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Viral Infections and Treatment

edited by

Helga Rubsamen-Waigmann Karl Deres Guy Hewlett Reinhold Welker Bayer Healthcare Wuppefial, Germany

MARCEL

MARCELDEKKER, INC. DEKKER

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NEWYORK BASEL

Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide speciﬁc advice or recommendations for any speciﬁc situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identiﬁcation and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-4247-8 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc., 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 Distribution and Customer Service Marcel Dekker, Inc., Cimarron Road, Monticello, New York 12701, U.S.A. tel: 800-228-1160; fax: 845-796-1772 Eastern Hemisphere Distribution Marcel Dekker AG, Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright # 2003 by Marcel Dekker, Inc.

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PRINTED IN THE UNITED STATES OF AMERICA

INFECTIOUS DISEASE A N D THERAPY Series Editor

Burke A. Cunha Winthrop- UniversityHospital Mineola, and State University of New York School of Medicine Stony Brook, New York

I.Parasitic Infections in the Compromised Host, edited by Peter D. Walzer and Robert M. Genta 2. Nucleic Acid and Monoclonal Antibody Probes: Applications in Diagnostic Methodology, edited by Bala Swaminathan and Gyan Prakash 3. Opportunistic Infections in Patients with the Acquired Immunodeficiency Syndrome, edited by Gifford Leoung and John Mills 4. Acyclovir Therapy for Herpesvirus Infections, edited by David A. Baker 5. The New Generation of Quinolones, edited by Clifford Siporin, Carl L. Heifetz, and John M. Domagala 6. MethiciI Iin-Resistant Staphylococcus aureus: CIinical Mlanagement and Laboratory Aspects, edited by Mary T. Cafferkey 7. Hepatitis B Vaccines in Clinical Practice, edited by Ronald W. Ellis 8. The New Macrolides, halides, and Streptogramins: Pharmacology and Clinical Applications, edited by Harold C. Neu, Lowelil S. Young, and Stephen H. Zinner 9. Antimicrobial Therapy in the Elderly Patient, edited by Thomas T. Yoshikawa and Dean C. Norman 10. Viral Infections of the Gastrointestinal Tract: Second Edition, Revised and Expanded, edited by Albert Z. Kapikian 11. Development and Clinical Uses of Haemophilus b Conjugate Vaccines, edited by Ronald W. Ellis and Dan M. Granoff 12. Pseudomonas aeruginosa Infections and Treatment, edited by Aldona L. Baltch and Raymond P. Smith 13. Herpesvirus Infections, edited by Ronald Glaser and James F. Jones 14. Chronic Fatigue Syndrome, edited by Stephen E. Straus 15. lmmunotherapy of Infections, edited by K. Noel Masihi 16. Diagnosis and Management of Bone Infections, edited by Luis E. Jauregui 17. Drug Transport in Antimicrobial and Anticancer Chemotherapy, edited by Nafsika H. Georgopapadakou

18. New Macrolides, Azalides, and Streptogramins in Clinical Practice, edited by Harold C. Neu, Lowell S. Young, Stephen H. Zinner, and Jacques F. Acar 19. Novel Therapeutic Strategies in the Treatment of Sepsis, edited by David C. Morrison and John L. Ryan 20. Catheter-Related Infections, edited by Harald Seifert, Bernd Jansen, and Barry M. Farr 21. Expanding Indications for the New Macrolides, Azalides, and Streptogramins, edited by Stephen H. Zinner, Lowell S. Young, Jacques F. Acar, and Harold C. Neu 22. Infectious Diseases in Critical Care Medicine, edited by Burke A. Cunha 23. New Considerations for Macrolides, Azalides, Streptogramins, and Ketolides, edited by Stephen H. Zinner, Lowell S. Young, Jacques F. Acar, and Carmen Ortiz-Neu 24. Tickborne Infectious Diseases: Diagnosis and Management, edited by Burke A. Cunha 25. Protease Inhibitors in AIDS Therapy, edited by Richard C. Ogderi and Charles W. Flexner 26. Laboratory Diagnosis of Bacterial Infections, edited by Nevi0 Cimolai 27. Chemokine Receptors and AIDS, edited by Thomas R. O’Brien 28. Antimicrobial Pharmacodynamics in Theory and Clinical Practice, edited by Charles H. Nightingale, Take0 Murakawa, and Paul G. Ambrose 29. Pediatric Anaerobic Infections: Diagnosis and Management, ‘Third Edition, Revised and Expanded, ltzhak Brook 30. Viral Infections and Treatment, edited by Helga Riibsamen-Waigmann, Karl Deres, Guy Hewleit, and Reinhold Welker 31. Community-Acquired Respiratory Infections, edited by Charles H. Nightingale, Paul G. Ambrose, and Thomas M. File, Jr.

Additional Volumes in Production

Preface

The obliteration of diseases that impinge on our health is a regal yardstick of civilization’s success, and those who accomplish that task will be among the true navigators of a brave new world. Michael B. A. Oldstone, Viruses, Plagues and History, New York: Oxford University Press, 1998 Viruses are regarded by some as thieves, parasites, and murderers. Others regard them as the ultimate examples of informational nanotechnology. This book is concerned with the dark side of viruses and the diseases they cause. However, it should not be forgotten that it is because viruses are so technologically advanced that we have been able to make so much progress in our understanding of genetics, cell biology, biochemistry, and molecular biology. Paradoxically, the knowledge given us by the study of viruses has also led to the discovery of many antiviral compounds. Viral infections have long been regarded as untreatable. In the middle of the last century, the science of virology was in its infancy and viral chemotherapy was but a gleam in the eye of leading microbiologists like Robert Doerr, who fervently believed in the existence of chemotherapeutic agents that would be effective against a large number of virusiii

iv

Preface

related diseases [1]. However, the realization that viruses usurped the metabolic machinery of the cell raised the intellectual barrier that a ‘‘virotoxic’’ agent would also be toxic toward the normal cell. Thus, therapeutic nihilism became an accepted philosophy within the majority of medical circles. Successful control of some viral diseases came about only through the advent of prophylactic vaccination for now nearly extinct viruses such as smallpox and polio. Unfortunately, not all viral diseases lend themselves to vaccination. For example, researchers have tried for decades to develop vaccines against herpes simplex viruses but without much success. At the turn of the new millennium, a glimmer of understanding emerged as to why previous attempts had failed: herpesviruses have evolved strategies to interact with the mechanisms that alarm the immune system and inactivate or divert the signals. Similarly, major efforts to develop a vaccine against human immunodeﬁciency virus have thus far failed to reach their target, partly because of the enormous genetic drift of the virus and partly because of the lack of knowledge about the immune reactions that have to be triggered in order to create an effective vaccine against a virus that attacks and modulates the immune system itself. Therefore, although our understanding of immune mechanisms in the ﬁght against viruses is increasing, some viruses may never be controlled by vaccines but will almost certainly require the development of low-molecular-weight antiviral compounds or novel immunomodulating principles before they succumb. A major conceptual breakthrough in chemotherapy occurred in the late 1940s when Hitchings and Elion realized that the rate of nucleic acid synthesis in infected tissues is much higher than that of the normal host tissue. This led to the idea of antimetabolite chemotherapy for cancer and parasitic diseases that ultimately led to antiviral agents such as iododeoxyuridine, deoxycytidine, hydroxyethoxymethylguanine, and azidothymidine. The latter two are better known as acyclovir and AZT, respectively. Although a thiosemicarbazone derivative was the ﬁrst lowmolecular-weight antiviral compound used in humans—as a prophylactic treatment for smallpox contacts in the early 1960s [2]—herpesvirus disease was the ﬁrst viral infection for which a truly efﬁcient treatment was developed, in the form of acyclovir. When AIDS was recognized as a viral disease in the early 1980s, treatment at ﬁrst seemed unlikely if not impossible. However, only four years after the virus had been discovered, the ﬁrst drug, azidothymidine, had been introduced onto the market. We now have several antiviral drugs in our arsenal, and not all are antimetabolites: two novel drugs against inﬂuenza have been developed that interact with, and inhibit, the viral neuraminidase,

Preface

v

whereas the viral protease and reverse transcriptase of HIV are principal targets for current treatments with non-nucleosidic inhibitors. Some of the nucleoside analogs developed for HIV therapy have been found to be effective against hepatitis B as well. Ribavirin, in combination with interferon, has also been successfully introduced for the treatment of hepatitis C and can be regarded as an effective immunomodulatory principle. Daunting as it seemed for years, in the year 2003 effective drug therapy does exist for a variety of viral diseases, just as Doerr and colleagues predicted 60 years ago. Concomitant with this, we have also witnessed the development of a new era of diagnostic tools. Polymerase chain reactions combined with appropriate methods for sequence detection now allow not only the diagnosis of a viral disease but also the determination of a viral subtype. In addition, methods that allow the quantiﬁcation of the virus in body ﬂuids or tissues in order to determine viral loads are now standard in clinical trials, and drugs are being approved on the basis of showing efﬁcacy in reducing this particular parameter. Thus, the demonstration of efﬁcacy of an antiviral drug no longer depends entirely on clinical endpoints once a relationship has been established between the reduction in viral load and resolution of clinical signs has been established. All these advances in understanding and treating viral diseases are relatively recent developments and are proceeding at a rapid pace. Thus there is often a gap in knowledge about the diagnostic and therapeutic options currently available for clinical management. It is our hope that this book will ﬁll that gap, and we have tried to present in a practical and cohesive manner all the latest developments and their use. The introductory chapter gives an overview of the most important human viral pathogens and their transmission as well as emerging viral pathogens. At the time of writing, severe acute respiratory syndrome (SARS) dominates the media; it is an example of the unpredictability of emerging viral diseases and illustrates the potential for explosive dissemination of viruses throughout our global community. Not only does the novelty of the SARS-associated virus make it difﬁcult to assess the long-term signiﬁcance of the outbreak, but also the SARS epidemic itself exposes the desperate need for rapid diagnosis and reporting of unusual outbreaks and for international cooperation in investigating such occurrences. The main structure of this book consists of speciﬁc sections on infections of the respiratory tract, the skin and mucosa, and the liver as well as special sections on human immunodeﬁciency virus and on

vi

Preface

herpesvirus infections of the immunocompromised host. We have included chapters on what are currently regarded as the most clinically relevant viruses—inﬂuenza viruses, respiratory syncytial virus, rhinovirus, the herpes simplex viruses, varicella zoster virus, papillomaviruses, hepatitis viruses, human immunodeﬁciency virus, human cytomegalovirus, Epstein-Barr virus, and the recently identiﬁed human herpesviruses 6, 7, and 8. Each chapter describes the respective diagnostic measures guiding therapy, approved therapeutic agents, their mode of action, and their clinical applications. This book is intended not only for infectious-disease physicians and virologists who want to update their knowledge on transmission, diagnosis, and treatment of viral diseases but also for the nonspecialist who wishes to obtain a greater understanding of the clinically important viral pathogens. It is clear that there is still a less than complete understanding in the community of the burden of mortality and morbidity caused by viruses. HIV continues to devastate the world population and now ranks equal to tuberculosis as a global killer. The spread of the virus is still exponential in many areas of the world, and a constant 35,000–40,000 people are being newly infected in both the United States and Europe each year. Persistent viral infections develop unusual patterns of disease and unusual severity under conditions of immunosuppression by HIV, be it herpesvirus, cytomegalovirus, or papillomaviruses. Similarly, many of the viruses discussed in this book cause considerable, even life-threatening, problems in immunocompromised states after transplantation or in neonates. Therein lies the reason for our putting this book together, and it is our profound hope that it will serve the reader well. We hope that it will be a constant companion for physician, student, and layperson alike. We are grateful to everyone who has accompanied us on the sometimes tortuous path of producing this volume, especially the contributors and the editorial staff of Marcel Dekker, Inc. Helga Ru¨bsamen-Waigmann Karl Deres Guy Hewlett Reinhold Welker References 1.

Doerr R. The chemotherapy of viral diseases. In: Doerr R, Hallauer C, eds. Handbook of Virus Research: 1st Suppl. (in German). Vienna: SpringerVerlag, 1944:271–348.

Preface 2.

vii

Bauer DJ, St. Vincent L, Kempe CH, Downie AW. Prophylactic treatment of smallpox contacts with N-methylisatin b-thiosemicarbazone. Lancet 1963; ii(7306):494–496.

