintains several detailed documents on scientific peer review meetings at http://cms.csr.nih.gov/Peer ReviewMeetings/ and also provides comprehensive advice for reviewers at http://cms.csr.nih.gov/ResourcesforApplicants/P olicyProcedureReview+Guidelines/Guidelines+for+Review+ of+Specific+Applications/. Included at this site is a document that details the evaluative and scoring processes and provides links to all relevant federal guidelines http://cms.csr.nih.gov/ AboutCSR/OverviewofPeerReviewProcess.htm. In addition there is a video of a peer-review meeting at http://cms.csr.nih.gov/ResourcesforApplicants/PolicyProc edureReview+Guidelines/OverviewofPeerReviewProcess/ InsidetheNIHGrantReviewProcessVideo.htm. This WWW page also includes links to actual Summary Statements that detail the results of the peer review of three different grant applications.

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52.7.1

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Streamlining

The NIH engages in a process of reducing the number of applications that are thoroughly discussed during a review meeting. This process is called Streamlining and is a formal procedure by which applications considered non-competitive for an award are not discussed in detail during the peer review meeting. Streamlining is undertaken only to facilitate the peer review process. An unscored application is not necessarily lacking in scientific or technical merit. However, due to the competitive nature of the grant application and scientific peer review processes, the reviewers did not consider the application competitive for an award. Every member of the review panel must agree to the list of streamlined applications. If even one reviewer does not agree to streamline an application, it will receive an evaluative discussion. Applicants who have a streamlined application receive a summary statement with written critiques, but without a resume or score since no evaluative discussion or score processes took place. More information can be found at: http://grants.nih.gov/grants/peer/ peer.htm.

52.7.2

Assigning Priority Scores

Assigned priority scores for non-streamlined applications can range from 1 to 5 in 0.1 increments and are linked to the evaluative adjectives as follows: outstanding (1.0–1.5), excellent (1.5–2.0), very good (2.0–2.5), good (2.5–3.5), acceptable (3.5–5.0), and nrfc (not recommended for further consideration). More information can be found at http://cms.csr.nih.gov/ ResourcesforApplicants/PolicyProcedureReview+Guidelines /Guidelines+for+Review+of+Specific+Applications/. Priority scores are assigned by each non-conflicted reviewer to each application. Reviewers not assigned to an application are expected to listen carefully to the comments of the assigned reviewers during the review process. Assigned reviewers are to recommend priority scores that reflect their comments about the application. Any reviewer who plans to score outside the range of the other reviewers is requested to make this known to the rest of the panel and to provide a reason for the different score.

52.7.3

Budget Recommendations

Reviewers discuss the scientific and technical aspects of, and score, each application before making any budget recommendations. This process assures that the applications are evaluated and scored only on the basis of their scientific and technical merit. No budget considerations are made for streamlined applications. Reviewers consider the reasonableness of the proposed budget and duration in relation to the proposed research. Key review elements include: the duration of the work, the effort (time) requested for each staff member, and the strength of the justifications for the requested budget (i.e., is the justification

inadequate, excessive, or appropriate for the proposed work). Reviewers are asked to make specific recommendations when modifying the budget. The SRO provides additional advice concerning the discussion of modular budgets http://grants1. nih.gov/grants/funding/modular/modular.htm.

52.7.4

Post-Review

NIH and NIAID peer reviewers prepare written evaluations of their assigned applications and present them during the peer review meeting. The written evaluations, and oral discussions, of each application form the basis of the Summary Statement prepared by the SRO after the review. The summary statement contains the reviewer evaluative critiques, a summary of the oral discussion with the final evaluative decisions about the scientific and technical merit of the application, and budget recommendations. The summary statement is available to applicants through the eRA Commons within weeks after the review.

52.8

Conclusion

In conclusion, the NIH recognizes that application development and submission processes takes time and can be challenging. Applicants at large organizations are urged to seek assistance from their institution Office of Sponsored Research (or similar group). Investigators at smaller institutions can take advantage of information available from the NIH WWW and from NIH outreach programs in various cities that explain the submission process and that address investigator and administration officer questions.

52.9

Additional Resources

52.9.1 Registration and Application Submission Process Details 52.9.1.1

Registration

Potential grantees must first register themselves with the systems below. This one-time registration is absolutely necessary for the individual and for the applicant’s institution/ organization. Different information is required for the applicant’s organization. While only the institution must register with Grants. gov, both applicants and institutions will register with the eRA Commons. 1. Grants.gov: This is the Federal government’s single on-line portal developed to allow applicants to find and to apply for Federal grant funding. It is used by all NIH ICs and all 26 federal grantmaking agencies. The applicant’s organization (“Applicant Organization/Institution”) must register first at http://www.grants. gov/applicants/get_registered.jsp

52. Competitive Grant Application

i. This one-time only registration is for your institution/ organization/business. ii. Obtain an EIN (Employer Identification Number) from the Internal Revenue Service (new businesses: http://www.irs. gov/businesses/small/article/0,,id=102767,00.html). iii. Request a DUNS (Dunn & Bradstreet) number (https:// eupdate.dnb.com/requestOptions.html). iv. Register with the US Government’s “Central Contractor Registry” (CCR); identify the eBiz Point of Contact (POC). v. Register the authorized organization representatives (AORs): individuals who can submit your application officially on behalf of your organization. vi. NOTE: This process can take ~2 to 4 weeks; or up to 8 weeks for newly established businesses. 2. eRA Commons: This is the NIH Electronic Research Administration system that allows applicants and grantees to electronically receive and transmit application and award information. Both the applicant organization and the PI must register first: Organization: this is one-time only, for the applicant’s organization/institution and the organization’s Signing Official (SO; also the AOR) i. If already registered, see (and confirm) your institution on the list of registered organizations: http://era.nih. gov/commons/index.cfm. ii. If not listed, the institution official should check the following website: https://commons.era.nih.gov/commons/ registration/registrationInstructions.jsp. iii. Create separate accounts for the PD/PI, or affiliate them. iv. eRA Commons and Grants.gov registration can be completed simultaneously (as soon as a DUNS No. is received). v. Allow 2 to 4 weeks to complete. 3. PD/PI: must register through the organization/institution’s SO i. A PI and a SO need separate accounts in eRA Commons because each has different privileges. ii. A PI has one account throughout his/her career. This same account may be affiliated with multiple institutions/organizations. iii. Allow 3 to 5 business days to complete.

52.9.1.2

Pre-Application

First, decide on the submission method: a) Will it be forms-based? If so, see the “Software Requirements” below. b) Will it involve a system-to-system (XML Data Stream) transfer? c) Will you or the institution be using a Third Party? (A commercial service provider may submit on behalf of your organization).

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Second, note the software requirements: http://www.grants. gov/resources/download_software.jsp a) The website above allows free downloads. b) NOTE: Adobe eForms replaced PureEdge software Summer/Fall 2007. This is platform independent, requiring only the free Adobe reader Finally, think ahead to the application deadline. Consider: a) your institution’s review and approval process b) any collaborator’s review and approval process; multiply by number of collaborators c) unforseen and forseen events/holidays/meetings/committee service, etc d) avoid the last minute submission rush at your institution’s Office of Sponsored Research

52.9.1.3 The Application Process Applying for the first time to the NIH for funding, whether it is for one of the many training programs available, or for an independent or collaborative research project grant, can be challenging. The electronic grant development and application processes are described in http://era.nih.gov/Electronic Receipt/prepare_app.htm#1. The two main application guides with instructions specific to completing the SF-424 application are the General Instructions Guide (Version 2/2A) and the General + SBIR/STTR guide. Application guides are in three parts: • Part I: Instructions for Preparing and Submitting an Application • Part II: Supplemental Instructions for Preparing the Human Subjects Section of the Research Plan • Part III: Policies, Assurances, and Definitions. All applicants should note that each FOA is linked to an appropriate guide that explains, in a step-wise manner, how to enter information into the form, and that also explains the form contents. There also is help available via the NIH Electronic Receipt website (http://era.nih.gov/ElectronicReceipt/ prepare_app.htm#1) and a particularly useful link has information on “avoiding errors” (http://era.nih.gov/ElectronicReceipt/ avoiding_errors.htm). NIH Electronic Application Submission: This section is designed to assist the potential grantee in navigating the NIH electronic Grant Application Process (http://era.nih.gov/ElectronicReceipt/). The transition plan timeline can be found at: http://era.nih.gov/ ElectronicReceipt/strategy_timeline.htm. This electronic transition replaced the paper PHS 398 grant application form with the SF-424 electronic application form. All mechanisms submitted via the electronic process, require electronic submission of the Standard Form (SF)-424-R&R; (Research & Related) through Grants.gov. Updates on this new process are available at http://era.nih. gov/ElectronicReceipt/listserv.htm.

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Begin with the following set of instructions: • Prepare to apply: • http://era.nih.gov/ElectronicReceipt/preparing.htm • Find opportunity and download application package: • http://era.nih.gov/ElectronicReceipt/find_app.htm • Prepare application: • http://era.nih.gov/ElectronicReceipt/prepare_app.htm • Submit application to Grants.gov: • http://era.nih.gov/ElectronicReceipt/submit_app.htm • Note: ONLY an AOR submits not the individual investigator(s) • Check submission status in commons • http://era.nih.gov/ElectronicReceipt/check_submission. htm • Check assembled application • http://era.nih.gov/ElectronicReceipt/check_submission. htm • Submission complete

52.9.2 DHHS, NIH Regulations, Policies and Offices that Affect the Submission, Evaluation, and Management of Awards The NIH Grants Policy Statement (GPS; http://grants.nih.gov/ grants/policy/nihgps_2003/index.htm) makes available to NIH grantees, in a single document, the policy requirements that serve as the terms and conditions of NIH grant awards. This document also is useful to those interested in NIH grants by providing information about NIH—its organization, its staff, and its grants process. The NIH GPS has three parts: Part I: NIH Grants—General Information; Part II: Terms and Conditions of NIH Grant Awards, and Part III: Points of Contact. The Office of Laboratory Animal Welfare (OLAW; http:// grants2.nih.gov/grants/olaw/olaw.him) provides guidance and interpretation of the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals, supports educational programs, and monitors compliance with the Policy by Assured institutions and PHS funding components to ensure the humane care and use of animals in PHS-supported research, testing, and training, thereby contributing to the quality of PHS-supported activities. The DHHS Office of Human Research Protections (OHRP; http://www.hhs.gov/ohrp/assurances/assurances_index.html) provides clarification and guidance to research institutions, develops educational programs and materials, and promotes innovative approaches to enhancing human subject protections. Basic HHS Human Subjects policy is in the Code of Federal Regulations (Title 45, Part 46; http://www.hhs.gov/ ohrp/humansubjects/guidance/45cfr46.htm). Guidance to investigators applying for federal funding for research involving human subjects—including decision trees to assess whether human subjects research is involved in a particular application or proposal—are found at the OER website (http://grants.nih.gov/grants/ policy/hs/index.htm) and also are included in the PHS 398 Part II instructions (http://grants.nih.

P. R. Jackson and H. Hornbeak

gov/grants/funding/phs398/instructions2/phs398instructions. htm and in Part 2 of the Grants.gov Application Guide for the new SF424 form (http://grants.nih.gov/grants/funding/424/ index.htm), which specify what information must be included in the Human Subjects section of a grant application. Investigators involved in conducting research involving an intervention with a living human or who provide coded human data or specimens and who collaborate on other activities related to the conduct of the research are involved in human subjects research and must follow the appropriate regulations (see: http://grants.nih.gov/grants/policy/hs/specimens.htm). However, an investigator solely providing de-identified coded human data or specimens collected for purposes unrelated to the research may not be involved in HS research. Decision trees that help to clarify the latter point are posted at: http:// grants.nih.gov/grants/policy/hs/ PrivateInfoOrBioSpecimens DecisionChart.pdf and at http://www.hhs.gov/ohrp/human subjects/guidance/decisioncharts.htm. The NIH Office of Extramural Research (OER; http:// grants2.nih.gov/grants/oer.htm) issues policies and guidelines for extramural research grants administration. This office has primary responsibility for the development and implementation of NIH grants policy, the monitoring of compliance with Public Health Service policy on Humane Use and Care of Laboratory Animals, the coordination of program guidelines, and development and maintenance of information systems for grants administration. NIH Select Agent Policy: The use of select agents and toxins is governed by a number of federal laws and regulations. These apply to applicants seeking funding for grants and contracts. http://grants.nih.gov/grants/policy/select_agent/#policies. The NIH GPS also includes a section on select agents and compliance with these laws and regulations: http://grants. nih.gov/grants/policy/select_agent/GPS_Select_Agent.pdf. Non-U.S. organizations also must comply with select agent policies. Frequently asked questions for the NIAID Select Agent Policy for Foreign Institutions are located at http:// www.niaid.nih.gov/ncn/qa/selagentfor.htm. A summary of NIH requirements for complying with select agent and toxin use in NIH grant applications, both domestic and non-United States, is included at http://www.niaid.nih. gov/ncn/grants/selectterm.htm. This site also contains links to lists of select agents from the NIAID, the Centers for Disease Control and Prevention (CDC) and the U.S. Department of Agriculture (USDA), as well as a flowchart for select agent research awards and procedures to be followed. NIH guidelines for research involving recombinant DNA and gene transfer, including transgenic/knockout animals: http://www4.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlnes_ lnk_2002z.pdf. The NIH guidelines on the proper use and disposal of transgenic animals, plants and other forms of recombinant DNA (rDNA) in research can be found in NIH guidelines for research involving recombinant DNA molecules. http://www4.od.nih. gov/oba/rac/guidelines/guidelines.html.