Contents

Preface Contributors 1.

Emerging and Reemerging Viral Pathogens Ulrich Desselberger

iii xi 1

Infections of the Respiratory Tract 2.

Inﬂuenza: The Virus, the Disease, and Its Control Thorsten Wolff and Rene´ Snacken

39

3.

Respiratory Syncytial Virus Philip R. Wyde and Pedro A. Piedra

91

4.

Rhinovirus Ronald B. Turner and Frederick G. Hayden

139

5.

Herpes Simplex Virus Kimberly A. Yeung-Yue, Gisela Torres, Mathijs H. Brentjens, Patricia C. Lee, and Stephen K. Tyring

165

6.

Varicella-Zoster Virus Jashin Joaquin Wu, Kimberly A. Yeung-Yue, Mathijs H. Brentjens, and Stephen K. Tyring

193

7.

Human Papillomaviruses Guy Hewlett, Philip S. Shepherd, and Jenny C. Luxton

227

ix

x

Contents

Infections of the Liver 8.

Hepatitis A Virus Verena Gauss-Mu¨ller and Reinhart Zachoval

259

9.

Hepatitis B Virus Guido Gerken and Christoph Jochum

277

Hepatitis C Virus Miriam Kerstin Huber, Ulrike Sarrazin, and Stefan Zeuzem

295

10.

Human Immunodeﬁciency Virus 11.

12.

13.

HIV Infection: Epidemiology, Pathogenesis, and Principles of Antiretroviral Therapy Reinhold Welker and Helga Ru¨bsamen-Waigmann

369

Diagnosis and Management of HIV Infection Using Immunoassays and Molecular Technologies Rainer Ziermann, Charlene E. Bush-Donovan, and David A. Hendricks

433

Nucleoside/Nucleotide Inhibitors of HIV Reverse Transcriptase Erik De Clercq

485

14.

Non-Nucleoside Inhibitors of HIV Reverse Transcriptase Matthias Go¨tte and Mark A. Wainberg

505

15.

HIV Protease Inhibitors Richard Ogden

523

16.

Emerging Therapies for HIV Infection Julie M. Strizki

555

Systemic Herpesvirus Infections and the Immune-Compromised Host 17.

Human Cytomegalovirus: Diagnosis, Pathophysiology, and Treatment Hermann Einsele and Gerhard Jahn

587

18.

Epstein-Barr Virus: Pathogenesis and Treatment Nancy Raab-Traub and Shannon C. Kenney

623

19.

The Human Herpesviruses HHV-6, HHV-7, and HHV-8 Dharam V. Ablashi and Gerhard R. F. Krueger

659

Index

707

Contributors

Dharam V. Ablashi, D.V.M., M.S., Dip.Bact.* Adjunct Professor of Microbiology and Director, Herpesvirus Program, Georgetown University Medical School, Washington, D.C., and Advanced Biotechnologies Inc., Columbia, Maryland, U.S.A. Mathijs H. Brentjens, M.D. Department of Dermatology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Charlene E. Bush-Donovan, Ph.D. Director of Research, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A. Erik De Clercq, M.D., Ph.D. Professor, Rega Institute for Medical Research, Catholic University of Leuven, Leuven, Belgium Ulrich Desselberger, M.D., F.R.C.Path., F.R.C.P.(Glasgow, London){ Consultant Virologist and Director, Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, England

* Retired { Current afﬁliation: Virologie Mole´culaire et Structurale, UMR 2472, CNRS, Gif-sur-Yvette, France.

xi

xii

Contributors

Hermann Einsele, M.D. Division of Hematology/Oncology, Department of Internal Medicine, Universita¨tsklinikum Tu¨bingen, Tu¨bingen, Germany Verena Gauss-Mu¨ller, Ph.D. Professor, Institute of Medical Molecular Biology, University of Lu¨beck, Lu¨beck, Germany Guido Gerken, M.D. Director, Gastroenterology and Hepatology Clinic, Center for Internal Medicine, University of Essen, Essen, Germany Matthias Go¨tte, Ph.D. Assistant Professor, Departments of Medicine and Microbiology and Immunology, McGill University, and Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada Frederick G. Hayden, M.D. Stuart S. Richardson Professor of Clinical Virology and Professor, Departments of Internal Medicine and Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. David A. Hendricks, Ph.D. Senior Research Fellow and Director, Medical and Scientiﬁc Affairs, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A. Guy Hewlett, Ph.D. Principal Scientist, Department of Virology, Bayer HealthCare, Wuppertal, Germany Miriam Kerstin Huber, M.D. Department of Gastroenterology, Johann-Wolfgang Goethe University Clinic, Frankfurt/Main, Germany Gerhard Jahn, M.D. Professor, Institute of Medical Universita¨tskinikum Tu¨bingen, Tu¨bingen, Germany

Virology,

Christoph Jochum, M.D. Department of Gastroenterology and Hepatology, Center of Internal Medicine, University of Essen, Essen, Germany Shannon C. Kenney, M.D. Professor, Lineberger Comprehensive Cancer Center, Department of Medicine and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

Contributors

xiii

Gerhard R. F. Krueger, M.D., Ph.D. Professor, Division of Allergy and Clinical Immunology, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Patricia C. Lee, M.D. Associate Professor, Department of Microbiology/Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Jenny C. Luxton, Ph.D. Department of Immunobiology, Guy’s, King’s and St. Thomas’ Medical and Dental Schools, London, England Richard Ogden, Ph.D. Senior Director, Scientiﬁc Development, Agouron Pharmaceuticals, Inc., A Pﬁzer Company, La Jolla, California, U.S.A. Pedro A. Piedra, M.D. Associate Professor, Department of Molecular Virology and Microbiology and Department of Pediatrics, Baylor College of Medicine, Houston, Texas, U.S.A. Nancy Raab-Traub, Ph.D. Professor, Lineberger Comprehensive Cancer Center, Department of Medicine, and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Helga Ru¨bsamen-Waigmann, Ph.D. Vice President, Division of Antiviral Research, Anti-Infective Research, Bayer HealthCare, Wuppertal, and Professor, Department of Biochemistry and Virology, University of Frankfurt, Frankfurt, Germany Ulrike Sarrazin, M.D. Clinic for Internal Medicine II, Saarland University Hospital, Homberg, Germany Philip S. Shepherd, M.B., C.H.B. M.Sc., M.R.C.P., F.R.C.P. Senior Lecturer and Honorary Consultant, Department of Immunobiology, Guy’s, King’s and St. Thomas’ Medical and Dental Schools, London, England Rene´ Snacken, M.D., M.P.H. Head, Inﬂuenza Branch, Department of Epidemiology, Scientiﬁc Institute of Public Health, Brussels, Belgium Julie M. Strizki, Ph.D. Principal Scientist, Antiviral Therapeutics, Schering–Plough Research Institute, Kenilworth, New Jersey, U.S.A.

xiv

Contributors

Gisela Torres, M.D. Clinical Research Fellow, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Ronald B. Turner, M.D. Professor, Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. Stephen K. Tyring, M.D., Ph.D. Professor, Department of Dermatology and Department of Microbiology/Immunology, University of Texas Medical Branch, Galveston, Texas, U.S.A. Mark A. Wainberg, Ph.D. Professor, Department of Microbiology, McGill University, and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada Reinhold Welker, M.D. Senior Scientist, Department of Virology, Bayer HealthCare, Wuppertal, Germany Thorsten Wolff, M.D. Germany

Group Leader, Robert Koch-Institut, Berlin,

Jashin Joaquin Wu, M.D. Department of Internal Medicine, Baylor College of Medicine, Houston, Texas, U.S.A. Philip R. Wyde, Ph.D. Professor, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A. Kimberly A. Yeung-Yue, M.D. Dermatology Clinical Research Fellow, Center for Clinical Studies, University of Texas Medical Branch, Galveston, Texas, U.S.A. Reinhart Zachoval, M.D. Professor, Department of Internal Medicine II, Grosshadern Medical Center, Munich, Germany Stefan Zeuzem, M.D. Professor, Department of Internal Medicine II, Saarland University Hospital, Homburg, Germany Rainer Ziermann, Ph.D. Principal Staff Scientist, Medical and Scientiﬁc Affairs, Bayer HealthCare Diagnostics, Berkeley, California, U.S.A.

1 Emerging and Reemerging Viral Pathogens Ulrich Desselberger Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, England*

1

INTRODUCTION

Over the last 25–30 years numerous viral and other microbial pathogens have been discovered to be etiological agents of human disease. This chapter presents a review on the viral pathogens found to cause novel or reemerging human disease. Animals were found or suspected to be the source of human infection in a number of cases. Prospective, laboratorybased, epidemiological surveillance is of paramount importance for early detection and management of emerging or reemerging infectious diseases. In 1990 the Institute of Medicine of the United States, in Washington, D.C., convened a committee under the cochairmanship of Joshua Lederberg and Robert Shope that conducted an extensive study on emerging microbial threats to health and the conditions under which they occurred. In the ensuing report [1] a large number of microbes (bacteria, * Current afﬁliation: Virologie Mole´culaire et Structurale, UMR 2472, CNRS, Gif-sur-Yvette, France.

1

2

Desselberger

TABLE 1 Major Etiologic Agents of Viral Infectious Diseases in Humans Identiﬁed Since 1972a Year 1972

Agent

1973

Small round structure viruses (SRSVs; caliciviruses); Norwalk virus Rotaviruses

1975 1975

Astroviruses Parvovirus B19

1977 1977

Ebola virus Hantaan virus

1980

Human T-cell lymphotropic virus-1 (HTLV-1)

1982 1983 1984 1985 1988

HTLV-2 Human immunodeﬁciency viruses (HIV-1, HIV-2) Puumulavirus SRSV: sapporovirus Human herpesvirus-6 (HHV-6)

1989

Hepatitis C virus (HCV)

1990 1990

Human herpesvirus-7 (HHV-7) Hepatitis E virus (HEV)

1991 1991 1992

Hepatitis F virus (HFV) Aichi virus Dobravavirus

1993

Sin nombre virus

1993 1994

Hepatitis G virus (HGV) Sabia virus

Human disease Diarrhea (outbreaks)

Major cause of infantile diarrhea worldwide Diarrhea (outbreaks) Aplastic crisis in chronic hemolytic anemia; ﬁfth disease; fetal infection Ebola hemorrhagic fever Hemorrhagic fever with renal syndrome (HFRS) Adult T-cell leukemia/ lymphoma; tropical spastic paraparesis (TSP)/HTLV-1 associated myelopathy (HAM) Hairy T-cell leukemia Acquired immunodeﬁciency syndrome (AIDS) Nephropathia epidemica Diarrhea (outbreaks) Exanthema subitum (roseola infantum); pneumonitis, excephalitis in bone marrow transplant recipients and AIDS patients Parenterally transmitted non-A, non-B hepatitis Exanthema subitum, others? Enterically transmitted non-A, non-B hepatitis Severe non-A, non-B hepatitis Diarrhea Hemorrhagic fever with renal syndrome Hantavirus pulmonary syndrome (‘‘Four corners disease’’) Non A-C hepatitis? Brazilian hemorrhagic fever and necrotizing hepatitis

Emerging and Reemerging Viral Pathogens

3

TABLE 1 Continued Year

Agent

Human disease

1994

1995

Human herpesvirus-8 (HHV-8) or Kaposi’s sarcoma-associated herpesvirus (KSHV) Borna disease virus

1995 1996

Hendravirus Prion (BSE?)b

1996 1996 1997 1997 1997 1998 1999 1999 2000 2001

Whitewater Arroyo virus Inﬂuenza A virus (H7N7) Inﬂuenza A virus (H5N1) Transfusion-transmitted virus (TTV) Enterovirus 71 (EV71) Nipahvirus Inﬂuenza A virus (H9N2) West Nile encephalitis virus Cantalago virus Metapneumovirus

Kaposi’s sarcoma; body cavitybased lymphoma; Castleman’s disease Neuropsychiatric disorders (disputed for human) Meningitis, encephalitis New variant Creutzfeldt-Jakob disease (nv-CJD) Hemorrhagic fever Conjunctivitis (England) Inﬂuenza (Hong Kong) No ﬁrm association yet

2001

SEN virus (TTV-related)

Epidemic encephalitis Meningitis, encephalitis Inﬂuenza (Hong Kong) Encephalitis (New York) Vesicular rash (Brazil) Respiratory tract infection (children) No ﬁrm association yet

a

Date of discovery assigned on the basis of the year of isolation or identiﬁcation of etiological agent. b Prions are proteinaceous infectious particles lacking nucleic acid (224), not viruses. Source: Refs. 8,11,12 (updated).

rickettsiae, chlamydiae, viruses, fungi, parasites) were identiﬁed that were either already known as human pathogens but appeared to be associated with changed disease patterns and/or increased case or infection rates, or were recognized as new human pathogens. Factors involved in their emergence or reemergence were population increase and increasing urbanization, industrial and economic development including man-made perturbation of the environment, global travel and mass movements (refugees), and civil unrest and wars but also microbial genomic change and adaptation [1–12]. It was further recognized that gradually improved surveillance using clinical, pathological (laboratory), epidemiological, and public health approaches has led to rapid identiﬁcation of newly emerging or reemerging infectious agents. Advances in the science of virology have allowed the introduction of a

4

Desselberger

number of molecular diagnostic techniques that have greatly enhanced the discovery of previously unknown viruses pathogenic for humans [13]. Numerous journal publications and books [e.g., 2,6,14,15] and even a dedicated journal, Emerging Infectious Diseases, have described the emergence of new pathogens in the 1990s. The major viral pathogens identiﬁed as causes of human disease since 1972 are listed in Table 1. (This time point is arbitrary, because shortly before 1972 Lassa virus, Marburg virus, and others had emerged.) In the following pages, selected aspects of diagnosis, epidemiology, and treatment and prevention of infection with these new or reemerged viruses are brieﬂy described.

2 2.1

SPECIFIC PATHOGENS Small Round Structured Viruses (Caliciviridae)

Norwalk virus (NV) was identiﬁed as the ﬁrst small round structured virus (SRSV) when it caused outbreaks of acute gastroenteritis in 1972 [16]. In 1990 the NV genome, a single-stranded RNA of positive sense and approximately 7.5 kilobases (kb) in size was cloned [17], and subsequently its genome and those of other emerging related viruses were sequenced [e.g., 18–21], allowing their classiﬁcation as members of the Caliciviridae family. Human gastroenteric caliciviruses occur in two genera [Norwalk-like viruses (genogroups I and II) and Sapporo-like viruses] [22]. These genera have recently been designated as Norovirus and Sapovirus, respectively [22a]. Several genogroups of SRSV have been found that in part cocirculate and even coinfect [23,24]. Calicivirus recombinants have been observed to occur naturally [25]. The virus structure, that of a nonenveloped viral particle, has been established by cryoelectron microscopy and image reconstruction [26]. Expression of the NV capsid protein from baculovirus recombinants in insect cells [27] and its use as an antigen in enzyme-linked immunosorbent assays (ELISAs), have allowed assessment of the agerelated seroprevalence of speciﬁc antibody to the virus [28–30], which starts to infect children early in life, often asymptomatically, and is causing much more widespread human infection than previously thought [31]. However, recently human caliciviruses were also recognized as a major cause of apparent gastroenteritis in children by using the now widely available reverse transcription-polymerase chain reaction (RT-PCR) for diagnostic purposes and outbreak investigation (screening) [32–34]. The evidence of close genetic relationships between porcine enteric caliciviruses and Sapporo-like viruses and between

Emerging and Reemerging Viral Pathogens

5

bovine caliciviruses and Norwalk-like viruses suggests that animals are potential reservoirs for human infections [35, 35a–c].

2.2

Rotaviruses (Reoviridae)

Rotaviruses were discovered as the cause of infantile human gastroenteritis in 1973 [36,37] after similar viruses had already been recognized as the cause of acute gastroenteritis in a variety of young animals (mice, monkeys, calves) in the 1960s. Since their discovery, human rotaviruses have been found to be the main etiological agent of gastroenteritis in infants and young children worldwide [38]. At least seven groups (A–G) and, within group A, various types exist (classiﬁed as G and P types). Group A rotaviruses cocirculate at any one time [38,39]. Whereas G1P[8], G2P[4], G3P[8], and G4P[8] represent the majority of viruses cocirculating in temperate climates, G5 and particularly G9 viruses have been found in recent years, ﬁrst in Asia, Africa, and South America, but G9 also in Europe and North America [40–44]. After many years of research, a live-attenuated, tetravalent (TV), rhesus rotavirus (RRV)–based, human reassortant vaccine was developed [45–47] that was licensed in the United States in August 1998. Guidelines for its application appeared in March 1999 [48]. In the period between 1 September 1998 and 7 July 1999, when 1.8 million doses of the vaccine were administered in the United States, gut intussusceptions were observed in 15 infants, of whom 13 developed the condition after the ﬁrst dose of the three-dose RRV-TV vaccine course, and 12 developed the symptoms within one week of any dose (see Refs. [49] and [50] for further details). Although the number of reported cases was within the expected range by chance during the week following the receipt of any dose, the well-known incompleteness and delays of reporting through the Vaccine Adverse Event Reporting System (VAERS) led the U.S. Centers for Disease Control (CDC) and the American Academy of Pediatrics (AAP) to recommend postponing administering RRV-TV to infants between July and November 1999. A study with evidence supporting an association between vaccination with RRV-TV and intussusception has been published [51], but the true vaccine-attributable risk is still under investigation. The original CDC Advisory Committee on Immunization Practices (ACIP) guidelines [48] have been revoked, and the vaccine was taken off the market in October 1999 [52]. Alternative concepts for developing candidate rotavirus vaccines are under active investigation [53].

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Astroviruses (Astroviridae)

Human astroviruses were ﬁrst detected in 1975 by electron microscopy (EM) in stool specimens of infants and children with diarrhea [54] and named after the pathognomonic star-shaped appearance of some of the particles in electron micrographs [55]. These viruses are now classiﬁed within a separate family, the Astroviridae [56]. They are nonenveloped particles and possess a genome of single-stranded RNA of positive polarity and 6.8 kb size with three open reading frames (ORFs) of which the second encodes the single capsid protein [57]. So far eight different human astrovirus types have been recognized [58,59], and Noel et al. [60] have shown a very good correlation of serotypes and genotypes for seven astrovirus types, using ELISAs and RT-PCR, respectively. Astroviruses have also been recognized as the cause of major outbreaks of gastroenteritis [e.g., 61,62]. In hospitalized children in Australia, astroviruses were found to be the second most frequent viral cause of diarrhea, after rotaviruses [63]. 2.4

Parvovirus B19 (Parvoviridae)

Parvovirus B19 was discovered as an agent infecting humans in 1975 [64]. The viral particles measure only 18–26 nm in diameter, are nonenveloped, and have an icosahedral symmetry. Their genome consists of linear, single-stranded DNA of both polarities and is approximately 5 kb in size. For their replication, parvoviruses have an almost absolute requirement of rapidly dividing cell populations such as those found in bone marrow and embryonic tissues [65]. This explains parvoviruses as the cause of aplastic crises in chronic hemolytic anemia (sickle cell anemia) patients [66] and of intrauterine infections followed sometimes by hydrops and early abortion as a clinical outcome but mostly remaining an inapparent infection [67,68]. Parvovirus B19 is also the etiological agent of the childhood disease erythema infectiosum (ﬁfth disease) [69]. 2.5

Ebola Virus (Filoviridae)

The Ebola virus was found to be the cause of a hemorrhagic fever with high mortality in Central Africa in the mid-1970s [70,71] and reemerged there in the mid-1990s [72,73]. Early recognition of Ebola virus as the causative agent of the outbreaks in Kikwit hospital in Zaire in 1995 was due to a broad international collaboration of physicians, virologists, immunologists, molecular biologists, pathologists, epidemiologists, and public health doctors. Ebola virus (like Marburg virus) is a member of the

Emerging and Reemerging Viral Pathogens

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Filoviridae family and has now been thoroughly characterized at the molecular level (for review, see Ref. [73]). Recently reverse genetics systems for Ebola virus have been devised by two groups [74,75]. An intensive search for a true animal reservoir of these viruses is ongoing [76,77]. 2.6

Sabia Virus, Whitewater Arroyo Virus (Arenaviridae)

Other emerging hemorrhagic fever viruses were observed in different parts of the world. Examples include Sabia virus, associated also with severe hepatitis [78,79], and Whitewater Arroyo virus [80,81]. Both of these viruses are members of the Arenaviridae family, of which the best known member is Lassa virus, detected as the cause of severe hemorrhagic fever in 1970 [82]. 2.7

Bunyaviruses (Bunyaviridae)

Viruses of the Bunyaviridae family were discovered several times as novel etiologic agents of human diseases. In 1977 Hantaan virus was found to be the cause of hemorrhagic fever with renal syndrome (HFRS) [83], which by then had been known as Korean hemorrhagic fever for more than 20 years [84]. Nephropathia epidemica, a mild form of HFRS occurring in Scandinavia, was found in 1984 to be caused by a bunyavirus, Puumala virus [85,86]. Another hantavirus, Dobrava virus, was shown to be the cause of severe HFRS in the Balkans [87]. Rodents (mice, rats, etc.) were recognized as the main, asymptomatic reservoir of bunyaviruses [84,88,89]. In 1993 another member of the Bunyaviridae family, sin nombre virus, was identiﬁed as the cause of the hantavirus pulmonary syndrome (HPS or ‘‘four corners disease’’) [89]. It was shown that climatic changes in the Southwestern United States (due to el Nin˜o) had led to an increase in food for rodents and a subsequent increase in their numbers, making it more likely for humans in close contact with the countryside (hiking, summer houses, etc.) to become infected [89,90]. For sin nombre virus, coevolution of virus and host species was extensively documented, each rodent species acting as the primary reservoir for only a single speciﬁc hantavirus [90,91]. Since then hantaviruses have been found throughout wide areas of the United States and also in Central and South America. The data suggest that hantaviruses and their rodent hosts have coevolved over 20–30 million years [90]. Bunyaviruses are widespread in animal reservoirs [90,92,93]. Transmission in animals seems to occur mainly horizontally by infectious excretions, and transmission to humans by inhalation of contaminated dust. Although bunyaviruses contain three segments of negative-stranded RNA as their genome,

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reassortment has so far not been found to be a major factor in the emergence of new strains (in contrast to inﬂuenza viruses, where reassortment events have repeatedly led to the emergence of pandemic strains; see below). A reverse genetics system was devised by Bridgen and Elliott [94] that is starting to help unravel the molecular biology of the virus. 2.8

Human T-Cell Leukemia Viruses (Retroviridae, Oncovirinae)

Two types of human T-cell leukemia viruses (HTLVs) have been recognized as the cause of human disease: type 1 (HTLV-1) in 1980 as being closely associated with T-cell lymphoma-leukemia (the ﬁrst human tumor ﬁrmly associated with a viral infection [95]) and type 2 (HTLV-2) in 1982 as being associated with atypical hairy T-cell leukemia [96]. In addition, HTLV-1 was shown to cause tropical spastic paraparesis (TSP) [97], also termed HTLV-1-associated myelopathy (HAM) [98]. These HTLVs are members of a large subfamily (Oncovirinae) of the Retroviridae family, which have for some time been known to be associated with a large number of tumors in animals (Rous sarcoma virus, mouse mammary tumor virus, etc.). HTLV-1 infections were discovered in Japan but are now found worldwide. Like HIV, HTLV-1 is transmitted sexually (mainly male to female), through infected blood, or vertically from mother to child. Although not of recognized animal origin, HTLV-1 has been transmitted to rats and rabbits, with some animals developing features of the human disease [99]. 2.9

Human Immunodeﬁciency Virus (Retroviridae, Lentivirinae)

A virus isolated in 1983 from a homosexual patient was ﬁrst termed lymphadenopathy-associated virus (LAV) [100]. Soon afterward the unequivocal association of LAV infection with the acquired immunodeﬁciency syndrome (AIDS) was demonstrated, and the virus was renamed human immunodeﬁciency virus (HIV). At least two types (HIV-1, HIV-2) and within them a large variety of subtypes/clades (HIV-1: clades A–J in group M, groups N and O; HIV-2: clades A–E) exist and cocirculate. The origin of HIV as a human pathogen has for a long time been an enigma. However, the ﬁndings that HIV-2 isolates are closely related to simian immunodeﬁciency virus (SIV) isolates from sooty mangabeys (SIVsmm) [101] and that HIV-1 isolates are closely related to SIVs obtained from chimpanzees (SIVcpz) [102] strongly suggested that chimpanzees and sooty mangabeys are the animal reservoirs for a zoonosis in humans.