52. Competitive Grant Application

NIH data and resource sharing: The NIH mission to improve public health through research also includes a longstanding legislative mandate to make available to the public the results of research activities that it supports and conducts with federal dollars. Likewise, through the GPS (http://grants.nih. gov/grants/policy/nihgps_2003/index.htm) the NIH expects that PDs and PIs who receive NIH funding will share those resources, including animal models and data, and encourages them to make the results and accomplishments of their activities available to the public. NIH Data Sharing policy: The NIH expects and supports the timely release and sharing of final research data from NIHsupported studies for use by other researchers. Data sharing allows scientists to expedite the translation of research results into knowledge, products, and procedures to improve human health. In NIH’s view, all data should be considered for data sharing. Data should be made as widely and freely available as possible while safeguarding the privacy of participants, and protecting confidential and proprietary data. Relevant information is at (http://grants2.nih.gov/grants/policy/data_ sharing/data_sharing_guidance.htm. To facilitate data sharing, investigators submitting a research application requesting $500,000 or more of direct costs in any single year to NIH are to include a plan for sharing final research data for research purposes, or state why data sharing is not possible. NIH Model Organism Sharing Policy: The Model Organisms for Biomedical Research Policy (http://www.nih.gov/ science/models/) and an information page (http://grants.nih. gov/grants/policy/model_organism/index.htm) are available. To extend NIH resource sharing policies, investigators submitting an NIH grant application (including competing renewals) or contract proposal are expected to include in the submission a specific plan for the timely sharing and distribution of unique model organisms and related research resources generated through the use of NIH funding so other researchers can benefit from these resources. Alternately, they must state appropriate reasons for why such sharing is restricted or not possible. The model organism sharing plan is not subject to a cost threshold and is expected in all applications where the development of model organisms is anticipated. NIH Principles and Guidelines Sharing of Biomedical Resources: The NIH considers the sharing of unique research resources (also called research tools) an important means to enhance the value of NIH-sponsored research as stated in this policy document http://ott.od.nih.gov/policy/ research_tool.html.

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The NIH believes that restricting the availability of unique resources can impede the advancement of further research. Therefore, after the resources are developed with NIH funds and after the associated research findings are reported through publication, or after they are provided to NIH, the resources also are to be made readily available for research purposes to qualified individuals within the scientific community. This requirement is in the following documents: NIH GPS—Sharing of Unique Research Resources: http:// grants.nih.gov/grants/policy/nihgps_2003/NIHGPS_Part7. htm#_Toc54600134, and Principles and Guidelines for Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating Biomedical Research Resources (64 FR 72090, December 23, 1999), http://www.ott.nih.gov/policy/ rt_guide_final.html Inventions and Patents: The NIH Office of Technology Transfer (OTT), http://ott.od.nih.gov/index.html, evaluates, protects, licenses, monitors, and manages NIH and Food and Drug Administration (FDA) discoveries, inventions, and other intellectual property according to the Federal Technology Transfer Act and other laws. With respect to patents, the OTT is involved the development of technology transfer policies and in patent prosecution and licensing agreements for the NIH, the FDA, the CDC and other components of the Department of Health and Human Services (DHHS). Incentives for the practical application of research supported through Federal funding agreements are provided through the Bayh-Dole Act of 1980 (US Public Law 96517; 35 U.S.C. 200-212) and the related EO 12591 (April 10, 1987). To retain rights and title to inventions made with Federal funds, grantees must comply with regulations that ensure the timely transfer of the technology to the private sector, while protecting limited rights of the Federal government. The regulations apply to any invention conceived, or first actually reduced to practice, in the performance of work under the Federal award, and they apply to all types of recipients of Federal funding. NIH grantees may retain intellectual property rights to subject inventions provided they do the following: • Report all subject inventions to NIH. • Make efforts to commercialize the subject invention through patent or licensing. • Formally acknowledge the Federal government’s support in all patents that arise from the subject invention. • Formally grant the Federal government a limited use license to the subject invention.

Chapter 51 Selecting the Appropriate Funding Mechanism Priti Mehrotra, Hortencia Hornbeak, Peter R. Jackson, and Eugene Baizman

51.1

Introduction

Now that the strategies needed to build a sustainable research career have been discussed, it is important to be cognizant of the mission of the funding agency, the National Institutes of Health (NIH), to select the most appropriate funding mechanism. This chapter provides details of the most important NIH grant funding mechanisms and subsequent sections explain in much more detail the preparation and submission of the application, and cover the NIH peer review process. With the goal of improving public health, NIH funds the most meritorious scientific research projects within available funding limits. Peer review is the process that evaluates each submitted application for its scientific and technical merit. In general, the scientific merit of the proposed research project is the most important factor that determines whether it is funded. However, it is important to focus not only on elegant science, but also on the institute’s mission and its programatic needs. The impact of the proposed study on public health, the feasibility of the proposed study, and selection of the appropriate funding mechanism also are very important for building a successful and sustainable personal research portfolio. Therefore, it is important to write a successful NIH grant application with a clear understanding of NIH administrative grant award policies and procedures, and this section provides the potential applicant with some help navigating the system. Because the NIH supports different types of grants (and contracts), it may be difficult to select the appropriate funding mechanism when writing an application. Therefore, this chapter helps with that selection and provides the investigator with an overview of some common funding mechanisms. In determining the most appropriate funding mechanism, investigators first should be aware of the requirements of each From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

mechanism, and then assess their ability to compete successfully for funds. Some award mechanisms are specific for PhDs and others for MDs. Figure 51.1 illustrates a career path for PhDs and Figure 51.2 for MDs. Note that there also are award mechanisms common to both career paths. Potential applicants should choose the award type that matches their area(s) of interest. To select the most appropriate award options, refer to the career-stage graphics (Figure 51.1 and Figure 51.2). As mentioned, NIH uses variety of award mechanisms to meet its mission and programs. NIH institutes and centers (ICs) may vary in the way they use grant mechanisms. However, it should be noted that not all ICs accept applications for all mechanisms. Thus, potential applicants should periodically check the National Institute of Allergy and Infectious Disease grants and contracts website at http://www.niaid.nih.gov/ncn/ grants/default_grants.htm for more details. All grant applications submitted to the NIH must be submitted in response to a funding opportunity announcement (FOA), which is a notice in Grants.gov for a federal grant funding opportunity. FOAs are released by NIH ICs and are published simultaneously in the NIH Guide for Grants and Contracts (http://grants2.nih.gov/grants/guide/index.html) and at Grants. gov (http://www.grants.gov). Potential applicants can obtain an application package as well as general instructions for completing an electronic grant applications package for a particular FOA (http://era.nih.gov/Electronic Receipt/find_app). Investigators may submit their research as an investigator-initiated grant application, known as “unsolicited” research, or submit in response to FOAs known as a program announcement (PA), a request for application (RFA), or a request for proposals (RFP). As mentioned earlier, institutes publish PAs and RFAs in the NIH Guide to Grants and Contracts and in Grants.gov as FOAs. Contract RFPs are published in FedBizOps (http://www.fedbizops.gov). It is important to determine and choose the award mechanism that fits the investigator’s research goal and also addresses the mission and programmatic needs of the participating ICs. Therefore, it is wise to contact the program officer at the IC to discuss the appropriateness of the funding mechanism as it 487

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Figure 51.1. Potential career path for an investigator with a PhD (See Color Plates).

relates to the proposed area(s) of science and the investigator’s stage along the career path.

51.2 Investigator-Initiated Research (Unsolicited Applications) The principal investigator (PI) of an investigator-initiated application has the responsibility of selecting the subject area, which should be exciting, significant, and important. PIs should capitalize on their strengths and find creative, novel and/or innovative ideas within their areas of expertise that would make an impact on public health. Once a

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topic has been selected, the PI should assess the literature to ensure the topic fills a research gap, search databases to see what has already been done, and assess other resources available (see Part V, Identifying Research Resources and Funding Opportunities) to leverage one’s research prowess and expertise. Then, the researcher should decide the type of funding mechanism that fits, based on his or her career level and the needs of the research. Generally, PIs submit a basic research project grant (R01), which gives a substantial level and duration of funding support. Potential applicants for their first independent research grant should apply through a NIH-wide parent PA that covers investigator-initiated research. Applicants also should check institute-specific PAs, because some institutes do not participate—or may not accept—applications in all research areas in response to a parent announcement, or they may have institute-specific requirements. A comprehensive list of all funding mechanisms used by NIH or NIAID can be found at http://www.niaid.nih.gov/ncn/ grants/mechan.htm or http://grants.nih.gov/grants/funding/ac. pdf. Listed below are some of the main NIH and NIAID investigatorinitiated grant funding mechanisms with brief descriptions.

51.2.1

Research Grants (R Series)

NIH has developed the parent announcement for investigator-initiated or unsolicited applications. Several parent announcements are available and are listed below. This list is by no means exhaustive. For a full list, see the website: http://grants1.nih.gov/grants/guide/parent_announcements. htm. Note that in general, all NIH grants that are funded successfully are awarded to the investigator’s institution, which administers them on behalf of a principal investigator.

Figure 51.2. Potential career path for investigator with an MD (See Color Plates).

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a. NIH Research Project Grant Program (R01): This mechanism provides support for a specified and focused investigator-initiated research project for 1 to 5 years. Applicants should provide preliminary data to support their research and there can be more than one principal investigator on an R01 grant; see http://www.niaid.nih. gov/ncn/newsletters/2007/0329.htm. This is a very competitive grant mechanism and the budget varies based on the proposed research. For more information see the PA: http://grants.nih.gov/grants/guide/pa-files/PA-07-070. html. b. NIH Small Grant Program (R03): This mechanism provides limited funding for a short period of time to support a variety of projects including pilot or feasibility studies, secondary analysis of existing data, small, self-contained research projects, development of new research technology, etc. Funding is limited to up to 2 years in direct costs generally up to $50,000 per year. For more information see the PA: http://grants.nih. gov/grants/guide/pa-files/PA-06-180.html. c. NIH Academic Research Enhancement Award (AREA; R15): AREA grants support small research projects, new or expanded, in the biomedical and behavioral sciences conducted by students and faculty in health professional schools and other academic components that have not been major recipients of NIH research grant funds. Direct costs are limited to $150,000 over the entire project period—up to 3 years— with a limited eligibility. For more information see the PA: http://grants.nih.gov/grants/guide/pa-files/PA-06-042.html. d. NIH Exploratory/Developmental Research Grant Award (R21): This mechanism is for new, innovative, exploratory and developmental research projects. Preliminary data are generally not required for this mechanism; however, supportive data are helpful. Funding is limited to up to 2 years and the combined budget for direct costs for the 2-year project period usually may not exceed $275,000. For more information, see the PA: http://grants.nih.gov/ grants/guide/pa-files/PA-06-181.html. e. NIH Clinical Trial Planning Grant Program (R34): This is a planning grant mechanism designed to support the development of clinical protocol and the preparation of essential documents for clinical trial implementation grants. A R34 planning grant is awarded for 1 year up to $150,000 in direct costs. For detailed information about NIAID investigatorinitiated clinical trial planning and implementation grants, see the website: http://www.niaid.nih.gov/ncn/clinical/R34.htm. f. Small Business Technology Transfer (STTR; R41/R42): This mechanism is designed to stimulate a partnership of ideas and technologies between innovative small businesses and research institutions through federally funded research or research and development (R&D). This mechanism assists the small business and research communities in commercializing innovative technologies. A phase I grant (R41) is to establish the technical and scientific merit and feasibility of the proposed research (R) or R&D efforts. A phase II (R42) is to continue the research or R&D efforts

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initiated in phase I. Eligibility is limited to U.S. investigators. For more information, see the PA: http://grants.nih.gov/ grants/guide/pa-files/PA-07-281.html. g. Small Business Innovative Research (SBIR; R43/44): This mechanism stimulates technological innovation in the private sector by supporting research or R&D for for-profit institutions for ideas that have potential for commercialization. Phase I (R43) is to establish the technical and scientific merit and feasibility of the proposed R/R&D efforts. Phase II (R44) is to continue the research or R&D effort initiated in phase I. Eligibility is limited to U.S. investigators. For more information see the PA: http://grants.nih.gov/grants/ guide/pa-files/PA-07-280.html. h. Resource Grants: The following represent some of the more frequently used types of grant programs that provide research-related support or access to resources. A comprehensive list of all funding mechanisms used by NIH and NIAID can be found at: http://www.nlm.nih.gov/ep/ Grants.html and http://www.niaid.nih.gov/ncn/grants/ mechan.htm. • NIH Support for Conferences and Scientific Meetings (R13 and U13): These mechanisms support high quality conferences and scientific meetings that are relevant to NIH and NIAID scientific mission and to the public health. Potential applicants should request advanced permission from the funding IC to submit an application. NIAID funds scientific meetings through conference grants (R13) or cooperative agreements (U13) for up to 5 years. Non-U.S. institutions are not eligible to apply. Follow the guidelines provided in the PA: http://grants.nih. gov/grants/guide/pa-files/PA-06-041.html. • Resource-Related Research Projects (R24): This mechanism supports research projects to enhance the capacity of resources that serve biomedical research. It is generally used to provide resources where multiple fields of expertise are needed to focus on a single complex question in biomedical research. Currently, there is no PA for this mechanism. Potential applicants should visit the IC website and contact the program officer for that science area.