Emerging and Reemerging Viral Pathogens

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There is evidence for SIV–host coevolution [103]. The striking diversity within and between clades is achieved by the accumulation of point mutations and by frequent recombinatory events in regions of the world where HIVs of different clades cocirculate in sufﬁcient numbers and frequencies, thus increasing the chance of coinfection [104–106]. Even intergroup recombination has recently been found to occur in nature [107]. This chapter is clearly not the place to review the replication, pathogenesis, diagnosis, and treatment of HIV in detail [103,108–110]. However, it should be noted that highly active antiretroviral therapy (HAART), which has beneﬁted many HIV-infected patients since 1996, has exerted a strong selective pressure on HIV, leading to the emergence of drug-resistant HIV mutants (in both the reverse transcriptase and protease genes). Although genotypic resistance assays were helpful in the formulation of antiretroviral regimes [111], increasingly (up to 27% of), new infections occur with HIV variants that are already resistant against one or several of the licensed drugs [112–115; D. Pillay, personal communication]. Thus, there is an accelerated evolution and emergence of drug-resistant viruses. 2.10

New Herpesviruses (Herpesviridae)

Since 1988, three new human herpesviruses (HHV-6, HHV-7, and HHV8) have been discovered. HHV-6, initially termed human B-lymphotropic virus (HBLV), was discovered in patients with lymphoproliferative disorders [116] but was later found to be the cause of the infancy and childhood disease exanthema subitum/roseola infantum [117,118]. Most cases of exanthema subitum are caused by the variant HHV-6B [119]. The primary infection with this ubiquitous virus mainly occurs within the ﬁrst three years of life [120], not infrequently associated with encephalitis [121,122]. Reactivation of HHV-6 in allogeneic bone marrow transplant patients was found to be associated with fever, skin rashes, pneumonitis, encephalitis, bone marrow suppression, and graft-versus-host disease [123–125]. In 1990, a new human herpesvirus was isolated from a healthy carrier and termed HHV-7 [126]. As with HHV-6, infection with HHV-7 seems to occur ubiquitously and to cause infection, mainly in children [127–129]. At present, the extent of the involvement of HHV-7 in human disease is not clear, although the virus has been isolated from patients with exanthema subitum [118,130] and pityriasis rosea [131]. In 1994, cDNA sequences with homology to herpesvirus sequences were classiﬁed as those of a new type of human herpesvirus, called

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HHV-8. Subsequently, HHV-8 was found to be ﬁrmly associated with the occurrence of Kaposi’s sarcoma (angioplastic sarcoma) and therefore also termed Kaposi’s sarcoma associated herpesvirus (KSHV) [132]. In 1996, a cell line (BCBL-1) propagating KSHV/HHV-8 was described [133]. Phylogenetically, KSHV is a g2-herpesvirus [134] related to the EpsteinBarr virus (EBV), a g1-herpesvirus. Knowledge of the epidemiology and transmission of the virus is still rudimentary. KSHV occurs in several variants, based on sequence differences at the left and right ends of the viral genome [135,136]. Several subtypes have been distinguished, subtype B being found mainly in Africa and subtypes A and C also found in Africa but mostly in Europe [137,138]. Seroprevalence data show differences among geographical regions, also depending on the antigen used in serological assays. In general, there is an age-related increase of antibody prevalence. Transmission is mainly sexual, due to the occurrence of the virus in seminal ﬂuid [139]. However, horizontal transmission among children has also been observed [140,141]. Parenteral transmission seems possible, but this observation needs to be supported by further studies [141]. A causative role of KSHV in a rare non-Hodgkin’s lymphoma, body cavity based lymphoma, and another a typical lymphoproliferative disorder, Castleman’s disease (a multicentric B-cell lymphoma), is also likely [138]. 2.11 2.11.1

New Hepatitis Viruses Hepatitis C Virus (Flaviviridae)

The study of hepatitis virology has moved tremendously fast since 1989 when, entirely by the use of molecular techniques, hepatitis C virus (HCV) was discovered as the main cause of transfusion-transmitted, nonhepatitis A, non-hepatitis B (non-A, non-B) viral infections [142]. The virus belongs to the Flaviviridae family but has now been classiﬁed as a separate genus (Hepacivirus) [143]. A ﬁrst diagnostic test was developed in 1989 [144], but reliable second and third generation tests became generally available in 1991 and were then immediately made obligatory for the screening of every blood donation. That measure virtually closed a previously predominant transmission pathway (blood, blood products), and needle sharing among intravenous drug users remained the main transmission route. Transmission by sexual contacts and vertical transmission are relatively rare events compared to the frequency of these transmission pathways being used by other bloodborne viruses (HBV, HIV). The infection resolves in only 20% of the cases; in 80% a chronic hepatitis results, along with an increased risk for the development of hepatocellular carcinoma (HCC). Chronic HCV infection is very

Emerging and Reemerging Viral Pathogens

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difﬁcult to manage. HCV-infected individuals with chronic liver disease are the most frequent subpopulation of patients becoming candidates for liver transplants. The seroprevalence of HCV infection in blood donors worldwide is between 0.02% and 1.2%, with higher rates in Japan, Spain, Hungary, Italy, and Saudi Arabia [145]. Exceptionally high donor infection rates of almost 20% have been recorded in Egypt [146]. Hepatitis C viruses are highly heterogeneous, and at least six different HCV types with 11 subtypes are recognized at present [147]. Treatment is by a-interferon [148,149], more recently in combination with other antiviral agents (ribavirin, lamivudine), but is of limited success; there is a high relapse rate after cessation of treatment. Different HCV types vary in responsiveness to interferon treatment, with type 1 strains being more resistant than type 2 and 3 strains [149]. 2.11.2

Hepatitis E Virus (Caliciviridae)

Hepatitis E virus (HEV) infection became known as a separate entity in the late 1980s and was termed epidemic non-A, non-B or enterically transmitted non-A, non-B (ENANB) hepatitis. It followed a transmission pathway and caused disease similar to hepatitis A but was not reactive with HAV-speciﬁc serological assays. In 1990 Reyes et al. [150] succeeded in cloning and sequencing part of the genome of this virus. The complete sequence of a number of isolates has now been determined [151–156], and the ENANB virus has been renamed hepatitis E virus (HEV). Its genome is similar to that of caliciviruses; however, the order of genes in HEV is not identical, and therefore HEV may be placed into a separate family at some stage [143]. The genomes of several HEV strains from Asia and Mexico have been entirely sequenced, and partial sequences are available for some other strains [157]. In all parts of the genome the Mexican strain was most different from the sequences studied [152]. Although moderate genetic heterogeneity has been identiﬁed among HEV strains, evidence for serological heterogeneity is limited [158]. The course of infection in experimentally infected primates is similar to that in humans [159,160]. The incubation period is 3–8 weeks, followed by an increase of liver enzyme concentrations in the blood. Peak viremia and shedding of HEV in feces occur during the incubation period and the very early acute phase of disease. In most cases the infection resolves completely. However, the severity of HEV infections is on average somewhat greater than the severity of HAV infections. Mortality of hepatitis E has varied in different reports and has been as high as 1%, compared to 0.2% of hepatitis A [161]. More important is the severity of hepatitis E in

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pregnant women, the mortality in pregnancy increasing with each trimester and possibly reaching 20% [161–163]. The reason for the excessive mortality of hepatitis E in pregnancy is at present unknown. The diagnosis is based on HEV-speciﬁc IgM and IgG ELISAs, using recombinant expressed capsid antigen [158,162,164]. The age-speciﬁc clinical attack rate with its peak among young adults is striking. In endemic areas the age-related prevalence of HEV antibodies reaches only 40% [165–167]. There is at present no vaccine, but protection of monkeys against experimentally induced hepatitis E by vaccination with recombinant HEV proteins has yielded encouraging results.

2.11.3

Hepatitis F Virus (Paramyxoviridae?)

In 1991, Phillips et al. [168] described 10 cases of a syncytial giant cell hepatitis observed sporadically in the United States between 1979 and 1988 and associated with a severe clinical course. The virological studies suggested a paramyxovirus as putative cause because paramyxoviruslike nucleocapsids were found by electron microscopy in patients’ livers (8/10). Chimpanzees inoculated with infected liver homogenate raised an antibody response that was cross-reactive with measles virus and parainﬂuenza virus type 4; however, the animals did not develop a hepatitis. Although this putative viral infection ﬁgured as hepatitis F at the time, the work by Phillips et al. [168] has not been pursued further.

2.11.4

Hepatitis G Virus (Flaviviridae)

Hepatitis G virus (HGV), also a member of the Flaviviridae family, was discovered in 1993 from cloned cDNA fragments in blood donations. Subsequently, sequences of this virus were found in 1–3% of all blood donations in different parts of the world; it seemed to replicate in liver cells and was thought to be associated with hepatitis. However, a close association of infection by this virus with liver disease has so far not been secured [169,170], and replication in the liver has not been conﬁrmed (P. Simmonds, personal communication). Therefore testing of blood donations for the presence of HGV sequences is at present not mandatory. Recently, two reports appeared demonstrating signiﬁcantly higher survival rates in HIV-infected patients coinfected with the HGV (GB virus C) (170a, 170b). The HIV load was signiﬁcantly lower in the coinfected patients (170b). The mechanisms underlying this remarkable effect remain to be explored.

Emerging and Reemerging Viral Pathogens

2.12

13

Transfusion-Transmitted Virus (Circoviridae)

In 1997 transfusion-transmitted virus (TTV) was discovered as the cause of some cases of hepatitis transmitted by infected blood donations [171]. The prevalence of TTV antibodies in various populations was found to be very variable [172]. TTV is the ﬁrst human-transmitted member of the Circoviridae family [143] (P. Simmonds, personal communication). The virus was found as a coinfection in HCV-infected patients but the two viruses reacted differently upon treatment with interferon [173]. In an American study, one-third of healthy blood donors were found to be infected with TTV. A connection with disease is still being debated [174]. Signiﬁcant numbers of chickens, pigs, cows, and sheep were found to be infected [175]. Erker et al. [176] found sequence diversities of up to 30% among more than 10 full-length genomic sequences of TTV isolates. This very substantial amount of variation suggests that there are at least three types of the virus. Ball et al. [177] followed up several chronically HCVinfected patients longitudinally and found in some a stable form of TTV over several years; however, in others there were ﬂuctuating levels of at least seven distinct variants of the virus over a 5-year period. The natural history of TTV is rapidly expanding; the high prevalence of TTV worldwide with apparently no signiﬁcant associated disease is astonishing [174]. Another virus of this family, the recently discovered SEN virus, was found to be common in people at high risk for bloodborne viral infections in Taiwan but not to be signiﬁcantly associated with hepatitis [177a].

2.13

Inﬂuenza Viruses (Orthomyxoviridae)

Type A and B inﬂuenza viruses regularly cause outbreaks of severe respiratory disease in large segments of the world’s population during winter and spring. Several inﬂuenza A virus pandemics have been described (caused by subtype H1N1 in 1918, H2N2 in 1957, H3N2 in 1968, and H1N1 in 1977). Inﬂuenza viruses have a wide animal reservoir, and, by the mechanism of reassortment, animal type A inﬂuenza viruses have contributed genes, for instance those coding for hemagglutinin H3, to viruses that became human pandemic viruses. It has been shown that human H1N1 and also H3N2 inﬂuenza A viruses can infect pigs and, vice versa, that related pig viruses can infect humans. By contrast, avian inﬂuenza viruses, representing by far the greatest diversity and biggest reservoir of inﬂuenza A viruses, are thought to circulate only within their original host or closely related species. Contribution of avian genes into viruses able to replicate in humans until recently was thought to be possible only by reassortment, pigs being the likely host, because they

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were shown to be able to replicate avian inﬂuenza viruses to a certain extent (‘‘mixing vessel’’ theory of Scholtissek et al. [178]). Recently, however, different events were recorded. During 1997 at least 18 people in Hong Kong came down with inﬂuenza-like symptoms, some of them with severe generalized disease, which was found to be caused by inﬂuenza A viruses of the H5N1 subtype. Six out of 18 patients with conﬁrmed H5N1 inﬂuenza died. Six out of 18 patients with conﬁrmed H5N1 inﬂuenza died. Viral isolates were very closely related in all their eight RNA segments to viruses isolated from chickens on several farms in and around Hong Kong in the spring of 1997. From the molecular data it could be excluded that reassortants between animal and human strains had been formed. This was the likely reason for the inability of these viruses to spread from human to human and thus widely within the human population. In fact, human-to-human transmission was never convincingly recorded. The recent isolation of H5N1 inﬂuenza A viruses from humans was seen as the possible advent of a new pandemic strain. The ﬁrst virus was isolated in May 1997 in Hong Kong from a 3-year-old boy who subsequently died of Reye’s syndrome after treatment with aspirin. A very close surveillance ensued from this case, and in the following 8 months a total of 16 more conﬁrmed and two suspected human cases of infection with inﬂuenza A virus of subtype H5N1 were observed. Twelve of the 16 patients became ill in December 1997, and rapid diagnosis was the result of intensiﬁed surveillance in hospital and healthcare centers. Seven patients were under the age of 5 years, three between 5 and 14, and six over 14 years. Whereas in patients under 5 the infection was generally mild, in the older patients there was a high rate of complications such as gastroenteritis and renal and liver dysfunction [179]. There was an H5N1 epidemic in chickens between March and May 1997 in Hong Kong and southern China. Extensive epidemiological surveillance has so far not revealed signiﬁcant spread in humans. Most important, a human-to-human infection has not been deﬁnitely proven. A close virological investigation, including partial sequencing of the whole genome, has shown that in at least the ﬁrst cases the H5N1 isolate was a true avian isolate, i.e., all eight segments were of avian origin [180,181]. This ﬁnding greatly decreases, but does not exclude, the likelihood that these viruses may spread widely in humans. The World Health Organization (WHO) and various nations have worked out plans to cope with the sudden emergence of a new pandemic inﬂuenza virus strain. In the United Kingdom, both the Department of Health and the Public Health Laboratory Service have such plans in place. With the emergence of the ﬁrst isolate in May 1997 in Hong Kong,

Emerging and Reemerging Viral Pathogens

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stage 1 of the plan was activated, entailing constant review of the situation and signifying increased surveillance of both humans and animals. By the end of 1997, the Hong Kong government took the bold step of killing the chicken population of Hong Kong (approximately 1.5 million) [182,183]. WHO sent a fact-ﬁnding mission to southern China whose participants found relatively intensive surveillance practices in place. No human cases or seroepidemiological evidence of wider spread of this virus in animals or humans has been found so far. The ﬁnding of 16 cases of human infection with H5N1 strains that are ‘‘pure’’ avian viruses is in itself a highly unusual event, and these isolates will be scrutinized very intensely for factors that might have changed their host tropism and allowed the emergence of pathogenicity for humans. The human isolates were found to remain pathogenic for chickens [180,181]. It is possible that an unusual sequence around the trypsin cleavage site of the H5N1 viruses is in part responsible for the wider host spectrum [180,181,184,184a]. A mutation in the PB2 gene was found to be correlated with pathogenicity in mice [184a]. In March 1999 two isolates of inﬂuenza A virus of subtype H9N2 were obtained from two children with inﬂuenza in Hong Kong [185]. Genetically all the genes except for hemagglutinin (HA) and neuraminadase (NA) were very similar to those of the H5N1 viruses, suggesting that these genes may be important for efﬁcient transmission from birds to humans [184,186]. Thus, the animal inﬂuenza virus reservoir is a permanent threat for transmission of infectious agents to humans. An inﬂuenza A virus of subtype H7N7 was isolated in 1998 from an inﬂamed eye of a woman in Oxfordshire who was keeping ducks on a pond. No such virus was isolated from the woman’s ducks, but molecular analysis showed the human isolate to be of animal origin [187] and to be closely related to an H7N7 virus isolated from a turkey in Ireland in 1995 [188]. There is the sword of Damocles hanging over the population of Hong Kong, southern China, and the whole world that avian inﬂuenza viruses carrying H4–H14, which have so far not circulated in humans, might reassort somewhere with inﬂuenza A viruses that replicate well in humans, leading to a new pandemic strain that could spread rapidly throughout the world (similarly to the ‘‘Asian’’ and ‘‘Hong Kong’’ inﬂuenza viruses in 1957 and 1968, respectively). Such an event could indeed occur relatively easily in southeast Asia, where humans and domestic animals live in very close proximity, often under the same roof [184,189]. Thus, although at present there is no acute cause for alarm, a high degree of vigilance is clearly indicated.

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New Paramyxoviruses (Paramyxoviridae)

In September 1994 an outbreak of severe respiratory disease affected 18 horses, their trainer, and a stablehand in Queensland, Australia. Fourteen horses and one human died. A novel virus was isolated from those affected and named equine morbillivirus (EMV) or Hendra virus [190]. In the following year several other humans became infected with this new virus, most of them developing meningitis and encephalitis [191]. Serological evidence showed that a paramyxovirus related to EMV was present in Pteropus, a species of fruit bat [190,192]. In the autumn of 1998 through the spring of 1999 an outbreak of encephalitis in pig farmers and slaughterhouse workers in Malaysia and Singapore occurred, with more than 250 cases and over 100 deaths. There were also sick animals (pigs, dogs, and cats). A virus was grown from patient material that formed syncytia in Vero cells and yielded positive immunoﬂuorescence with a Hendra virus–speciﬁc antiserum. The virus was termed Nipah virus and found to be homologous to 89% at the nucleic acid level with Hendra virus. However, it is clearly different from other paramyxovirus genera, so a new genus is proposed [193,193a]. Recently a cytopathic infectious agent was isolated from the kidneys of an apparently healthy tree shrew (Tupaia belangen) that had been captured in the area of Bangkok. The virus turned out to be a paramyxovirus termed tupaia paramyxovirus (TPMV), and partial sequences of its genome (more than 4000 nucleotides) showed that this virus had the highest homologies with Hendra virus [194]. This supports the hypothesis that new human morbilliviruses are likely to be derived from animal reservoirs. 2.15

Human Metapneumovirus (hMPV)

Metapneumovirus is a second genus of the subfamily Pneumovirinae of the Paramyxoviridae family. Until recently it was considered to infect only birds, e.g., the turkey rhinotracheitis virus (TRTV). In June 2001 a report was published of novel viral isolates obtained from acute respiratory tract infections in children in the Netherlands during the winter [195]. The genomes of these viruses termed hMPV had 60–80% sequence homology with genes of TRTV (not all genes of hMPV have been identiﬁed yet). Preliminary serological data suggest that by the age of 5 years, >70% of children had been infected with this virus; practically 100% of adults are seropositive [195]. Preliminary data from England of clinical pediatric respiratory samples that had been negative for other known viruses yielded 10% positivity for incidence of infections with this virus and the virus detected in the respiratory tract of immunocompro-

Emerging and Reemerging Viral Pathogens

17

mised adults (P. Cane, personal communication), and the virus has now also been found in Australia [195a] and Canada [195b]. 2.16

Enterovirus 71 and Aichivirus (Picornaviridae)

Enterovirus 71 (EV71), one of the major causative agents of hand, foot, and mouth disease (HFMD), is also sometimes associated with severe central nervous system disease. HFMD epidemics were recorded in Malaysia and Japan in 1997 and in Taiwan in 1998. They resulted in sudden death among young children, often from encephalitis, and were mainly due to the A-2 B genotypes of this virus [196]. Central nervous system complications were observed in previous HFMD outbreaks [197– 199]. By contrast, in large HFMD epidemics in Japan in 1973 and 1978, there was hardly any CNS involvement [200]. In 1989, an outbreak of gastroenteritis occurred in the Aichi prefecture, Japan, for which a new enterovirus, termed Aichi virus, was found to be the causative agent [201,202]. This virus was recently deﬁned as a new genus (Aichivirus) of the Picornaviridae family [143,203]. The virus is also found to cause sporadic cases of diarrhea, mainly in travelers in Southeast Asia [202,204]. 2.17

West Nile Fever Virus (Flaviviridae)

West Nile (WN) virus has long been known for its wide host spectrum (including mammalian, avian, amphibian, and insect species) and was found to infect humans in many parts of Africa, eastern Europe, and Asia [205]. It emerged in the western hemisphere in 1999 when it infected 59 mainly elderly people in the New York City area; those infected developed fever, headache, rashes, myositis, polyneuropathy, meningitis, or meningo-encephalitis, and seven died [206,207]. The virus was also isolated from sick crows and other birds and mosquitoes [208] and found to be closely related genetically to a Middle Eastern virus isolate [206]. Despite extensive efforts to eliminate the virus by vector control, it reappeared in 2000 in New York City and New Jersey [209] and spread into the bird population of the east coast of the United States. Molecular analyses of a number of WN virus isolates support the model of migrating birds as hosts that spread the virus to local mosquito populations along their migrating routes; the spread into North America may also have been due to an infected traveler [205]. Physicians in the eastern United States have now been asked to consider WN virus infection in their differential diagnosis in hospitalized patients with encephalitis (particularly when occurring in conjunction with muscle weakness) and in adults with viral meningitis [207]. By 2002, WN virus

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had spread in animals and humans throughout the midwestern United States and California [207a].

2.18

Cantalago Virus (Orthopoxviridae)

A virus isolated from vesicular lesions of cattle and milkers in Cantalago County, Rio de Janeiro State, Brazil, turned out to be an orthopoxvirus and was genetically most closely related to the vaccinia virus strain (VVIOC) used for over 20 years in Brazil. The data suggest that Cantalago virus derived from VV-IOC after long-term persistence in an as yet unidentiﬁed animal reservoir [210].

2.19

Borna Disease Virus (Bornaviridae)

Borna disease virus (BDV) has for a long time been recognized as the causative agent of chronic neurodegenerative disease in horses, sheep, cattle, and other vertebrates. It also replicates in experimentally infected rats. The virus is the prototype of the Bornaviridae family in the order Mononegavirales [211]. After infection in the periphery (olfactory epithelium), the virus is transported intra-axonally toward the CNS, which it infects, with a preference for the limbic system [212]. The disease, a nonpurulent encephalomyelitis, develops by a T-cell-dependent immune mechanism and is characterized clinically by movement abnormalities and by hyperactive and abnormal behavior followed by apathy, somnolence, and depression. In the neonatal rat a chronic persistent infection without immune response and without clinical signs develops (persistent tolerant infection of the newborn). These different features of disease in animals have led to the development of animal models for neuropsychiatric disorders [213]. The virus came to wide attention around 1995 when antibody to the virus was found in the sera of patients with mood disorders [214]. The advent of RT-PCR led to reports of human BDV isolates being obtained from blood and tissues of psychiatric patients [215,216], but these data require broader conﬁrmation [217,218]. A recent review [217] concluded that ‘‘a critical evaluation indicates that no laboratory has to date been able to present solid evidence that BDV is infecting humans.’’ The validity of testing for BDV antibody in human sera has recently been questioned [219], but highavidity antibodies against BDV-speciﬁc peptides have been found in human sera [219a].

Emerging and Reemerging Viral Pathogens

TABLE 2

19

Zoonotic Origin of Emerging Viral Pathogens in Human Disease

Virus (genus or type species)

Human disease

Animal source

Probabilitya

Calicivirus Rotavirus Ebola virus Sin nombre virus HIV HEV Inﬂuenza virus

Diarrhea Diarrhea Hemorrhagic fever HFRS, HPS

Swine, cattle Swine, cattle Monkeys Rodents

L L P C

AIDS Hepatitis Inﬂuenza

Hendravirus

Meningoencephalitis

Monkeys Swine Pigs Horses Birds Fruit bat

L/C P reassortants L reassortants L C L

Nipahvirus

Encephalitis

Metapneumovirus

Respiratory tract infection Encephalitis Vesicular rash Encephalomyelitis

Tree shrew Pigs Dogs Birds

L L P ??P

Birds Cattle Horses

C C ??P

Cattle

P

West Nile virus Cantalagovirus Borna disease virus BSE agentb a b

nvCJD

P, possible; L, likely; C, conﬁrmed. See Table 1, footnote b.