51.2.2 NIH Research Training and Research Career Development Opportunities (F, K, and T series) In general, NIH provides research-training opportunities and career development awards to research doctorates or health professional doctorates to gain education and experience in the biomedical field. NIH and NIAID have a variety of training programs for early to mid-career stage investigators. Focused training is a fundamental part of the critical path toward developing a sustainable research effort and portfolio. NIAID awards grants for different career stages and types of research, e.g., basic, clinical, and patient-oriented research

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and also offers training and career development in biodefense and emerging infectious diseases. Extramural Research Training & Research Career Opportunities: NIH and NIAID support a variety of predoctoral and postdoctoral training opportunities to non-federal scientists at universities, hospitals, and at other research institutions throughout the U.S. (extramural; outside of NIH) and in numerous countries. The main goals of the training program are to have highly trained scientists meet the nation’s future needs and to develop tomorrow’s scientists in the biomedical field. During the past several decades, NIAID has faced and continues to address many global challenges, e.g., HIV/AIDS, pandemic influenza, SARS, drug-resistant tuberculosis, other emerging and reemerging infectious diseases, allergic diseases, potential biowarfare threats as part of Project Bioshield, and the development of radiation countermeasures. Below are some of the examples of training and career grants that are funded by NIH or NIAID. For further information go to the NIAID or the Office of Extramural Research websites: http:// www.niaid.nih.gov/ncn/training/advice/training_dev_grants. htm or http://grants1.nih.gov/training. All NIAID training and career development awards (except for the pathway to independence award mechanism, K99/ R00) require either U.S. citizenship or legal residence (“green card”). Potential candidates should check the eligibility and visa requirements for each mechanism in the PA. Potential applicants also should see frequently asked questions at: http://grants.nih.gov/training/q&a.htm and the new investigators program web page at: http://grants.nih.gov/grants/new_ investigators/index.htm. a. Fellowships (Fs): The National Research Service Award (NRSA) provides support to pre- and postdoctoral trainees for independent research projects in basic or clinical scientific areas within the NIAID mission. These are individual awards under a mentor; most awardees are PhDs. If the candidates are predoctoral students and are members of an underrepresented minority group, have a disability, or come from a disadvantaged background, the academic institution can nominate them for a fellowship that provides up to 5 years of support for biomedical, behavioral sciences, or health services research. Training can be provided at domestic or non-U.S. institutions. For more information see: http:// www.niaid.nih.gov/ncn/training/advice/fellowships.htm. • NRSA Pre-doctoral Fellowships (F31): This is an NIHwide program that provides funding under the National Research Service Award to predoctoral students with disabilities, from disadvantaged backgrounds, or from underrepresented racial and ethnic groups. It provides a stipend and a limited amount of tuition funds. Potential applicants must be enrolled or accepted into a PhD or MD, PhD program in biomedical and behavioral sciences or health services research. For further information and eligibility check the PA: http://grants1.nih. gov/grants/guide/pa-files/PA-07-002.html.

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• NRSA Postdoctoral Fellowships (F32): This program is for postdoctoral candidates only and provides fellowships to individuals who have a doctoral degree from an accredited institution. The award supports promising applicants who have the potential to become productive, independent investigators, but may not be used to support studies leading to the MD, DO, or other similar healthprofessional degrees, or residency training. Research clinicians must devote full time to their proposed research training and confine clinical duties to those activities that are part of the research-training program. For further information and eligibility check the PA: http://grants1. nih.gov/grants/guide/pa-files/PA-07-107.html. ● NRSA Senior Fellowships (F33): This program generally supports faculty, MDs or PhDs on sabbaticals. Potential applicants must have at least 7 years of research experience after the doctoral degree and the proposed research must expand experience and offer career benefits. This program is not designed for postdoctoral level scientists seeking to enhance their research experience prior to independence. For further information and eligibility check the announcement: http://grants1.nih.gov/grants/guide/pa-files/PA-07172.html. b. Career Development Awards (Ks): This program awards individuals with MD and PhD degrees who wish to develop careers in biomedical research. These are more senior awards than the fellowship awards mentioned above. Some awards are mentored and others are not. Below are some specific career award mechanisms and detailed information on the Ks Kiosk can be found at: http://www.niaid.nih.gov/ ncn/training/advice/career_dev.htm. ●

For Individuals with a PhD Degree (Figure 51.1): For the PhD, there are several award mechanisms from which to choose. Most awards support individuals who have been accepted or are ready for a faculty position. Some award mechanisms listed below may be eligible for candidates with an MD degree. Potential candidates should check each announcement for eligibility. i. Mentored Research Scientist Development Award (K01): This mechanism is for new faculty members who need additional supervised research experience. Both MDs and PhDs in the fields of epidemiology, modeling, and outcomes research are eligible to apply. For detailed information see the PA: http://grants1.nih. gov/grants/guide/pa-files//PA-06-001.html. ii. Independent Scientist Award (K02): This program provides support to more established “senior” investigators who have a doctoral degree and have an independent research project grant (R01 or equivalent). This award protects at least 75% of the effort for the awardees to focus on research program development. Most successful candidates are assistant professors or just-promoted associate professors. For detailed

51. Appropriate Funding Mechanism

iii.

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information see the PA: http://grants1.nih.gov/grants/ guide/pa-files/PA-06-527.html. Senior Scientist Award (K05): This mechanism provides time and salary support to senior and established investigators. Not all NIH institutes participate in this program. For detailed information check the PA: http:// grants.nih.gov/grants/guide/pa-files/PA-00-021.html. Research Scholar Development Award (K22): This program provides research support to both MD and PhD candidates during the transition from the postdoctoral position to an assistant professor at an academic institution. Candidates with more than 5 years of research experience in a postdoctoral position are not eligible to apply. This mechanism is funded for 2 years with no renewal option. The application has two phases. In phase 1 the application is submitted for peer review of scientific merit. To qualify for phase 2, the applicant must be offered a position as an assistant professor with an independent laboratory, available start-up funds, and minimal teaching or other responsibilities. This award is used differently by the ICs and centers that participate. Potential applicants should review the PA at http:// grants.nih.gov/grants/guide/pa-files/PAR-07-347.html and the institutes’ website for preferences. Mentored Quantitative Research Development Award (K25): This program is for junior faculty members with an advanced degree in engineering or quantitative science such as a PhD or MSEE who would like to apply their experience to biomedical science. There are limited eligibility requirements; for detailed information, check the PA: http://grants1.nih.gov/grants/ guide/pa-files/PA-06-087.html. Other K Awards: There are other K awards for individuals interested in stem cell research or mouse pathobiology (K18). Detailed information may be found at http://grants.nih.gov/grants/guide/pa-files/ PAR-02-069.html and http://grants.nih.gov/grants/ guide/pa-files/PAR-99-065.html websites respectively. The academic award (K07) is used to recruit research faculty into areas where there is a growing need for the research and instructional capabilities. For further information see the PA: http://grants.nih. gov/grants/guide/pa-files/PA-00-070.html. ●

For Individuals with an MD Degree or Equivalent Clinical Degree (Figure 51.2): Individuals with a health professional doctorate degree, the MD have an opportunity to choose from different career development award mechanisms. Most of these awards support individuals who have completed their clinical training and have accepted a faculty position. i. Mentored Clinical Scientist Development Award (K08): This program provides awards to candidates interested in basic research project and have a health professional doctoral degree such as

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the MD, DO, DVM or equivalent degree, and a professional license to practice in the United States. For further information see the PA: http://grants1. nih.gov/grants/guide/pa-files/PA-06-512.html. Mentored Clinical Scientist Development Program (K12): This program provides awards to specific institutions to establish career development programs for clinicians to become independent patient-oriented researchers. Some, but not all, NIH institutes participate in this program. For further information check the institute’s website and their PA. Mentored Patient-Oriented Research Career Development Award (K23): This program provides awards to candidates interested in patientoriented research projects. Candidates must have a health professional doctorate degree or its equivalent. Candidates with PhD degrees are eligible if the degree is in a clinical field and they usually perform clinical duties. For further information check the PA: http://grants1.nih. gov/grants/guide/pa-files/PA-05-143.html. Mid-Career Investigator Award in PatientOriented Research (K24): This program is for established mid-career clinicians or PhDs who are committed to patient oriented research and are willing to serve as a mentor primarily for junior scientists engaged in clinical research. The principal investigator should have concurrent research support, such as R01, or pharmaceutical company funding, or the equivalent. For further information check the PA: http://grants1. nih.gov/grants/guide/pa-files/PA-04-107.html. NIH Pathway to Independence (PI) Award (K99/R00): This is a transition award for postdoctoral researchers moving to assistant professor positions. The program is for both clinical or research doctorate degree candidates with no more than 5 years of postdoctoral research training at the time of initial application or subsequent resubmissions. The award provides funds for up to 5 years and it consists of two phases. The first phase provides 1 to 2 years of mentored support for postdoctoral research scientists (K99) and the second phase provides up to 3 years of independent support (R00) contingent on securing an independent research position. U.S. citizenship and green card are not required for this mechanism. For eligibility check the NIH PA: http://grants.nih.gov/grants/ guide/pa-files/PA-06-133.html.

c. Training Grants (Ts): The national research service award (NRSA) institutional research training grant (T32) and NRSA short-term institutional research training grant (T35) awards fund training programs for pre- and postdoctoral candidates in basic or clinical scientific areas

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for MDs, PhDs, and DVMs. These are multi-slot awards administered by U.S. institutions only. Senior investigators at academic institutions generally apply for T32 grants and trainees work in a mentor’s laboratory. For further detail check the website: http://www.niaid.nih.gov/ncn/training/ advice/t32_qualifying.htm. ●

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Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grants (T32): This funding mechanism provides awards to support institutional training grants at eligible institutions for graduate and postdoctoral researchers in biomedical and behavioral science. The goal of the program is to prepare a highly diverse group of individuals for careers that have a significant impact on the healthrelated research needs of the nation. Trainees must be citizens or non-citizen nationals of the United States, or have been lawfully admitted in the United States for permanent residence and have a valid “green card” or other legal document of such status in the United States. Individuals on temporary or student visas are not eligible for support by the NRSA. For further information check the PA: http://grants1.nih.gov/grants/guide/pa-files/PA07-172.html. Ruth L. Kirschstein National Research Service Award Short-Term Institutional Research Training Grants (T35): This grant mechanism exclusively provides support for intensive, short-term research training for students in health professional schools during the summer. In addition, the short-term institutional research training grant may be used to support other types of predoctoral and postdoctoral training in focused, often emerging scientific areas relevant to the mission of the funding NIH institute or center. For further information see the PA: http://grants1.nih.gov/ grants/guide/pa-files/PA-05-117.html.