2.20

Transmissible Bovine Spongiform Encephalopathy and New Variant Creutzfeldt-Jakob Disease (nv-CJD)

During the mid-1980s, a rapid spread of bovine spongiform encephalopathy (BSE) was observed in British cattle herds. The likely origin of this infection was the scrapie agent, one of the prion agents [220], originating from meat and bone meal used for cattle feed in 1981–1982 when a previously established processing step of extraction with solvents and hot steam was omitted. It was then calculated that an incubation period of 4–5 years had to be taken into account, and it was hypothesized that 5 years would have to pass after a ruminant protein ban was set in force in July 1988 before the epidemic would peak. The peak of the epidemic occurred in 1994. During the epidemic, BSE cases within a herd remained constant at approximately 4%, but over the years more herds became

SRSVs (NLVs, SLVs) Rotavirus Astrovirus Parvovirus B19 Ebola virus Hantavirus HTLV-1 HIV HHV6/7 HCV HEV SNV HHV-8 Hendra virus Nipahvirus Inﬂuenza virus WNV Metapneumovirus Alchi virus X

X

X

X

X

X X

Genetic changes/ evolution of viruses

X X X X

X

X

X

Globalization: industry, trade, travel

X

X X

Changed behavior: IVDU, etc

X?

X

X

X X

Changes in environment: deforestation, urbanization, industrialization

X?

X?

X

X?

X X X

Contaminated food or water

X

Civil unrest, wars, refugee camps

X?

X?

X

Anti-microbial resistance

X

X X

X

X

Immunosuppression

X X X X X X X X X X X

X X

X X X

Improved surveillance: clinical, diagnostic, epidemiological

Factors Contributing to Emergence of Viral Pathogens in Humans (Other Than Animal Reservoir)

Emerging viruses

TABLE 3

20 Desselberger

Emerging and Reemerging Viral Pathogens

21

infected. The reason for this ﬁnding was likely the continuation of cattleto-cattle recycling for food purposes, which may have gone on for some time after the initiation of the ruminant protein ban. In early 1996 several cases of a rapidly progressive form of Creutzfeldt-Jacob disease (CJD) in humans were described that also seemed to differ pathologically from previously identiﬁed forms [220,221]. Some molecular data point to the possibility that the causative agent of this so-called new variant CJD (nvCJD) may be the BSE agent [222,223]. However, the epidemiology of nvCJD has so far not conﬁrmed this hypothesis [224,225]. By 21 March 2002, there were 109 conﬁrmed cases of nvCJD counted and seven awaiting conﬁrmation [226]. The predictions of how many cases may become apparent over the next 3–5 years vary grossly [227]. One major unknown in this calculation is the uncertainty about the variability of the incubation period. There has been enormous progress in recognizing the nature of the transmissible agent and the molecular genetics of the prion–host relationship but major riddles remain [224,227a].

3

ANIMAL RESERVOIRS FOR EMERGING VIRUSES

Long before 1972, animal reservoirs for human viral infection were a well-known fact (e.g., rabiesvirus, herpesvirus B, inﬂuenza viruses, bunyaviruses, ﬂaviviruses). It is remarkable that many of the new emerging viruses do or may originate from an animal reservoir. In Table 2 an attempt is made to assess the signiﬁcance of animal reservoirs for human infection by emerging viruses. Although in detail some of the considered possible links may not stand up to scrutiny, the concept is compelling and should lead to close surveillance of animal as well as human populations for emerging diseases and their causes.

4

SIGNIFICANCE OF OTHER FACTORS INVOLVED IN VIRAL EMERGENCE AND REEMERGENCE

Besides animal reservoirs, numerous other factors mentioned in the Introduction contribute to the emergence and or reemergence of viral (and other) infections. Table 3 is an attempt to allocate certain factors to the viral infections reviewed in the preceding sections. The evolution of viruses, forces driving globalization, changes in the environment, food contamination, and immunosuppression are recognized as major factors. It is very clear that improved tools of surveillance have signiﬁcantly helped to recognize emerging infections early.

22

Desselberger

The issue of emerging, antiviral drug-resistant virus mutants is already of major concern for the highly active antiretroviral therapy (HAART) of HIV infection [112–115]; is well described for herpes simplex viruses, cytomegalovirus, inﬂuenza viruses, and others; and is likely to become a very signiﬁcant factor in virus evolution as increasing numbers of antiviral drugs are being developed and applied. In ﬁghting emerging infections, the balance of treatment with antiviral agents, immunization procedures, and exposure prophylaxis will have to be constantly reviewed and redeﬁned. More detailed considerations on this topic will be found in other parts of this book. 5

EMERGING NONVIRAL PATHOGENS

Besides viruses, many other microorganisms (bacteria, fungi, parasites) have emerged as human pathogens. These have been reviewed elsewhere [e.g., refs. 1,6,11,15]. 6

CONCLUSIONS

Increases in population sizes, global travel, and changes in the ecology all contribute to the assumption that more new infectious diseases will arise in the future. It is likely that there are more viruses around causing hepatitis than are recognized at present, and the number of human retroviruses (including endogenous retroviruses) is likely to be underrecognized. Clinical attentiveness; good laboratory facilities, including the application of molecular identiﬁcation techniques; and comprehensive epidemiological surveillance systems for infections in both humans and animals, which often form a reservoir for human infections, have to be combined for early recognition of emerging infections. Lack of facilities can have adverse consequences and can lead to misdiagnoses. Finally, economic enablement in the public health sector will be necessary to allow early recognition and comprehensive management of emerging infections. Given the record of emerging and reemerging microorganisms as the cause of infectious diseases over the last three decades there is every prospect of this continuing for some time, and high vigilance in detecting them is of paramount importance. Acknowledgments The author gratefully acknowledges the support of Lynne Bastow and Narguesse Stevens, who typed and processed the manuscript, and the

Emerging and Reemerging Viral Pathogens

23

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2 Inﬂuenza: The Virus, the Disease, and Its Control Thorsten Wolff Robert Koch-Institut, Berlin, Germany

Rene´ Snacken Scientiﬁc Institute of Public Health, Brussels, Belgium

1

INTRODUCTION

Inﬂuenza is a highly contagious acute respiratory disease that has global signiﬁcance because it affects all age groups and can recur in any individual. The etiologic agent of the disease, inﬂuenza virus, was ﬁrst isolated in 1933 [180] and has served since then as a paradigm of an important viral pathogen. Thus, fundamental principles such as antigenic drift and shift have been recognized with inﬂuenza viruses. Molecular analyses have revealed that the unique potential of inﬂuenza viruses to cause epidemics annually and pandemics occasionally is based on an amazing variability of its segmented negative-strand RNA genome. This is reﬂected in the existence of several antigenically distinct subtypes, a wide host range that comprises a variety of mammalian and avian species, and the propensity to escape from selective conditions such as neutralizing antibodies or antiviral drugs by rapid mutation. Thus, inﬂuenza viruses have in the past equally concerned basic life 39

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scientists, clinicians, public health authorities, and pharmaceutical companies. Undoubtedly this will continue in the future, because inﬂuenza viruses have a large natural host reservoir in feral birds that provide an ineradicable pool for viral genes that may emerge in future novel pandemic virus strains. Here we discuss current knowledge and recent developments in the different ﬁelds of molecular biology, antiviral therapy, and immunoprophylaxis that together promise improved understanding, control, and management of inﬂuenza in the future.

2

THE BURDEN OF INFLUENZA

Inﬂuenza epidemics and pandemics impose a considerable socioeconomic burden on individuals and society, which is due to morbidity and mortality and direct medical costs as well as to indirect economic losses related to work absenteeism and decrease in productivity [62,108,187]. Whereas uncomplicated inﬂuenza is a self-limited disease, severe respiratory or systemic complications may develop that require in-patient medical attention (see Sec. 6). A recent analysis reported an average of 49 pneumonia and inﬂuenza-associated hospitalizations per 100,000 persons among all age groups during the years 1970–1995 in the United States (average number of annual inﬂuenza-associated hospitalizations: 114,000) [178]. The relative risk for hospitalization is highest among the very young (birth to 1 year) and the elderly (>65 years) [18]. Inﬂuenza and pneumonia together were ranked the seventh most frequent cause of all deaths in the United States in 1999 [2]. However, precise quantiﬁcation of the impact of inﬂuenza on mortality is difﬁcult, because the infection is not routinely conﬁrmed by laboratory diagnostics and because impact estimates are not transposable from country to country. Inﬂuenzaassociated mortality is usually expressed as the number of deaths during seasonal virus circulation that exceeds a projected baseline level of expected deaths that occur in the absence of inﬂuenza. The available U.S. data indicate that between 1972 and 1992 inﬂuenza was responsible on average for 21,300 fatalities per year, with great variation among seasons (range: 0–46,200 deaths) [177]. In interpandemic years, more than 90% of all inﬂuenza victims were older than 64 years, indicating a distinct need for improvement of immunoprophylaxis in this age group [18]. In total, interpandemic inﬂuenza years accounted for many more deaths than pandemic-associated fatalities. Immunization and the use of antiviral agents for prophylaxis and treatment can only reduce the impact of the disease; vaccine and antiviral agents are not exclusive but complementary for controlling the disease.

Inﬂuenza

3

41

INFLUENZA VIRUSES—STRUCTURAL AND MOLECULAR PROPERTIES

Inﬂuenza viruses are divided into types A, B, and C on the basis of antigenic differences of their matrix- and nucleoproteins. Inﬂuenza viruses are systematically speciﬁed by indication of the particular virus type (A, B, or C), the species, the geographic site from which the virus was isolated, a strain number, and the year of isolation, as exempliﬁed in A/Swine/Iowa/15/30. For human strains, indication of the host species is omitted as in A/HK/1/68. The inﬂuenza A viruses are epidemiologically most relevant and are considered as the prototype of the Orthomyxoviridae. These viruses not only infect and replicate in humans but have also been isolated from pigs, horses, minks, and marine mammals as well as from domestic and wild aquatic birds. Only type A inﬂuenza viruses are further categorized into subtypes that reﬂect antigenic differences in their two major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Currently, we know of 15 different HA subtypes (numbered H1–H15) and nine NA subtypes (N1– N9), which have all been found in viruses isolated from feral birds. Human inﬂuenza A virus strains that have caused epidemics and pandemics in the last century belonged to the groups H1N1, H2H2, and H3N2. However, future epidemic strains may carry other HA subtypes as suggested by the recent sporadic occurrences of human infections by H5N1 and H9N2 viruses [26,102]. Inﬂuenza B viruses can cause similar severe disease symptoms; however, the host spectrum of inﬂuenza B viruses is restricted mainly to humans, which lowers their capacity for genetic alterations. This chapter focuses on type A and B inﬂuenza viruses, because inﬂuenza C viruses are less prone to antigenic changes and are clinically less relevant. In the laboratory, inﬂuenza viruses can be propagated in embryonated chicken eggs or a variety of standard tissue culture cell lines such as Madin-Darby canine kidney (MDCK). Virus titers are usually quantiﬁed either by plaque assays on tissue culture cells or by hemagglutination of erythrocytes [6]. Accordingly, virus titers are given either in plaque-forming units (PFUs) or in hemagglutination units (HAUs). Moreover, hemagglutination of erythrocytes by inﬂuenza viruses can be inhibited by HA-speciﬁc antibodies in immune sera. Thus, titers of antisera can be expressed as their activity in hemagglutination inhibition (HI) [221]. Inﬂuenza A and B viruses are characterized by an outer membrane envelope and a genome that consists of eight single-stranded RNA segments of negative polarity (complementary to mRNA sense). The

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FIGURE 1 Structure of inﬂuenza viruses. (A) Electron micrograph of inﬂuenza B viruses (magniﬁcation 6140,000). (Courtesy of Hans Gelderblom, RKI Berlin.) (B) Schematic drawing of a virion particle and of viral structural proteins.

diameter of spherical virion particles (Fig. 1) is in the range of 80–120 nm. The genomic viral RNAs (vRNAs) are replicated in the nucleus of the infected cell via synthesis of positive-strand copy RNA (cRNA) intermediates that serve as template for the production of new vRNA molecules [99,107]. The viral RNA segments are between 2.4 and 0.9 kilobase (kb) in size, adding up to a total of about 13.6 kb (type A) and 14.6 kb (type B), respectively (Table 1). The coding capacities of these viruses comprise 11 known proteins each, which is relatively few in comparison to large DNA viruses such as herpes- or poxviruses, which encode more than 300 viral gene products. The vRNA segments carry short stretches of conserved nucleotides (nts) at their 50 (type A: 13 nts; type B: 11 nts) and 30 ends (type A: 12 nts; type B: 9 nts), respectively. These sequences are in part complementary, and thus the ends of viral RNAs can engage in base-pairing interactions resulting in a partially double-stranded promoter structure that is recognized by the viral RNA polymerase. More than half of the genomic information is dedicated to the four replicative proteins PA, PB1, PB2, and NP [86]. The former three proteins assemble into a trimeric RNA-dependent RNA polymerase that provides the enzymatic functions for replication and transcription. The nucleoprotein NP and the polymerase encapsidate the genomic RNA, forming a viral ribonucleoprotein (vRNP). Within the virion, the vRNPs are embedded into a layer of the viral M1 matrix protein that is associated with a few copies of the viral NS2/NEP protein, a putative viral RNA export factor. The viral envelope contains three viral transmembrane proteins: the trimeric hemagglutinin (HA), the tetra-

Inﬂuenza

TABLE 1

43 Inﬂuenza A and B Viral Genomesa A/PR/8/34 Encoded protein (AA)

B/Lee/40

vRNA (nts)

Encoded protein (AA)

Segment

vRNA (nts)

1

2341

PB2, 759

2396

PB2, 769

2

2341

PB1, 757

2368

PB1, 752

PB1-F1, 87

3

2233

PA, 716

2304

PA, 726

4

1778

HA, 566

1882

HA, 584

5

1565

NP, 498

1841

NP, 560

6

1413

NA, 454

1557

7

1027

M1, 252 M2, 97

1191

NA, 466 NB, 100 M1, 248

8

890

NS1, 230

1096

BM2, 109 NS1, 248

NS2/NEP, 121

NS2/NEP, 122

Functions(s) Subunit of viral RNA polymerase; capbinding Catalytic subunit of viral RNA polymerase Mitochondrial localization; induction of apoptosis Subunit of viral RNA polymerase Surface glycoprotein; receptor binding, membrane fusion Nucleoprotein; encapsidation of viral genomic and antigenomic RNA Neuraminidase Putative ion channel Matrix protein Ion channel; acidiﬁcation of virions, protecting HA conformation Structural protein Post-transcriptional regulator: inhibition of pre-mRNA splicing, polyadenylation and PKR activation Nuclear export factor

a The lengths of the eight viral RNA segments and the encoded polypeptides of the inﬂuenza A/PR8/34 and B/Lee/40 viruses are given in nucleotides (nts) and amino acids (AA), respectively. The functions of the gene products are indicated in the rightmost column.

meric neuraminidase (NA), and the M2 ion channel (type A) or NB protein (type B). Inﬂuenza viruses express one major nonstructural polypeptide in infected cells that is designated NS1. The NS1 protein has several regulatory functions at the post-transcriptional level, as it inhibits host

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cell RNA processing and transport [25,143,214]. Furthermore, the NS1 protein antagonizes activation of unspeciﬁc cellular defense mechanisms by its ability to bind to double-stranded (ds) RNA that can be generated during replication of the negative-sense vRNA through the plus-sense cRNA intermediates [70,106]. This property prevents both the activation of the dsRNA-activated protein kinase (PKR), which downregulates cellular translation, and activation of the transcriptional regulators NF-Kb, ATF-2/c-Jun, and IRF-3, which induce expression of type I interferons (IFNs) [55]. IFNs are secreted from infected cells and induce in neighboring cells the expression of gene products that establish an antiviral status [183]. Inﬂuenza viruses apparently counteract activation of this defense mechanism through NS1-mediated masking of dsRNA. Consequently, an inﬂuenza virus with a genetic knockout of the NS1 gene was completely avirulent in animal studies and replicated only in interferon-deﬁcient hosts to considerable titers [56]. There are two viral proteins that are uniquely found in either type A or type B inﬂuenza viruses. A very recent discovery is the expression by inﬂuenza A viruses of the 87 amino acid PB1-F2 protein that is transported into mitochondria and has the ability to induce apoptosis [24]. The capability for PB1-F2 expression is not conserved in all virus strains and has been shown to be dispensable for efﬁcient viral replication in tissue culture cells. It remains to be determined if and how the PB1-F2 protein contributes to viral virulence and/or pathogenesis. Only inﬂuenza B viruses express the BM2 polypeptide that is incorporated into the virion, but its function has not been recognized yet [141]. The main target tissue of human inﬂuenza viruses is the epithelial cell layer that lines the respiratory tract. The viruses initiate infection by binding of the HA to sialic acids attached to cellular surface sialoglycoproteins or sialoglycolipids followed by internalization of the virus through receptor-mediated endocytosis [114] (Fig. 2). Two events that are crucial for the establishment of the infection occur in the low-pH environment (pH 5–6) of the endosome. First, the ion channel activity of the M2 protein facilitates inﬂux of protons into the interior of the virion, which destabilizes the tight association of the viral vRNPs with the M1 protein [83,154]. It is the acidiﬁcation through the M2 ion channel that is blocked by amantadine and rimantadine, the ﬁrst discovered class of compounds with anti-inﬂuenza virus activity (see Sec. 7). Second, the HA undergoes a major structural rearrangement leading to the exposure of a short a-helical hydrophobic domain that initiates the fusion of viral and endosomal membranes [179].

Inﬂuenza

45

FIGURE 2 Replication cycle of inﬂuenza viruses in an infected cell. See text for a description of the steps involved. The virus and the cellular structures are not drawn to scale.

After fusion, the vRNPs are released into the cytoplasm and are transported to the nucleus, where they serve as templates for transcription of viral mRNA transcripts. Synthesis of viral mRNAs is primed by capped oligonucleotides of 10–13 bases that the polymerase acquires by nucleolytic cleavage from the 50 ends of cellular mRNAs [156]. Polyadenylation at the 30 end occurs through a ‘‘stuttering’’ mechanism at an oligo-U signal in the vRNA template [109]. Mature viral transcripts are exported to the cytoplasm, where they are translated. Inﬂuenza viruses efﬁciently downregulate cellular protein synthesis, whereas translation of viral transcripts is maintained at high levels [57]. Replication of the negative-strand RNA genome through synthesis of positive-strand cRNA intermediates is a primer-independent process and continues for several hours [174]. Newly replicated vRNPs are exported to the cytoplasm in the late stage of infection, which is presumably mediated through the activity of the M1-NS2/NEP complex [140]. Recent analysis suggests that this step requires activation of the intracellular Raf/MEK/ERK signaling cascade [155]. Subsequently, the

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vRNPs assemble with other viral structural proteins at the plasma membrane into new virions that are released to the exterior by budding. The activity of the viral neuraminidase in removing sialic acids from cellular and viral glycoproteins is critical to prevent retention and aggregation of virions at the plasma membrane [151]. Therefore, the recently developed neuraminidase inhibitor substances inhibit the release of progeny viruses from the cell. A prerequisite for the initiation of a new replication cycle is proteolytic cleavage activation of the viral HA by secreted proteases. For most human inﬂuenza virus strains, cleavage of the HA precursor occurs at a single arginine residue, which generates the HA1 and HA2 subunits [97]. This is an essential step, because only cleaved HA molecules can undergo the pH-dependent structural rearrangement in the endosome that leads to membrane fusion and release of vRNPs (reviewed in Ref. 179). Only a few cellular proteases, including plasmin and tryptase clara, have been identiﬁed that function in HA cleavage activation in humans or animals [94]. In viral infections of tissue culture cells, trypsin is added as an adequate enzyme. Depending on the cell type and the strain, inﬂuenza virus infections normally result in lysis and death of the cell. Inﬂuenza viruses became amenable to targeted genetic alteration more than a decade ago [44]. However, efﬁcient procedures that allow rapid and systematic reverse genetic analysis through de novo generation of recombinant inﬂuenza A viruses from transfected cloned cDNA were established only recently [50,132]. In essence, tissue culture cells are cotransfected with four plasmids expressing the viral replicative proteins and eight plasmids, each of which encodes a complete viral gene segment in an RNA polymerase I expression cassette. The viral RNAs are transcribed within the cell and packaged into ribonucleoprotein by the viral NP and polymerase. This, in turn, allows expression of all viral gene products and the assembly of new infectious virus particles. It is expected that this methodical breakthrough will greatly stimulate and accelerate basic research on virus pathogenicity and host cell tropism. Moreover, these systems should also have great potential in the development of novel tailormade inﬂuenza vaccines and expression vectors.

4 4.1

EPIDEMIOLOGY, EVOLUTION, AND HOST RESERVOIRS OF INFLUENZA VIRUSES Epidemiology

Inﬂuenza A and B viral infections are a signiﬁcant cause of morbidity and mortality, because they affect all age groups and, in contrast to many

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47

other pathogens, can occur repeatedly in all persons. In epidemic years attack rates of inﬂuenza can reach 10–20% of the total population [35,61]. In this section we discuss epidemiological and molecular data that are relevant for the evolution, spread, and survival of these viruses in the population. Human epidemics in the last century were caused by three different HA subtypes of inﬂuenza A viruses that circulated during different decades. Thus, the epidemiology of these viruses is characterized by the predominance of one or two subtypes that can last for decades. The introduction of a new subtype that succeeds in establishing an independent evolutionary lineage is usually associated with a pandemic and often results in the extinction of the previous predominant strain. The H1N1 viruses probably appeared shortly before the 1918 pandemic and circulated until 1956 [190]. In 1957, the H1N1 subtypes were replaced by the H2N2 Asian pandemic viruses that had acquired the HA, NA, and PB1 segments from avian strains while maintaining the ﬁve other segments [90,173]. In 1968, these strains were replaced by viruses of the H3N2 subtype, which contained novel HA and PB1 genes derived from duck strains [46,90]. The year 1977 witnessed the reappearance of H1N1 viruses that were almost identical to strains that circulated shortly before the 1957 pandemic and thus appear to have been maintained in a frozen state for 20 years. H1N1 and H3N2 strains have cocirculated since that time. In countries with a temperate climate, inﬂuenza epidemics are observed almost exclusively in the winter months (November to April and May through September in the northern and southern hemispheres, respectively). In tropical areas, the inﬂuenza activity is less linked to the season and the virus can be isolated during the whole year. Globally, inﬂuenza epidemics move from north to south across the globe, crossing the equator twice annually [85]. Inﬂuenza activity is divided into ﬁve levels—no activity, sporadic cases, local outbreak, regional activity, and widespread activity—according to the number of isolated viruses and the number of observed cases of ﬂulike syndromes in comparison with the baseline used by the international surveillance network [49]. The main epidemiological factors related to the host are age, prior immunity, and indoor crowding. Health status does not affect the attack rate but is the main factor that underlies the high morbidity and mortality related to inﬂuenza. In the elderly, cardiopulmonary complication rates increase with age up to 70% in persons over 70 years of age [14] (see also Sec. 6). Attack rates are higher in young people, whereas complications and mortality are higher in the elderly, although their incidence rates are lower. Healthy babies are also affected, with an inﬂuenza-related hospitalization rate of 104/10,000 in children less than

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6 months of age and 50/10,000 in children between 6 and 12 months [133]. These rates are comparable to those observed in adults with high risk conditions [134]. Herd immunity determines the severity of the incidence rates in the community: The higher the levels of circulating neutralizing antibodies, the lower the infection rate [104]. Crowding greatly inﬂuences the attack rate, and the most common factor is the institutionalization of individuals who are in poor condition. Nosocomial outbreaks of inﬂuenza are common, even in pediatric wards, with high variable attack rates of up to 47% of hospitalized persons [219]. Inﬂuenza outbreaks with very high attack rates were also observed in conﬁned healthy populations: 42% in a U.S. Navy ship despite an optimal vaccination rate [43], 49% in a ski school hotel [110], and 72% in an aircraft [130]. Surveillance is of particular importance for the early detection of inﬂuenza activity, the identiﬁcation of circulating strains, and the estimate of the impact of the outbreak. National surveillance networks often merge both virological and epidemiological data, simultaneously including the activity of the respiratory syncytial virus (RSV). This latter virus is essentially known for causing bronchiolitis in infants, but it is also responsible for respiratory infections that can mimic inﬂuenza with a comparable morbidity and mortality [45]. Information provided by surveillance networks* from the general population and hospitals gives additional arguments for clinical diagnosis. Public health authorities are also informed about the scope and the impact of the outbreak, and the World Health Organization (WHO) collects useful data for deciding the composition of vaccines for the next season. Timeliness of the latter information is essential, because the vaccine composition has to be declared by WHO in February in the northern hemisphere and in September in the southern hemisphere, leaving 6 months for the pharmaceutical companies to prepare the appropriate trivalent vaccine. 4.2

Evolution

A characteristic feature that distinguishes inﬂuenza viruses from other human respiratory viral pathogens is their enormous spectrum of genetic diversity and changeability. For instance, the HA protein sequences of different subtypes differ by up to 60% although they probably have very similar spatial structures [139]. Numerous serological and nucleic acid sequencing studies have shown that several mechanisms account for the * http://www.cdc.gov/nip/ﬂu/News.htm#Bulletin in the United States and http:// www.eiss.org in Europe.