d. Women in Biomedical Careers: NIH has established an Office of Research on Women’s Health (ORWH), under the Office of the NIH Director. This office develops opportunities and programs to support the recruitment and retention of women to advance their careers in the biomedical field. NIH has a working group on women in biomedical careers to work with extramural community and the public on the issue of the advancement of women in research careers. Several initiatives have been issued by NIH ICs for advancing novel science in women’s health. For further information see the website: http://womeninscience.nih.gov. e. Research Supplements: This program supports underrepresented minority high school, undergraduate, graduate, and medical students, as well as postdoctoral scientists and faculty members. Awards are supplements to funded research grants and provide salary, supplies and travel. Funds are added to a grant for training researchers from underrepresented groups. Note: diversity supplements replace research

supplements for underrepresented minorities and research supplements for individuals with disabilities. For further information check the website: http://www.niaid.nih.gov/ ncn/training/advice/research_supp.htm and the PA: http:// grants2.nih.gov/grants/guide/pa-files/PA-05-015.html. f. NIH Loan Repayment Program (LRPs): The NIH loan repayment program is designed to help recruit and retain highly qualified clinicians, dentists, and other health professionals with doctoral-level degrees in biomedical and behavioral research careers. There are two kinds of LRPs: one for health professionals pursuing careers in clinical, pediatric, health disparities, or contraception and infertility research (Extramural LRP), and another for health professionals in research positions and fellowships (clinical and basic science) in the NIH intramural laboratories (intramural LRP). For more information about the NIH LRPs visit the website: http://www.lrp.nih.gov. g. Intramural Research Training & Research Career Opportunities: This program is open to scientists at all career levels and provides opportunities for non-US scientists to conduct collaborative research at the NIH. There are several research and training opportunities at the NIH intramural (within NIH) laboratories. These opportunities range from summer programs for high school students through employment for postdoctoral scientists. Below are examples of some of the training opportunities. For more information check the website: http://www.training.nih.gov. • Training in NIAID Intramural Laboratories: NIAID offers both basic and clinical research training programs for students and postdoctoral fellows, e.g., postdoctoral intramural research training award (IRTA) and National Research Council (NRC) research associateship program. There are two programs for non-U.S. scientists through NIH visiting programs: the visiting fellow program, which is open to applicants with a doctoral degree or equivalent and 5 years or less of research experience, and the visiting scientist program is open to applicants with a doctoral degree or equivalent and at least 6 years of research experience. For detailed information see the website: http:// www.training.nih.gov/postdoctoral. NIAID postdoctoral fellowship positions application can be submitted online. See: http://www.training.nih.gov/webforms/postdoctoral/ application/adIndex.aspx?strSearch=NIAID. • Office of Training and Special Emphasis Programs (OTSEP): This NIH Office actively recruits students interested in exploring careers in biomedical research. The OTSEP program is designed to facilitate training programs that promote diversity in NIAID laboratories. The intramural NIAID research opportunities program recruits underrepresented minority students interested in exploring career opportunities in allergy, immunology, and infectious diseases. The summer internship program in biomedical research provides an opportunity to high school and undergraduate students to join one of the

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NIH or NIAID laboratories to work with world-leading scientists in the field of biomedical research. Students 16 years of age or older who are U.S. citizens or hold a green card and are currently enrolled at least half-time in high school or an accredited U.S. college or university are eligible to apply. Students who have been accepted into a college or university may also apply. For more information check following websites: http://www3.niaid. nih.gov/about/organization/dir/otsep.htm and http://www. training.nih.gov/student.

51.3 Program Project/Center Grants (P series; Solicited or Unsolicited Applications) The P series grants are large, multidisciplinary, multi-project, long-term research programs with a common theme. NIH ICs issue FOAs to indicate their interest in funding these types of program grants. Multi-project grants may be investigator-initiated or submitted in response to an FOA. Potential applicants should not only follow the instructions provided in the PHS 398, but also should check the IC’s websites for additional instructions. Furthermore, before considering applying for a multi-project grant, potential applicants should speak with NIAID program staff. Pre-application approval is required for the acceptance of an investigator-initiated unsolicited program grant application. Some multi-project application mechanisms are not accepted as investigator-initiated applications, but are submitted in response to FOAs (RFAs or PAs). NIAID has special instructions for the preparation of the multi-project applications. For detailed information see the NIAID website at: http://www.niaid.nih.gov/ncn/grants/multi/index.htm. a. Research Program Project Grant (P01): This mechanism supports integrated, multidisciplinary, multi-project research projects, often long-term research programs with an objective or theme involving a number of independent investigators who share knowledge, experience, expertise, and common resources. Each project contributes or is directly related to the common theme of program, thus forming synergistic research activities and projects directed toward a well-defined research program goal. NIAID usually accepts unsolicited program project applications with at least two interrelated research projects having a common theme and shared resources, cores or facilities. In general, there is no specific budgetary limit for these applications unless specified in FOA. Note: If the application is not in response to an RFA, potential applicants should discuss ideas with the program officer and should obtain pre-approval for the submission of the application. Applications for more than $500,000 in direct costs in any given year require a pre-approval (prior to the submission) from the funding institute.

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b. Center Core Grants (P30): This multi-project mechanism supports shared resources and facilities for a multidisciplinary research team or a group of investigators focusing on a common research topic to extend and enhance the effectiveness of the research. The core center grants provide accessible resources to extramural community (see Part V, Identifying Research Resources and Funding Opportunities), fostering interaction and collaborations between investigators at multiple institutions to promote a multifaceted approach to a common theme. Generally, this mechanism is available only through a RFA or a PA. To be eligible for a core center grant, the potential applicant’s institution already must have a substantial program base and investigators that will benefit from shared resources. For eligibility, visit the IC websites and and FOAs (RFA or PA). c. Specialized Center Grants (P50): This award mechanism supports multi-project grants in all aspects of R&D, from basic to clinical, and may involve ancillary support activities such as patient care. Like P30 grants, this mechanism is available only through an RFA. Investigator-initiated applications are not accepted for this mechanism. There is substantial program staff involvement after the award. Centers also may serve as regional or national resources for special research. For further information and instructions for preparation of a multi-project center grant see the NIAID website: http://www.niaid.nih.gov/ncn/grants/ multi/index.htm. d. Exploratory Grants (P20): This mechanism often is used to support planning activities associated with large multiproject program project grants, e.g., P50 center grants, which are usually by RFA only.

51.4 Responding to an Institute-Specific FOAs (Solicited Applications) Institute-specific FOAs, formerly known as RFAs, RFPs, and PAs, state the areas in which institutes or centers have interest and would like applications to be submitted. Applying in response to an initiative could be advantageous to potential applicants studying similar scientific areas, because there is often set-aside money allocated to fund these applications. However, competition for set-aside funds is vigorous, and such a strategy may or may not enhance one’s chances of getting funds. NIAID promotes basic and applied research in scientific areas that pose an emerging opportunity. NIAID also must respond to new, emerging diseases, such as West Nile virus, pandemic influenza, re-emerging diseases, such as malaria and multi-drug resistant tuberculosis, as well as Project BioShield mandates, such as radiation countermeasures. NIAID program divisions fulfill their scientific requirements by collectively issuing RFAs, RFPs, and PAs in the NIH Guide at: http://grants.nih.gov/grants/guide/index.html and in

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Grants.gov http://www.grants.gov as FOAs and by supporting a broad array of investigator-initiated studies. a. RFA: RFAs are initiatives sponsored by one or more NIH institutes or centers to stimulate research in well-defined scientific areas identified by the funding institute. RFAs have a single receipt date, set aside funds, and indicate the number of awards likely to be made. In responding to an RFA, applicants must respond to the specifics of the announcement. Investigators should check the NIAID concepts page at http://www.niaid.nih.gov/ncn/budget/in-main.htm for highest priority areas. Applications are peer-reviewed based on the five NIH standard review criteria and RFA-specific review criteria, if any (see Part V, Preparing and Submitting a Competitive Grant Application). Competitive applications are funded based on scientific and technical merit, programmatic relevance, and availability of funds. Applicants should capitalize on their strengths when responding to an initiative and seek collaborations to build their sustainable personal research portfolio. Before writing the application, applicants should contact the program officer or the review staff listed in the announcement to discuss the science and the Institute’s priorities. NIAID RFAs are published as FOAs in Grants. gov: http://www.grants.gov. To find active NIAID initiatives, go to: http://www.niaid.nih.gov/ncn/budget/opps.htm. If an RFA uses more than one funding mechanism, Grants. gov will list a separate FOA for each mechanism. b. RFP: An RFP is an initiative sponsored by an NIH institute that requests proposals for a contract to meet a specific need of that institute. Proposals consist of two parts: technical and business. The technical part includes a description of the project, personnel, and facilities to carry out the proposed work. The business part includes cost information. RFPs, like RFAs, have a set proposal receipt date but are published in FedBizOps, the Federal Business Opportunities website at http://www.fbo.gov; sometimes they are listed in the NIH Guide to Grants and Contracts. Contract procurement legally binds the federal agency (NIH IC) and the offeror—the recipient of the funds—obligating the offeror to provide a product or service as defined by NIAID and obligates the institute to pay for that product or service. To find published initiatives, go to NIAID’s R&D contracts page: http://www.niaid.nih.gov/contract/default. htm. Potential offerors should check http://www.fedbizopps.gov routinely for RFPs and amendments. NIAID does not notify offerors directly of changes or amendments to the RFPs. RFPs are indexed on the NIAID website FedBizOpps, but amendments appear only on FedBizOpps. c. Institute-Specific PAs: PAs generally request grant applications, which are investigator–initiated grants in specific scientific areas. PAs are often broader in scientific scope than RFAs. Most PAs do not have set-aside funds, although some do. NIAID publishes PAs in the NIH Guide and in Grants.gov as FOAs. These initiatives have standard receipt dates, and usually open for 3 years. PAs also have special requirements for applicants. For further informa-

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tion check the NIAID funding opportunity website: http:// www.niaid.nih.gov/ncn/budget/opps.htm.

51.5

Support for International Research

The NIAID research mission of conducting and supporting research in infectious, allergic and immunologic diseases is of global importance. NIAID has two international offices to coordinate domestic and international efforts for emerging and reemerging infectious diseases. The Office of Global Research coordinates and supports collaborative studies in developing countries. This office responds to new disease outbreaks, and facilitates partnership with the Fogarty International Center, the World Health Organization, the State Department and the Center for Disease Control. The other office of the NIAID, the Office of International Extramural Activities, manages international research awards. NIAID has established various international research programs: NIAID intramural research training and collaborative research; non-U.S. investigator-initiated awards; U.S. investigator awards with international components; domestic training programs; bilateral scientific agreements with non-U.S. governments or organizations; multilateral programs with the World Health Organization, UNAIDS, and European Union; and most importantly, through interagency agreements with other federal agencies. NIAID has very active multidisciplinary research programs to build and maintain research capacity in developing countries. To see the complete list of NIAID international grants and contracts, visit the NIAID funding website, http://www. niaid.nih.gov/ncn/grants/int/default.htm; resources available to seek collaborations are described in Part V, Identifying Research Resources and Funding Opportunities. Most NIH grants are awarded to domestic institutions. However, non-U.S. investigators or grantee institutions do not need U.S. affiliation or citizenship to apply for research project grants (R01s), small grants (R03s), or exploratory/ developmental grants (R21s) unless specified in the FOA. However, some grant types do have a citizenship requirement, including small business and training grants. Non-U.S. investigators should read the guidelines and eligibility requirements at http://www.niaid.nih.gov/ncn/grants/basics/basics_a2.htm. In general, NIH does not award program projects, centers, resources, institutional national research services awards, business grants, or construction grants to non-U.S. institutions. It is very important for non-U.S. investigators to focus on their strengths, such as unique expertise, resources not available in the U.S. or regionally important diseases and patient populations. Applications are assessed on the relevance of the proposed research to NIH or NIAID missions, whether similar work is being done or can be done in the U.S., and whether there is a need for the research in a global health perspective. The NIH Fogarty International Center, the international component of the NIH, addresses global health challenges through innovative and collaborative research and training programs and supports and advances the NIH mission

51. Appropriate Funding Mechanism

through international partnerships. To address these needs, the Fogarty International Center (FIC) and NIAID support collaborations with domestic and international partners in international scientific research and training to reduce disparities in global health. Examine the brochure “International Opportunities in Biomedical Research and Training” at http://www.fic.nih.gov/news/publications/interop_03-2004. htm. The website describes programs and other international opportunities supported by the NIH. The NIH Fogarty International Center provides a variety of training programs in biomedical fields. Below are some of the Fogarty research training opportunities at http://www.fic.nih.gov/programs/ training_grants/index.htm. a. AIDS International Training and Research Program (AITRP): This program provides grant awards to U.S. institutions to support HIV-AIDS related research training with institutions in low- and middle-income countries. The grantees at U.S. institutions collaborate with non-U.S. institutions to develop joint research training programs. Details are at: http://www.fic.nih.gov/programs/training_ grants/aitrp/index.htm#introduction. b. FIC/Ellison Clinical Research for US Graduate Students: The Fogarty Center, in collaboration with the Ellison Medical Foundation and NIAID, provides 1-year clinical research training experiences for U.S. graduate students in developing countries. Details can be seen at: http://www. fic.nih.gov/programs/training_grants/fic_ellison.htm. c. Global Infectious Disease Research Training Program (GID): The Fogarty Center and the CDC have collaboratively developed this program to address research training related to infectious diseases that are predominantly endemic in developing countries. Details can be seen at: http://www.fic.nih.gov/programs/training_grants/gid.htm. d. Informatics Training for Global Health (ITGH): This program supports informatics training in low- and middleincome countries in partnership with U.S. institutions and investigators. Details can be seen at: http://www.fic.nih. gov/programs/training_grants/itgh/index.htm. e. International Research Ethics Education and Career Development Award: The Fogarty Center in partnership

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with several NIH institutions including NIAID support domestic and international educational and training opportunities in international bioethics related to research conducted in low- and middle-income countries. Details can be seen at: http://www.fic.nih.gov/programs/training_grants/ bioethics/index.htm. f. International Clinical, Operational and Health Services Research Training Award for AIDS and TB (ICOHRTA AIDS/TB): This program awards grants to U.S. investigators’ institutions to support clinical, collaborative, multidisciplinary, international, operational, and health services at U.S. and non-US collaborating sites and mentored research and training in low- and middle-income countries. Details can be seen at: http://www.fic.nih.gov/programs/training_ grants/icohrta/index.htm. g. International Clinical, Operational and Health Services Research Training Award for AIDS and TB (ICOHRTA AIDS/TB): This program awards grants to institutions located in low- and middle-income countries where AIDS, TB or both are widespread to strengthen their capacity to conduct clinical, operational and health services research. Details can be seen at: http://www.fic.nih.gov/programs/ training_grants/icohrta/aids_tb.htm. h. International Collaborative Genetics Research Training Program: This program promotes international collaborative activities between U.S. and non-U.S. investigators to build institutional infrastructure for the advancement of the genetic science and develop scientists and health specialist with expertise in human genetics. Details can be seen at: http://www.fic.nih.gov/programs/training_grants/ genetics.htm. i. International Research Scientist Development Award (IRSDA): This program provides opportunities to junior U.S. scientists and postdoctoral candidates to develop careers in international health research and establish collaborations in developing countries. The program is similar to NIH career development award (K01) but the main focus is the developing world. It supports basic, behavioral and clinical scientists. Details can be seen at: http:// www.fic.nih.gov/programs/training_grants/irsda.htm.

Chapter 50 Strategies for a Competitive Research Career Hortencia Hornbeak and Peter R. Jackson

50.1

Introduction

Choosing a career in biomedical research can be very rewarding and making discoveries can be exhilarating. In order to be successful in building a sustainable research portfolio, it is important to approach a research career as an entrepreneur. When pursuing a career as a biomedical entrepreneur, it is imperative to have a plan to secure funds over the span of the career, to have the expertise needed to remain competitive and to have links into existing structures to leverage resources and expertise. Similarly, in pursuing a research career, it is important to know how to navigate the labyrinth of information to identify funding sources that support specific research objectives, to understand application processes and associated policies, to be familiar with available resources and expertise, to establish collaborations and to secure the training necessary to be competitive. A successful biomedical research career requires the building of a sustainable research portfolio (Figure 50.1). As shown in Figure 50.1, there are four interlocking components that affect the development of a sustainable research portfolio. These include: (1) complementary funding to support research goals, (2) establishment and maintenance of collaborations to gain access to a broad range of expertise and perspectives, (3) training to keep abreast of new scientific developments and technologies, and (4) access to research administration infrastructure that provides the expertise and support necessary to conduct research.

50.2

Secure Complementary Funding

It is incumbent on the investigator(s) to define research goals as an individual or team (e.g. institution, regional effort) and seek support from those organizations that support research in the defined research area(s). From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

Each agency/organization that funds or supports biomedical research has a specific mission. These mission statements are made public through websites and other communication vehicles. Numerous public, private, national, and international agencies provide support for basic, behavioral, and clinical biomedical research in a broad spectrum of disease areas or in specific diseases, as well as technical assistance in support of research and research resources. Since the mission(s) of funding organizations define the area(s) of research they support and the nature of support (e.g., research, supply drugs, technical assistance), investigators may need to seek complementary research support to meet their research objectives. Specific information on funding opportunities and research resources is addressed in Part V Selecting the Appropriate Funding Mechanism and Identifying Research Resources and Funding Opportunities.

50.3 Identify and Seek Collaborative Opportunities A sustained, successful biomedical research career in the current trans-disciplinary research environment depends on collaborations among scientists with a variety of expertise. In addition to gaining new scientific and technical expertise, collaborations can provide opportunities for research support over a long period of time and perspectives for leadership positions in large networks or programs. In addition, collaborations provide opportunities to work with others who have valuable resources (e.g., unique patient populations, genetic databases, animal colonies) or expertise (e.g., statistics, informatics) that could serve as a base for future grant applications and additional collaborative projects. The National Institutes of Health (NIH) policy on multiple principal investigators (PI) (http://grants2.nih.gov/grants/multi_ pi/index.htm) allows individuals who share the authority and responsibility for leading and directing projects intellectually and logistically to be recognized officially as a PI. The multiple PI policy will allow the designation of more than 483

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ment of an effective e-mail message to an investigator seeking collaboration.

Figure 50.1. Four components required to build a sustainable research portfolio: complementary funding, collaboration, training, and access to research administration infrastructure (See Color Plates).

one PI on grants. The goal of the policy is to encourage team science and to recognize the contributions of collaborators. The first step in establishing collaborations is to identify scientists with similar scientific interests and their respective research projects. Resources and search tools are available that include: the technical literature, databases of funded projects, conference programs, academic libraries, and World Wide Web search engines. Several U.S. Federal agencies provide useful databases of funded programs/ projects. The NIH, CRISP Database (Computer Retrievable Information of Sponsored Projects; http://crisp.cit.nih.gov) can be searched by keywords and includes the names, titles and affiliations of PIs and the abstracts of projects funded by the NIH or other agencies within the Department of Health and Human Services. The National Science Foundation (NSF) also maintains a database of funded projects (http://www.nsf.gov/awardsearch/). The Department of Energy maintains an extensive compendium of research in the Biological and Environmental sciences (http://www. osti.gov/collections). In addition, research dissertations, that are often overlooked, can serve as resources and they can be located through the Center for Research Libraries (http://www.crl.edu/catalog/index.htm). When approaching another scientist to establish a collaboration, it is important to be appropriately informed, to approach the negotiations with confidence and to be clear about resources, technical expertise, and research objectives. When traditional face to face meetings to establish collaborations are not possible or convenient, researchers often rely on electronic communication options. There are techniques for developing an e-mail message that is effective, informative and sufficiently compelling to elicit a response. The following are tips for the develop-

(1) Clearly identify who you are and why you are writing. Describe your scientific interests, qualifications, research program, current team and facilities. Discuss how you learned about the potential collaborator’s research interests and very briefly state why collaboration is needed. (2) Expand upon why the collaboration is being sought. Describe the overall research objectives and methods of the proposed work and, if relevant, any preliminary results. Explain the overall plan for the composition of the proposed research team and the facilities. Explain the duties envisioned for the collaborator(s) and their eligibility requirements and make sure to include the benefits to the collaborator(s). (3) Specifically and clearly ask for collaboration. Sometimes this point is overlooked. The request needs to more fully develop the proposed duties of, and benefits for, the collaborator. This part of the message should provide contact information of the sender, should ask for feedback or a response by a particular date, and should ask for the email request to be passed along to other potential collaborators (if appropriate). The sender’s contact information should include the e-mail, phone, and fax, and appropriate website information (personal or institutional) and a list of relevant publications. (4) The format of the e-mail needs to be considered to help ensure the message is read. The subject line needs to convey a clear message and should not resemble a virus or Spam message. The message should not be attached to the e-mail, it needs to be included in the body of the e-mail. Attachments are sometimes not opened for fear of virus infection of computers. It is important to keep trying to develop collaborations even if there are rejections. Identify as many people to contact as possible and continue to refine the message(s), as needed. There have been, are, and will be many successful collaborations.

50.4 Identify and Seize Training Opportunities The second component of building a successful career as an investigator is securing continuous training to remain competitive. It is important to (1) keep abreast of new scientific developments in one’s area of interest, but also in emerging scientific areas, (2) be familiar with enabling technologies and determine if and how the new technology could impact your research, and (3) be aware of funding agency research and development budgets and research priorities so that your research will be directed or redirected to areas where support may be more likely. The American Association for the Advancement of Science publishes a yearly Research and

50. Strategies for a Competitive Research Career

Development Report (http://www.aaas.org/spp/rd/rd08main. htm) that includes federal agencies’ (e.g., NIH, NSF, DOD) research and development budgets. There are numerous national and international training opportunities. Resources for training and associated websites are described in Part V, Selecting the Appropriate Funding Mechanism and Identifying Research Resources and Funding Opportunities. The NIH supports extramural grant awards that provide research training and career development for PhDs and MDs from early to mid-career stages in the areas of basic, clinical, and patient-oriented research. The National Institute of Allergy and Infectious Diseases (NIAID) also offers training and career development in biodefense and emerging infectious diseases. In addition, scientists gain experience and perspectives through collaborations on grants supported by NIAID, NIH, and other funding agencies.

50.5 Gain Access to Research Administration Infrastructure There are numerous challenges associated with the administrative aspects of conducting biomedical research under a grant or contract. In order to be successful, an investigator needs to have access to a research administration infrastructure that supports their research portfolio. The administrative infrastructure should cover the spectrum of administrative responsibilities including: identification of funding opportunities, facilitation of application processes that meet funding agency submission requirements, compliance with funding agency policies and development of a sound financial management system. If an administrative infrastructure is not available within the investigators’ organization, there are national and international research administration organizations that provide perspectives and assistance in several aspects of research administration. These include: The National Council of University Research Administrators (NCURA; http://www.ncura.edu/) and SRA International

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(http://srainternational.org). The types of assistance provided by these organizations include: • adhering to funding agency guidelines • developing estimated costs which are fair and consistent with those charged to other agencies • determining perceived or actual financial interests of investigator(s) and the institution using appropriate facilities and administration rates • addressing issues of intellectual property rights • developing consulting agreements • establishing payment provisions • improving the quality and lowering the cost of research • increasing the amount of interdisciplinary research • facilitating training opportunities and technology transfer • increasing exposure to new scientific insights • gaining cost-effective access to external research It is important for a career scientist to be aware of the administrative requirements associated with conducting research; however, investigators should focus on their areas of expertise and research goals. Research scientists should not dilute their efforts by assuming responsibility for the multiple administrative aspects of managing research. Effective research administration requires multiple skill sets, which are best handled by personnel who have specific expertise in the administrative areas listed previously.

50.6 Overview of the Essential Components for Building a Sustainable Research Portfolio and Biomedical Research Career Subsequent sections provide insights into how to select the appropriate funding mechanism based on one’s training and research objectives, prepare and submit a competitive grant application, and, national and international research resources that can be used to leverage investigator or institutional resources and funding opportunities. Each section in Part V includes useful websites that provide additional information on each topic.

Index

A A-315675 182 A-549 cells 441 ACE, see Angiotensin converting enzyme 5-Acetamido-4-guanidino-6-(1, 2, 3-trihydroxypropyl)-5, 6-dihydro-4H-pyran-2-carboxylic acid, see Zanamavir Acetylsalicylic acid 369–371 ACH-2 280 Adamantanes 179, 180 amantadine 179, 180 rimantadine 179, 180 Amblyomma americanum 37, 42 American Association for the Advancement of Science (AAAS) 484 ε-Aminocaproic acid (E-ACA) 193–198 anti-HSV action 196, 197 anti-influenza activity 193–196 enhancement of antibiotic antibacterial efficacy 197, 198 enhancement of vaccination efficacy 194, 196 mode of antiviral action 193, 194 toxicity 194 usage for treatment of influenza in infants and children 195–196 usage for treatment of patients with herpetic infection 196, 197 Anaplasma phagocytophilum 37, 38 Angiotensin converting enzyme (ACE) 169 Animal efficacy rule 151, 152 Animal models, smallpox 152–155 Anthrax 19–36 adaptative immunity to anthrax 21 anthrax LT 26–31 anti-LT therapy 27, 28 genetics of 26, 27 spore resistance and LT 28, 29 apoptosis 29, 30 Factor II 26 hemolysins 31 lethal factor (LF) 26 chemical inhibitors of 28 mitogen-activated protein kinase kinases (MAPKK) 29 pathogenic factors 25–36 non-cytotoxic pathogenic mechanisms 32, 33 syndecans 32

proteases 31, 32 toxicity 32, 33 Antibiotics, resistance to 55 Antibodies anti-idiotypic 243 monoclonal 242, 243 polyclonal 241, 242 Anti-influenza activity 169–171 Antimicrobial misuse 101 Antimicrobial resistance 101–104 Anus platyrhynchus 132 APC engineering 350, 351 Apoptosis 29, 30, 440, 441 Aporphinoid alkaloids 199 cell growth inhibitory concentration 50 (CGIC50) 199 cytotoxic effect 199 cytopathic effect (CPE) inhibition test 199–201 inhibitors of enteroviral replication 199 inhibitory concentration 50 (IC50) 200 maximal tolerated concentration (MTC) 199, 200 quantitative suspension test 201 selectivity index (selectivity ratio, SI) 200–202 timing-of addition study 201, 202 virucidal effect 201 APRIL 343, 379, 380 APRIL-R 380 Arthritis collagen-induced 377 rheumatoid 377 ATP-binding cassette 56 Autoimmunity 377–384, 385 B Bacillus anthracis 19–23 Ames strain 21 BclA 19 capsule formation 19 exosporium 19 formaldehyde-inactivated spores (FIS) 21 gamma-D-glutamic acid, polymer of 19 519

520 Bacillus anthracis (cont'd) plasmids pXO1, pXO2 19 protective antigen (PA) 21 spore surface components 19 spores 19 Sterne strain 19 toxin production 19 virulence of 21 virulence factors 19 Bacillus cereus 19, 26 Bacillus thuringiensis 19 BACMA, see B-cell maturation antigen Bacterial LPS 246 Bacteriophage 67 BAFF 343, 344, 379–380 BAFF-R 344, 379–380 BARDA, see Biomedical advanced research and development authority Bartonella henselae 206, 207 Bartonella quintana 206, 207 Bcl-2 protein 440 BCMA 379, 380 BcR receptors 380, 381 BcR-mediated signaling 380, 381 Bifunctional peptide 243 Bioinformatics 429, 430, 433 Biomedical Advanced Research and Development Authority (BARDA) 157 Biomodels 243 Biotechnology Engagement Program (BTEP) 257, 260 Bioweapons 149 research funding 149 Blattobacterium 207 Blochmannia 207 Bordetella 227 phase variation 230 virulence factors 227 bvg-activated 227 Bordetella bronchiseptica 205, 206 Bordetella parapertussis 205, 206 Bordetella pertussis 205, 206 Borrelia burgdorferi 211 Brucella spp 47–54 genotypes 50 genetic relationships 50, 51 BTEP, see Biotechnology engagement program Buchnera aphidicola 207, 208 Bunyaviridae 435 Burkholderia mallei 207 Burkholderia pseudomallei 207 Burkholderia thailandensis 207 C C3 357 CAF, see CD8 antiviral factor Caf1 dimer 217–222 Calcium modulating cyclophilin ligand 343 Callosobruchus chinensis 209 CamL, see Calcium modulating cyclophilin ligand Candida 102

Index Capsular antigen F1 217–222 Carsonella ruddii 207 Caspases (2-, 3-, 7-, 8-, 9-, 10) 441 CCR2 260 CCR5 379, 260, 272, 291–293 CD4 cells 279 CD8 antiviral factor (CAF) 282 CD22 381 CD45 381 CD40-FasL 352 B-Cells 397–406 APRIL 343, 379, 380 APRIL-R 380 autoantibodies 377 B-cell activating factor (BAFF) 343, 379–380 BAFF-R 343, 379–380 B-cell deficiency 346 B-cell depletion 399–402 B-cell maturation antigen (BCMA) 343, 379, 380 B-cell receptors (BcRs) 380, 381 BcR-mediated signaling 380, 381 B-cell survival, regulation of 379, 380 B-cell tolerance 378, 379, 397–404 dysfunction in autoimmunity 377–384 germinal centers 378, 398 IL-10 380 marginal zone 379 MRL-lpr/lpr mice 380 (NZBxNZW) F1 mice 381 in pathogenesis of SLE 397 TACI 379, 380 Cell-to-cell fusion 436 Cellular networks 353 Cellular webs 353 Center for Research Libraries 484 Central Asia 251, 252 emerging and re-emerging diseases 251, 252 Chimeric Flavivirus Vaccines, ChimeriVax 459 construction of 462 clinical development 467–469 ChimeriVax-DEN 468 ChimeriVax-JE 467 ChimeriVax-WN 469 efficacy testing in animal models 465 immunogenicity of 465 ChimeriVax-dengue 465 ChimeriVax-JE 465 ChimeriVax-WN 465 protection 465 active immunization with 465–466 ChimeriVax-dengue 465 ChimeriVax-JE 466 ChimeriVax-WN 466 passive immunization with 465 ChimeriVax-JE, mice 465 GMO 466 preclinical testing of 463–467 recombination studies 466 ChimeriVax-JE and Kunjin recombinant 467 “Worst Case Scenario” 467

Index regulatory and commercialization status Table of 48.2 462 safety testing in animal models of 463 extraneural pathology of 464 genetic stability and vector tropism of 466 ChimeriVax-dengue 466 ChimeriVax-JE 466 ChimeriVax-WN 466 neuroinvasiveness of 464 ChimeriVax-dengue 464 ChimeriVax-JE 464 ChimeriVax-WN 464 YF17D 464 neurovirulence of 463 ChimeriVax-dengue, mouse, monkey 464–467 ChimeriVax-JE, mouse, monkey 464–467 ChimeriVax-WN, mouse and monkey 463 infection of crows (Corvus ossifragus) or domestic chicken with 467 PreveNile vaccine for horses 463 YF17D, mouse, monkey 463–467 viremia 464–465 ChimeriVax-dengue 464 ChimeriVax-JE 464 ChimeriVax-WN 464 YF17D 465 vector tropism 467 ChimeriVax-dengue 467 infection and transmission by Aedes aegypty and Aedes albopictus 467 ChimeriVax-JE 467 infection and transmission by Culex tritaeniorhynchus, Aedes aegypti and Aedes albopictus mosquitoes 467 ChimeriVax-WN 467 infection and transmission by Aedes aegypti 467 Chlamydia trachomatis 207, 243 Chorioallantoic membranes 171 Chronology of chimeric Flavivirus publication, table 48.3 468 Ciprofloxacin 101 Cis-acting control elements 57 Class switching recombination (CSR) 344 Common variable immunodeficiency (CVID) 344–346 Comparative Molecular Fields Analysis (CoMFA) 169–171 Comparative Molecular Similarity Analysis (CoMSiA) 169–171 Competitive Grant Applications 497–505 NIH Grantsmanship: Electronic Research Administration (eRA) 502–504 Dun & Bradstreet (DUNS) Number 503 Electronic Submission 502–504 eRA Commons 503 eRA Grants.gov 502–503 U.S. Central Contractor Registry (CCR) 503 NIH Grantsmanship: Offices 504, 505 Center for Scientific Review 500 DHHS Office of Human Research Protections 504 DHHS Office of Laboratory Animal Welfare 504 NIAID Division of Extramural Activities 500 NIH Office of Extramural Research 504 NIH Office of Peer Review 500 NIH Office of Technology Transfer 505

521 NIH Grantsmanship: Peer Review 500–502 NIAID All About Grants Tutorial 497–499 NIAID Application Checklists 498–499 NIH Peer Review Criteria 499–500 Peer Review Meetings 501 NIH Grantsmanship: Policy 500–505 DNA 504 NIH Application Submission 498, 500 NIH Conflict of Interest and Confidentiality 501 NIH Data Sharing 505 NIH Funding Opportunity Announcements 487, 497 NIH Grants Policy Statement 498 NIH Guidelines for Research Involving Recombinant NIH Model Organism Sharing 505 NIH New Investigator Program 499 NIH Notice of Grant Award 499 NIH Peer Review 500 NIH Select Agent Use 504 NIH Sharing Biomedical Resources 505 Complement 359 C3 357, 359–361 C3 activation/cleavage 359 C3aR 360, 361 Correia Element (CE) 58 Costimulator blockade 351, 352 Coxsackie B viruses 199–201 Coxsackievirus A9 199–201 CRAMP 61 α-Crystallin, see Rv2031c CSR, see Class switching recombination xCT receptor, see Kaposi’s sarcoma herpes virus CTLA-4-FasL 352 CVID, see Common variable immunodeficiency CXCR4 272, 279, 291, 293, 357 Cyanovirin-N 184 Cytokines/chemokines 437 Cytomegalovirus (MCMV) infection, in mouse 445 MCMV downregulation of NKG2D ligands 452 MCMV evasion of macrophages 446 MCMV evasion of NK cells 450 MCMV impairment of DCs function 447 D Daptomycin 103 DAS 181 184 1D-QSAR 163–177 2D-QSAR 163–177 3D-QSAR 163–177 4D-QSAR 163–177 Dual signaling 351 Death domain protein (FADD) Death-associated protein 6 (Daxx) 440, 441 Dendritic cells (DCs) 409–427, 436–437 activation of 410 by innate immune cells 412 by microbial components 410, 411 by product of dying cells 411 by tissue environment 412 biology 409–427 interaction with adaptive immune cells 413

522 Dendritic cells (DCs) (cont'd) maintenance of tolerance 413 maturation 437 protective immunity 409, 410 role in designing vaccines 417 ex vivo DC-based 418 targeting DCs in vivo 418 role in disease 416, 417 autoimmunity 416, 417 cancer 417 infection 417 secretion of chemokines by 411, 412 subsets of 413–416 blood 415, 416 myeloid 414, 415 Diaphorina citri 207 DUs, see Intravenous drug users, E EB, see Entry blocker Echovirus 199–201 Ectromelia virus 154–155 Efflux pumps 55–63 ATP-binding cassett superfamily 56 bacterial 55 FarA-FarB-MtrE efflux system 56 MacA-MacB efflux pump 56 major facilitator (MF) superfamily 56 MtrC-MtrD-MtrE pump 57 multi-drug and toxic compound extrusion (MATE) superfamily 56 Neisserial efflux pumps 56 NorM efflux pump 56 regulation of 56 resistance/nodulation/division superfamily 56 small multidrug resistance family 56 ELISA 243, 437 ELISA-PCR 437 Endothelial cells 437–440 human vein endothelial cells (HUVEC) 437, 438 Enteroviruses 199 Entry blocker (EB) 185 Epitopes 429, 430, 431, 432, 433 Ehrlichiae 37–46 Ehrlichia canis 37, 38 Ehrlichia chaffeensis 37, 38, 41, 43 Ehrlichia ewingii 37 Ehrlichia muris 40, 41 Ehrlichial developmental cycle 40 Ehrlichiosis 37–46 animal models 41 human monocytotroptic 37, 40, 41 immunity 42 immunopathology 42 Eigenvalues analysis (EVA) 169 Emerging and re-emerging diseases 251, 252 Caucauses 251, 252 Central Asia 251, 252 NIAID involvement in Central Asia and the Caucauses 251, 252 Encephalitis, tick-borne 237, 238 Endoplasmic reticulum (ER) stress 441

Index Erythromycin 102 Escherichia coli 70–73, 101, 365, 366, 371 Ethyl (5S, 3R, 4R)-4-(acetylamino)-5-amino-3-(ethylpropoxy) cyclohex-1-enecarboxylate, see Peramivir F Factor II 26 FarA-FarB-MtrE 56 Fas-associated factor (FAF) 441 Fas-associated phosphatase (FAP) 441 Fas-Fas ligand 441 FasL deficiency 387 Fatty acids 60 FL cells 199 Flaviviruses 459 Dengue 459 dengue fever (DF) 460 dengue hemorrhagic fever (DHF) 460 dengue hemorrhagic shock syndrome (DHSS) 460 estimated cases 460 fatality/mortality for 460 geographical distribution 459–460 immune enhancement of 460 serotypes of 459 symptoms of 460 vaccines for DNA vaccines 462 live attenuated vaccines 460 recombinant live vaccines 460 subunit vaccines 462 vector Aedes aegypti of 460, 467 Family of 459 Flaviviruses causing hemorrhagic fever 460 genome organization of 460 Japanese encephalitis (JE) 459 amplifying host for 459 estimated cases of 459 vaccines against 461 mouse brain-derived 461 naked DNA 461 SA14-14-2, 461 virus like particles, VLP 461 vector Culex tritaeniorhynchus, principal vector, other vectors Aedes aegypti and Aedes albopictus mosquitoes of 467 mosquito-borne 460 tick borne 460 West Nile 460 antibody treatment for 461 asymptomatic cases of 461 geographical distribution of 460 ribavirin treatment 461 symptomatic cases of 461 transplant patient infection with 461 vaccines 462 DNA 462 live attenuated chimera, Chimera YF/WN 462–469 live attenuated chimera D4/WN 462 live attenuated chimera, veterinary, Intervet 462 subunit 462 vector Culex genus for 460, 467 Yellow Fever (YF) 459

Index estimated cases of 459 geographical distribution of 460 Nobel Prize, Max Theiler, website for 461 protection against 461 vaccine for 461 vector Aedes, Haemagogus, and Sabethes genera of 459 YF 17D Vaccine 461 neurotropic AEs of 461 risk factors, thymus, advanced age for 461 serious adverse events SAEs, after YF vaccination 461 viscerotropic AEs of 461 Fluoroquinolone, C-8-methoxy 102 Fluoroquinolones 101–104 Fourier-transformation 166–167 Funded Research Resources 483–485 NIH CRISP Database 484 American Association for the Advancement of Science 484 Center for Research Libraries 484 National Science Foundation 484 U.S. Department of Energy 484 Funding Mechanisms 487–495 Career Development Awards with a MD Degree 490 Career Development Awards with a PhD Degree 490 Independent Scientist Award (K02) 490 Mentored Clinical Scientists Development Award (K08) 491 Mentored Clinical Scientists Development Program (K12) 491 Mentored Patient-Oriented Research Career Development Award (K23) 491 Mentored Quantitative Research Development Award (K25) 491 Mentored Research Scientist Development Award (K01) 490 Mid-career-Investigator Award in Patient-Oriented Research ((K24) 491 NIH Pathway to Independence (PI) Award (K99/R00) 491 Research Scholar Development Award (K22) 491 Senior Scientist Award (K05) 491 Center Core Grants (P30) 493 Conference Grants (R13) 489 Exploratory Grants (P20) 493 FedBizOp 494 Fellowships Awards (Fs) 490 Funding Opportunity Announcement (FOA) 493 Grants.gov 487, 494, 502 Intramural Research Training & Research Career Opportunities 492 Multiproject Grants 493 NIAID Grants and Contracts 487–495 NIH Academic Research Enhancement Award (AREA; R15) 489 NIH Clinical Trial Planning Grant Program (R34) 489 NIH Exploratory/Developmental Research Grant Award (R21) 489 NIH Guide for Grants and Contracts 487 NIH Loan Repayment Program (LRPs) 492 NIH Research Career Development Opportunities 489–493 NIH Research Project Grant Program (R01) 489 NIH Research Training Awards 489–493 NIH Support for Conference and Scientific Meetings (R13; U13) 489 NIH Small Grant Program (R03) 489

523 NRSA Pre-doctoral Fellowships (F31) 490 NRSA Postdoctoral Fellowships (F32) 490 NRSA Senior Fellowships 490 Office of Extramural Research 490 Office of Research on Women’s Health 492 Office of Training and Special Emphasis Programs (OTSEP) 492 Program Announcement (PA) 494 Request for Applications (RFA) 494 Request for Proposals (RFP) 494 Research Grants (R Series) 488, 489 Research Program Project Grant (P01) 493 Research Supplements 492 Resource Grants 489 Resource-Related Research Projects (R24) 489 Small Business Innovative Research (SBIR; R43/44) 489 Small Business Technology Transfer (STTR; R41/R42) 489 Solicited Applications 493 Specialized Center Grants (P50) 493 Support for International Research 494, 495 AIDS International Training and research Program (AITRP) 495 FIC/Ellison Clinical Research for U.S. Graduate Students 495 Global Infectious Disease Research Training Program (GID) 495 Informatics Training for Global Health (ITGH) 495 International Research Ethics Education and Career Development Award 495 International Clinical, Operational and health Services Research Training Award for AIDS and TB (ICOHRTA AIDS/TB) 495 International Collaborative Genetics Research Training program 495 International Research Scientist Development Award (IRSDA) 495 Training Grants (Ts) 491, 492 Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grants (T32) 492 Ruth L. Kirschstein National Research Service Award (NRSA) Short-Term Institutional Research Training Grants (T35) 492 Unsolicited Applications 488 Women in Biomedical Careers 492 G Gadd153/chop 441 Gallus domesticus 131 GAS, see Group A streptococcal sepsis Genetic mosaicism 68–70 Genomes, bacterial 205–214 compactization of genomes 210, 212 direct repeats 210 endosymbionts 207, 211 genome evolution 207, 208 genomic islands 208 genome pulsing 211 genome rearrangements 208, 209 genome reduction 205–207 genome reduction, mechanism of 207, 208 horizontal transfer 207, 209 integrase 208

524 Genomes, bacterial (cont'd) integration site 208 integron 208 inverted repeats 208 IS-elements 208, 209 movable genetic elements 208, 209 natural selection 208, 209, 212 parasitism 211 pathogenicity islands 208 phenotypic selection 209 plasmids 208 polynucleotide selection 209, 210 prophage DNA 208 pseudogenes 207, 208, 210 reductive evolution 211 repeated sequences 208 selfish DNA 210 selfish gene 210 symbiotism 212 target site 208 tRNA genes 208 tse-tse fly 207 Georgia, Republic of 257–261, 263–267 blood donor program 257, 258, 260, 261 disease surveillance system 253–255 Emory University, collaboration with 257 female sex workers (FSW) 257, 258, 259 Fogarty International Center, collaboration with 257 HBV 257 incidence 257, 258 prevalence 257 HCV 257, 260 Henry M. Jackson Foundation, collaboration with 257 hepatitis B Virus (HBV) 257, 258, 261 hepatitis C virus (HCV) 257, 258, 260, 261 genotype 260 recent/acute 260, 261 HIV 257–261 AB recombinant 258, 259 HIV subtypes, prevalence 258, 259 incidence of 257, 258 molecular epidemiology 257–259 subtype A 258, 259 subtype B 258, 259 HIV, epidemiology of 257, 258, 259 HIV, prevalence of 257 Infectious Diseases, AIDS and Clinical Immunology Research Center 257 intravenous drug users (IDUs) 257–261 Johns Hopkins University, collaboration with 257, 260 National Center for Disease Control and Medical Statistics (NCDC) 253, 254 SDF1 259 sexually transmitted disease (STD) 257, 258 State University of New York (SUNY), collaboration with 257, 259 Tuberculosis (TB) 257, 258, 263–267 active case finding 264 bacteriology 263 case detection rate 265 country coordinating mechanism (CCM) 265

Index directly observed treatment (DOT) 264 directly observed treatment short-course (DOTS) 263 DOT 263–266 DOTS-Spot Project 264 drug resistance survey (DRS) 266 drug resistant TB (DR-TB) 265 drug susceptibility testing (DST) 264 first line anti-TB drugs 264 Global Drug Facility (GDF) 264 Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) 264 GOPA 264–267 Green Light Committee (GLC) 266 HIV/tuberculosis 264 incidence 263 International Committee of the Red Cross (ICRC) 265 Medical Service Corporation International (MSCI) 264 Medicines Sans Frontiers (MSF) 264–266 Merlin 264–266 Millennium Development Goals (MDGs) 265 Multidrug-resistant TB (MDR-TB) 264 National Center for Tuberculosis and Lung Diseases (NCTBLD) 263 National Reference Laboratory (NRL) 264 National Tuberculosis Program (NTP) 263 NRL 264–267 passive case finding 263 second-line anti-TB drugs 264 sputum collection point 264 sputum culture 264 sputum-smear microscopy 264 State Medical Statistics Department 264 TB, see Tuberculosis TB case detection rate 265 TB incidence 263 United States Agency for International Development (USAID) 264 USAID, see United States Agency for International Development U.S. Civilian Research and Development Foundation (CRDF) 257, 259 U.S. Defense Threat reduction Agency (DTRA) 254, 255 Walter Reed Army Institute for Research, collaboration with 257, 258 WHO 263–267 World Health Organization (WHO) 263–267 Ziehl-Neelsen method 264 Germ line transcription (GLT) 344 Glaucium flavum L. Crantz 199 Glossina brevipalpis 207 gp 41 Env 272, 279 gp120 Env 272, 279 Glycation 365–371 covalent dimmers 367–369 human interferon-γ 365, 367 inhibition 369–371 recombinant proteins 365 GM-CSF, see Granulocyte-macrophage colony stimulating factor Gonococcus 55 GPI, see Glycophosphatidyl-inositol proteins

Index Granulocyte-macrophage colony stimulating factor (GM-CSF) 284, 437 Group A streptococcal (GAS) sepsis 15, 16 Gyrase 102 H Haemophilus influenzae 103 Hantavirus pulmonary syndrome (HPS) 435 Hantaviruses 435 Andes virus 435 cell receptors of 435, 436 Dobrava virus 435 Hantaan virus 435 New York virus 435 non-pathogenic 435 pathogenic 435 Prospect Hill virus 435 Puumala virus 435 Saarema virus 435 Seoul virus 435 Sin Nombre virus 435 Tula virus 435 HBV, see Hepatitis B virus HCV, see Hepatitis C virus Hematopoietic stem/progenitor cells (HSPC) 357 homing of 359 mobilization of 358 Hemorrhagic fever with renal syndrome (HFRS) 435 Hendra virus 303, 304 Henipaviruses 303, 304 Herpesviruses 199 herpesvirus-8 271–277 Hepatitis B Virus (HBV) 257, 258, 261 incidence 257, 258 prevalence 257 Hepatitis C virus (HCV) 257, 258, 260, 261 genotype 260 incidence 257, 258 prevalence 257, 260 recent/acute 260, 261 Hit, see Hierarchic informational technology HIV, see Human immunodeficiency virus hmAbs, see Human monoclonal antibodies H3N2 influenza virus 170, 171 Hologram QSAR (HQSAR) 169 HOOPF-Prints, see Hypervariable octameric oligonucleotide finger-prints HPS, see Hantavirus pulmonary syndrome HSPC, see hematopoietic/progenitor cells HspX, see Rv2031c HTLV-1, see Human T lymphotropic virus-1 Human embryonic kidney cells (HEK293) 441 Human immunodeficiency virus (HIV) 257–261, 271–277, 279–288, 299–301, 309–318 AB recombinant 258, 259 sCD4-17b 273, 274 dementia, HIV-associated (HAD) 291, 290 drug resistance 309–318 generation of 309–311 genes of 279

525 against protease inhibitors 312, 313 replication capacity 314 against reverse transcriptase inhibitors 311, 312 transmission of 313, 314 entry/fusion mechanism 272 envelope glycoprotein 293, 300, 301 neutralizing antibodies against 293 epidemiological surveillance in Lithuania 327–337 heteroduplex tracking assay (HTA) 289, 290 HIV subtypes, prevalence 258, 259, 313 incidence of 257, 258 HIV-1 variants, compartmentalization of 290, 291 Janus kinase/signal transducer and activator of transcription (JK/STAT) 285 minor cognitive motor disorder (MCMD), HIV-associated 290 molecular epidemiology 257–259 neutralizing antibody 273 prevalence 257 preventive research 319–325 NIAID role 322–326 Sub-Saharan Africa 321 subtype A 258, 259 subtype B 258, 259 TAR 279 Tat 279 Human interferon-γ 365–371 covalent dimmers 367–369 Escherichia coli 365–366, 371 fluorescence 367, 371 glycation 365, 367 LC/ESI-MS 366–368 post-translational processing 367 production 23 purification 366 Human monoclonal antibodies (hmAbs) 299–308 Human systemic autoimmune disease 380 Human systemic lupus erythematosus (SLE) 380, 397–406 pathogenesis, role of B-cells in 397 treatment of 399–402 anti-CD20 antibody (rituximab) 399 Human T lymphotropic virus-1 (HTLV-1) 283 HUVEC, see Human vein endothelial cells Hypervariable octameric oligonucleotide finger-prints (HOOF-Prints) 47 I Iceland: 1918 Spanish Flu epidemic 115–122 birth data 115 characteristics of the illness 119–121 dyspnea 120 hemorrhage 120 hemorrhagic complications 120 optochin, parotitis 120 palpitations 120 respiratory insufficiency 120 disease registry 118 familial aggregation, crowded settings 116 historical and medical data 115, 116

526 Iceland: 1918 Spanish Flu epidemic (cont'd) incubation period, attack rate, case fatality 116, 117 age-specific mortality 119 pregnancy (pregnant women) 119 sex distribution 120 quarantine 116, 118 IEDB, see Immune epitope database and analysis resource IFN-α 436 IFN-γ 437 IFN-γ/IFN-γR recognition system 216–218 IFN-stimulated genes (ISG) 440 IgA deficiency (IgAD) 344, 345 Immune Epitope Database and Analysis Resource (IEDB) 431, 432, 433 Immunity cellular 23 innate 436 protective 22 Immunodiagnostics 241–248 Immunogenesis, directed 243 Immunophosphorescence 233–240 PHOSPAN 235–239 Immunotherapy, passive 185, 186 Infectious diseases, system biology approach to 13–15 reference mouse panel 13–15 Influenza 179–192 antivirals against 179–192 avian (AI) 123–129, 131–141 bird species 137–139 DIVA system 125 domestic ducks 136, 137 ecology 131, 132 epidemiology 131, 132 gallinaceous poultry, AI lesions in 134 highly pathogenic AI (HPAI) 123, 131 HPAI H5N1 subtype 132–136 HPAI, pathobiology 134–136 HPAI, pathogenesis 131–134 low pathogenic AI (LPAI) 123, 131 pathology 131 prevention 124 vaccination 124–127 virulence 132 computational biology 110 epidemiology 109 evolutionary biology 111 influenza A virus 123 Influenza Genome Sequencing Project 109–113 bioinformatics 110 GenBank 110 sequencing 110 surveillance 111 whole virus sequencing 109 molecular epidemiology 112 pandemic 109, 112 seasonal 110–111 Interferons 186 Interferon-γ 282, 365–371 production of 23 β3 Integrins 435

Index Interleukins (ILs) IL-1 282 IL1β 437 IL-2 283 IL-6 282, 437 IL-8 438 IL-10 379, 380, 391, 437 IL-12p40 437 IL-12 283 IL-16 282 IL-1R/TLR recognition system 215–222 IL-8/CXCL-8 437 Intravenous drug users (IDUs) 257–261 IS481 227–231 transposition of 227–230 in E. coli 227, 228 in B. pertusssis 228–230 Ischemia/reperfusion injury 359 Isoniazid 102 Isotype switching 344 Ixodes ovatus 41, 44 J Janus kinase/signal transducer and activator of transcription (JK/STAT) 284 K Kaposi’s sarcoma-associated herpesvirus (herpesvirus-8) (KSHV) 271–277 entry/fusion mechanism 274 pathogenesis 276 xCT receptor for KSHV 274, 275 L β-Lactams 102 Levofloxacin 103 Lethal factor (LF) 26 LcrV 215–218, 222, 223 Lithuania 327–337 Alytus CF 332, 333 HIV, epidemiological surveillance 327–337 LL-37 60 Long-acting neuraminidase inhibitors (LANI) 182, 183 CS-8958 183 multivalent LANI 182, 183 Lupus-prone MRL-lpr mouse model 377 B-Lymphocytes, see B-Cells T-Lymphocytes, CD4, CD8 23, 437, 438 Lymphoma precursors 388 Lyn 381 M MacA-MacB 56 Macrophage inflammatory protein (MIP-1α/CCL3) 437 Macrophage inflammatory protein (MIP-1β/CCL4) 437 Macrophage inflammatory protein-1 (MIP-1) 282 MAPKK 29 MATE 56 MCDB 105 medium 437 MDM, see Monocyte-derived macrophages

Index Metaloporphyrins 234 Pt coproporphyrins 234 Pt uroporphyrins 234 Miconazole 102 Microarray 391, 233–240 immunophosphorescence 233–240 MIP-1, see Macrophage inflammatory protein-1 Membrane lipid rafts 360 Minor cognitive motor disorder (MCMD), HIV-associated 290 MLVA, see Multiple locus variable number tandem repeat (VNTR) analysis Monocyte chemoattactant protein (MCP-1/CCL2) 437 Morphology, viral 68 MRL-lpr/lpr mice 380 mtrE 57 Molluscum contagiosum virus (MCV) 145 Monocyte-derived macrophages (MDM) 280 Monocytes/macrophages, human 436 MPC, see Mutant prevention concentration mtrR locus 57, 58 mtrF 57 MpeR transcriptional regulator 57 MtrA transcriptional regulator 59 MtrR transcriptional regulator 59 MtrC-MtrD-MtrE efflux pump 57 Mutant prevention concentration (MPC) 101–103 Mutant selection window 101–104 Multi-drug and toxic compound extrusion (MATE) efflux pumps 56 Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) 47–54 MLVA design and multiplex PCR conditions 48, 49 Multiple sclerosis 378 Multiplicity of infection (MOI) 437 MxA protein 438 Mycobacterial biofilms 73–74 Mycobacterial nonsense suppressors 72 Mycobacteriophage 67–76 acquisition of host genes 70, 71 evolution 70 genetic mosaicism 68–70 genomes 67, 68 morphologies 68 Mycobacterium leprae 205 Mycobacterium tuberculosis 77–81, 101, 102, 205, 207 Mycobacterium smegmatiis 77, 74, 102 Mycoplasma contamination 437 Mycoplasma genitalium 207 N Nanoarchaeum equitans 207, 209 National Council of University Research Administrators 485 National Science Foundation 484 Natural killer (NK) cells 447 NK cell receptors 447 NF-kB 281 Neisseria gonorrhoeae 55–56, 101 Neisseria meningitides 56 Neuraminidase (NA), inhibitors of 179, 180 A-315675 182

527 long-acting NA inhibitors (LANI) 182, 183 CS-8958 183 multivalent LANI 182, 183 nucleoside analogues 183 T-705 183 peramivir 181, 182 ribavirine 183, 184 viramidine 183, 184 zanamavir 181 NF-κB 187, 379 NIH CRISP Database 484 National Science Foundation 484 U.S. Department of Energy 484 Center for Research Libraries 484 American Association for the Advancement of Science 484 Nipah virus 303, 304 NK cells, see Natural killer cells NKG2D ligands 450 Non syncytia-inducing virus (NSI) 283 Nonoxynol-9 60 Nor-M 56 Nucleosides 183 (NZBxNZW)F1 mice 381 O One-step replication cycle 202 Orthomyxoviruses 199 Oxoglaucine 199–202 P PA 21 Paramyxoviruses 199 PCR, see Polymerase chain reaction Penicillin 55 Penicillin-binding proteins 55 Peramivir 181, 182 Peripheral blood mononuclear cells (PBMC) 283, 441 PHA, see Phytohemagglutinin Phage display methodology 302 Phage integration 71, 72–74 plasmid vectors in phage integration 71, 72 Phamily (phams) 70 Phams 70 Phorbol 12, myristate-13, acetate (PMA) 280 PHOSPHAN 235–239 Phytohemagglutinin (PHA) 280 Picornaviruses 199 pilF 59 pilM 59 pilMNOPQ 59 PKC, see Protein kinase C Pla 215, 222, 223 Plaque inhibition test 199–201 Plasmacytoid lymphoma 385 Plasminogen factor (Pla) 215, 222, 223 PMA, see Phorbol 12, myristate-13, acetate PMBC, see Peripheral blood mononuclear cells Pneumonic plague 215, 220, 221, 223 anti-plague vaccine 215, 222, 223

528 Poliovirus type 1 199–202 Polymerase chain reaction (PCR) 282 ponA 59 Population analysis, bacterial 101, 102 Poxviruses 145–161 cowpox virus 152–153, 155 monkeypox 147–149 clinical disease 148 history 147, 148 person-to-person transmission 148 mousepox (ectromelia virus) 154–155 rabbitpox 154 VARV 147 Prochlorococcus marinus 207 Progesterone 60 Programmed cell death, see Apoptosis Protease, HIV 312, 313 Protease inhibitors 185 Protein chimerization 349 Protein kinase C (PKC) 281 Protein paint 351 Proteins auto-inhibition 352 cis loop-back proteins 352 fusion 349–356 glycophosphatidyl-inositol (GPI) 350 signal conversion 352 trans signal converter protein 351, 352 Pseudomonas aeruginosa 32 Q QSARS 163–177 automatic variable selection (AVS) 166, 167 atomic refraction 168, 171 bond nature 165 conformer 166 correlation coefficient 169 cross-validation 169 domain applicability (DA) 167, 168 field of potential information (FPI) 166 genetic algorithm (GA) 167 H-bond 166–168, 171 hierarchic informational technology (HIT) 171 hologram QSAR (HQSAR) 169 inertia ellipsoid 167 information potential 168 latent variables 167 lipophilicity 166 molecular design 166 partial atom charge 166 partial least squares (PLS) 168 pharmacophore 166 “productive” conformation 164, 171 rank correlation 167, 168 regression coefficients 164, 165 simplex descriptor (SD) 166, 167 simplex representation of molecular structure (SiRMS) 164–166 standard error of prediction 167, 169 stereochemical configuration 169

Index structural dissimilarity 167 structural parameters space (SPS) 168 test set 167 trend-vector procedure 167, 168 virtual screening 168 Quantitative suspension test 201 Quantitative trace loci (QTL) 13 R Rac-1/Rac-2 360 Receptor editing 379 Recombineering 73 Regulated upon activation normal T cell expressed and secreted (RANTES) 282, 437 Research Administration Organizations and Resources 485 National Council of University Research Administrators 485 SRA International 485 Research Resources 483–485 NIH CRISP Database 484 American Association for the Advancement of Science 484 Center for Research Libraries 484 National Science Foundation 484 U.S. Department of Energy 484 Research Resources and Funding Opportunities 507–517 AIDS Clinical Trials Group (ACTG) 511 Bill and Melinda Gates Foundation 514, 515 Centers for Disease Control and Prevention 513 ClinicalTrials.gov 512 CRISP 509 European Union 516 GenBank 509 Genetic Modification and Clinical Research Information System (GeMCRIS) 509 HIV Prevention Trials Network (HPTN) 511 HIV Vaccine Trials Network (HVTN) 511 Howard Hughes Medical Research Institute 515 Institute for Genomic Research 515 International Maternal Pediatric Adolescent AIDS Clinical (IMPAAC) 511 International Network for Strategic Initiatives in Global HIV Trials (INSIGHT) 511 Literature Searching and Databases Resources 507, 508 Medical Genetics Resources 508, 509 MedlinePlus 508 Microbicide Trials Network (MTN) 511 U.K. Medical Research Council Funding Opportunities 516 National Center for Biotechnology Information 508 National Center for Research Resources (NCRR) 512 National Institute of Biomedical Imaging and Bioengineering 512 National Library of Medicine 508 National Science Foundation 514 NIAID Division of Acquired Immunodeficiency Syndrome 510 NIAID Division of Allergy, Immunology, and Transplantation 511 NIAID Division of Microbiology and Infectious Diseases 510, 511

Index NIAID International Grants and Contracts 512 NIAID Resources 509–512 NIAID-Funded Research Networks and Other Programs 510–512 NIH Fogarty International Center 513 NIH Genome-Wide Database (dbGaP) 508, 509 Open Society Institute 517 PubMed 508 PubMed Central 508 Soros Foundation 516 U.S. Civilian Research and Development Foundation 514 Wellcome Trust 515 World Health Organization 517 Reverse transcriptase, HIV 279, 311, 312 Rheumatoid arthritis 380 Ribavirine 183, 184 Rickettsia prowachekii 207 Rifampicin 102, 104 Rituximab 399 RNA inhibitors 186 Rpf, see Resuscitation -promoting factor under Tuberculosis Rv2031c 94–96 S SARS-CoV 302, 303 SDF-1, see Stromal derived factor Secretion, type IV 38, 39 Sepsis, Group A streptococcal (GAS) 15, 16 Sexually transmitted disease (STD) 257, 258 SHP-1 381 Sitophilus ozyrae (SOPE) 207 SLA, see also Human systemic lupus erythematosus clinical disease 146 history 145 person-to-person transmission 146, 147 smallpox vaccines 150 smallpox vaccine, Acambis 2000 156 smallpox vaccine, MVA 156 smallpox antivirals 150 smallpox antiviral, CMX001 156 smallpox antiviral, ST-246 157 Smallpox 143–147 Sodalis glossinidius 207 Sparfloxacin 101 Splenocytes 23 SRA International 485 Staphylococcus aureus 101–104 STD, see Sexually transmitted disease Streptococcus pneumoniae 101–103 Streptomycin 102 Strategies for Sustaining a Research Program 483–485 contacting scientific experts 483, 484 establishing collaborations 483, 484 identifying funding sources 484 identifying training resources 484, 485 Stromal derived factor-1 (SDF-1) 357 Sub-Saharan Africa 321 Syncytia-inducing virus (SI) 283 Syndecans 32

529 T T-705 183 TACI 343, 345, 346, 379, 380 mutations 346 Tetracycline 56 TGF-β, see Transforming growth factor-β THP-1 cells 436 TNF-α, see Tumor necrosis factor-α TNFRp55 441 Topoisomerase IV 102 Transcriptional regulatory proteins 56 Transforming growth factor-β (TGF-β) 283 Trichomonas vaginalis 209 Treponema pallidum 207 Tuberculosis (TB) 67–76, 83–99, 257, 258, 263–267 anti-spore antibodies 22 diagnosis 91–99 antigenics 91, 92 immune response in 92–96 genomics 91, 92 tuberculin skin test 91–93 dormancy models for mycobateria in vitro 83–85 latent and acute diseases 91–99 non-culturable mycobacteria, formation and resuscitation 83–85 pathogenesis, macrophage models 78 pathogenesis, murine models 77, 78 persistence in tuberculosis 83 M. tuberculosis strain virulence 79–80 resuscitation -promoting factor (Rpf) 85–87 Rpf, mechanisms of action 85–87 lytic transglycosylases 86 methylerythritol cyclodiphosphate (MEC), role in resuscitation 87 murein hydrolysis by Rpf 85, 86 Rv2031c (α-crystallin, HspX) 94–96 Tumor necrosis factor-α (TNF-α) 281, 437, 437, 438 TNF-α receptor 1-associated death domain (TRADD) 440, 441 TNFα related apoptosis-inducing ligand (TRAIL) 440, 441 sTRAIL 441 TUNEL assays 441 U U1 280 U1026 187 U.S. Department of Energy 484 V Vaccinia immune globulin 150 VACV, see Vaccinia virus Vaccine anti-idiotypic 246–247 anti-plague 215, 222, 223 development 244–247 Vaccinia virus (VACV) 152, 429, 430, 431, 433 Vancomycin 101, 104 Variable number tandem repeats (VNTR) 47–54 automated genotype analysis 49 identification of VNTR sequences 48

530 Vero E6 cells 437 Vibrio cholerae O1 and O139 243 Viral attachment, inhibitors of 184, 185 cyanovirin-N 184 DAS 181 184 Viramidine 183, 184 Virulence factors 60, 227 bvg-activated 227 Viruses, emerging 302–304 escape mutants 303 treatment 302–304 conserved targets 302–304 multiple targets 302–304 vaccine immunogens 303 Vitamin B1 369–371 VNTR, see Variable number tandem repeats

Index W White tailed deer 42, 43 Wigglesworthia glossinidia 207 Y Yersinia pestis 205, 215–223, 238, 245, 246, 473–480 live Y. pestis vaccine 473–480 human humoral responses 476 human T cell-mediated responses 479 immunization in a murine model 473, 474 murine humoral responses 474, 475 murine T-cell-mediated responses 475 Yersinia pseudotuberculosis 205, 243 Z Zanamavir 181