Inﬂuenza

49

geno- and phenotypical ﬂexibility of inﬂuenza viruses. Antigenic drift is the most important principle during interpandemic periods by which inﬂuenza viruses escape increased immunity in the population toward an epidemic strain. This involves the appearance of inﬂuenza A or B virus variants with point mutations that can evade neutralization or clearance mediated by HA- and NA-speciﬁc antibodies generated after infection or immunization with their predecessor. Thus, individuals who have experienced infection with a given virus subtype can be reinfected by a drift variant. The ‘‘original antigenic sin’’ that gives a preferential response against prior infecting strains and may inhibit a speciﬁc response to the newly infecting virus might explain some poor reactivity, especially in the elderly. Structural analyses have shown that drift variants differ by only a few amino acids, which are conﬁned to ﬁve epitopes (A–E) within the globular head domain of the HA [212] and at least two epitopes on the NA [168]. A successful drift variant usually replaces the prevalent strain of the previous seasons and circulates on average for 2–5 years. Therefore, the composition of the inﬂuenza vaccines that contain components of recent inﬂuenza A/H3N2, A/H1N1, and B viruses is annually adjusted to ensure the best possible level of protection against circulating virus strains (see Sec. 8). On a molecular level, antigenic drift is facilitated by the relatively high error rate of the viral RNA polymerase, which appears to lack a proofreading function. This results in a mutation rate of about 1 6 10 5 base pairs per site per replication, which is in a range similar to those of other RNA viruses [184]. Thus, a given wild-type virus population usually contains at low frequency variants that may gain a replicative advantage under selective conditions such as the presence of neutralizing antibodies or antiviral substances. For the HA and NA genes, mutational rates of 6.7 6 10 3 and 2.6 6 10 3 substitutions per site per year, respectively, have been determined [48,218]. However, the genes of the internal viral proteins such as NP, M1, or NS1 that are believed not to underlie any immune surveillance also evolve, albeit at somehow slower rates [20,87,175]. A second important mechanism that contributes to genetic diversity among inﬂuenza viruses is the reassortment of viral gene segments. This is based on the capability of cells to produce progeny viruses that contain RNA segments from both parents after infection by two different virus strains [147]. It is important that when genes of the viral surface glycoproteins are involved, reassortment can lead to the emergence of viruses with novel subtypes of HA and/or NA proteins that are antigenically unrelated to strains that circulated previously among humans. Such a fundamental change in viral antigenicity that occurs in

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unpredictable intervals is termed antigenic shift. Shift variants such as the pandemic H2N2 and H3N2 viruses of the years 1957 and 1968 encounter a naive population that largely lacks immunological protection and is therefore highly susceptible to widespread infection. Thus, every human inﬂuenza virus strain that contains novel HA and/or NA subtypes must be considered a potentially dangerous pathogen. Apart from immunogenicity, reassortment can have further consequences that are unpredictable. For instance, upon crossing two virulent avian strains, both pathogenic and nonpathogenic reassortant viruses were recovered [167]. Thus, not only the presence of a speciﬁc segment (such as an HA containing a multibasic cleavage site) may be important for viral virulence, but also the constellation of reassorted genes. A third mechanism is the transfer in toto of a non-human strain into humans. This infection by an animal virus without prior reassortment has been rarely observed [27,91,165] but has to be considered as a pandemic threat because a reassortment with circulating strains could also occur in a human host. Although human-to-human transmission was not demonstrated in the A/H5N1 bird ﬂu infections in Hong Kong in 1997, mass slaughtering of domestic poultry prevented a possible reassortment with human inﬂuenza strains. Finally, there are few reports on viruses carrying genes with insertions of cellular or viral RNAs [13,92]. Thus, in contrast to some positive-strand RNA viruses such as polio virus, true recombination between RNA strands appears to play in general only a minor role in the evolution of inﬂuenza virus. However, recent phylogenetic analysis suggests that the HA of inﬂuenza viruses that circulated during the devastating Spanish inﬂuenza in 1918 may have originated from recombination between human and swine genes [59]. It has been proposed that the fusion of human and swine HA sequences granted the 1918 virus unique immunogenicity or tissue speciﬁcity that may have contributed to its extreme virulence. 4.3

Host Range of Inﬂuenza Viruses

As indicated above, inﬂuenza A viruses are not restricted to humans but have been isolated from a broad range of hosts including pigs, horses, minks, seals, whales, and a variety of avian species [217]. For inﬂuenza B and C viruses, humans appear to be the major host, although recently type B viruses were also isolated from diseased seals [144] and inﬂuenza C viruses have been found in pigs. All inﬂuenza A viruses are believed to have originated from the avian reservoir [88]. This hypothesis is supported by the ﬁndings that all 15 HA subtypes and nine NA subtypes currently known exist in avian inﬂuenza viruses that circulate in feral

Inﬂuenza

51

and domestic birds. Furthermore, phylogenetic analyses suggest that avian viruses are close to evolutionary stasis, because their proteins are highly conserved. In their natural hosts the viruses multiply predominantly in the cells lining the intestinal tract, from which they can be excreted in large quantities. From this reservoir, the viruses can enter, directly or after adaptation, other host populations in which they may establish independent evolutionary lineages. However, crossing of the interspecies barrier followed by continued circulation in the new hosts has been rarely observed. Avian inﬂuenza viruses replicate only poorly in humans [9]. One important factor in this species restriction appears to be the strong preference of avian inﬂuenza viruses for a2,3-linked sialic acid receptor determinants [117]. Such glycoconjugates are abundant in avian target cells but seem to be largely absent from epithelial cells in the human respiratory tract [7,32,89]. Instead, these cells carry a2,6-linked neuraminic acids that are recognized by human inﬂuenza viruses [7,33,164]. The basis for preferential binding to one or the other type of receptor is not a matter of a speciﬁc HA serotype but rather depends on the presence of distinct amino acids in the receptor binding site of the HA [117]. Indeed, reassortment of avian HA genes into virulent mammalian viruses was associated with alterations of the avian consensus sequence at amino acid positions 190, 225, and 226, resulting in increased binding to a2,6-linked sialic acids [115]. However, the recent occurrence of severe human infections by avian H5N1 viruses whose HA did not carry such adaptations suggests that such interspecies transmissions can be successful in spite of inappropriate receptor speciﬁcity [116]. Thus, additional factors may also deﬁne the host range of inﬂuenza virus strains. For instance, several internal viral genes including the NP, matrix, NS, and polymerase segments have been suggested to restrict the replication of avian viruses in monkeys and humans, although the precise mechanisms are not known yet [182,194,200]. How can the emergence of avian–human reassortant viruses be envisioned when humans are less susceptible to infection with avian strains and human viruses do not spread in birds? Although unlikely, it cannot be excluded that such reassortants are directly generated in either host. However, several ﬁndings suggest that reassortment of genes from avian and human viruses may more favorably occur in swine as an intermediate host that facilitates the adaptation of avian virus genes to a mammalian environment [170]. Pigs are relatively susceptible to infection with both avian and human virus strains [93] and contain both a2,3-and a2,6-linked sialic acid receptor determinants in their trachea cells [89]. Furthermore, in rural areas pigs are abundant hosts

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that can come into close contact with humans and birds, which would be a prerequisite for the acquisition of progenitor strains. In fact, H3N2 inﬂuenza viruses containing segments of avian and human strains have been shown to circulate in pigs [23] and have also been isolated from diseased children [27]. This demonstrates that avian–human reassortant viruses can be transmitted from the porcine population to humans and strengthens the suspicion that pigs may play an important role in the generation of novel virulent strains [172]. 5

TRANSMISSION AND PATHOGENESIS OF HUMAN INFLUENZA

Inﬂuenza viruses are most likely perpetuated by continuous human-tohuman transmissions, because there is no evidence for persistent infections in immunocompetent individuals. The viruses are usually disseminated in small droplets (<5 mm in diameter) that are expelled through sneezes and coughs. Although inﬂuenza virus infections can remain asymptomatic, the disease signs of ‘‘the ﬂu’’ usually occur within 1–3 days after infection. Depending on the immune status and age of the individual and the viral strain, symptoms may range from very mild respiratory complaints to severe systemic life-threatening disease. Infections by type A and B inﬂuenza viruses cannot be clinically distinguished, which is an important consideration if antiviral therapy is initiated with amantadine or rimantadine, which are inactive against inﬂuenza B. Furthermore, none of the acute inﬂuenza-associated symptoms are pathognomonic, because several other viral or bacterial pathogens can induce similar disease (see Sec. 6). The onset of disease signs correlates with virus shedding into nasopharyngeal ﬂuids and the secretion of proinﬂammatory cytokines, including type I interferons and interleukin-6. In experimentally infected adult volunteers, virus titers peaked after 2 days at 103–107 TCID50 and diminished during the following 4–6 days [76,131]. However, virus shedding may be prolonged in children and in immunocompromised individuals [51]. Replication of human inﬂuenza viruses is usually conﬁned to the superﬁcial cells of the respiratory tract. It is believed that initially cells of the tracheobronchial epithelium are infected. Subsequently, the infection can spread into the lower respiratory tract, resulting in partial or complete destruction of the ciliated and mucusproducing epithelial cells [84]. The loss of the ciliated epithelium may sensitize infected individuals for secondary bacterial infections that are a frequent complication of inﬂuenza (see Sec. 6). Viral budding occurs almost exclusively from the apical side of the polarized epithelium,

Inﬂuenza

53

which may restrict infection to the luminal side of the respiratory tract and prevent viral spread to other organs [17,53]. Not surprisingly, the magnitude of viral replication parallels the severity of fever and respiratory symptoms [76,131]. Whereas in uncomplicated inﬂuenza the median duration of fever is 3 days, it may take 8–10 days for the resolution of other disease signs. Remarkably, very little is known about singular factors that determine the virulence of human pandemic and epidemic strains. As discussed in Section 10, human strains with novel HA and NA subtypes can cause inﬂuenza pandemics. However, even in the absence of antigenic shift there appears to be a correlation of the prevalence of particular virus subtypes (or strains) and the severity of epidemics. For instance, a recent analysis of the 1972–1992 period found that inﬂuenzaassociated mortality and morbidity rates were on average threefold higher in years during which H3N2 viruses were the predominant subtype than in seasons that were dominated by inﬂuenza A/H1N1 or B viruses [177]. It is not known if this difference in virulence is due to a more successful immune evasion by the former strains, differences in transmission rates, or mediation by other discernible virus-encoded components. Extensive studies of avian inﬂuenza viruses have demonstrated that the insertion of basic amino acids at the HA cleavage site allows the virus to spread and replicate in multiple organs, resulting in a fulminant systemic infection with lethality rates of up to 100% [166]. The cleavage of HA with a multibasic processing site does not depend on secreted proteases conﬁned to the intestinal or respiratory tract but is mediated by ubiquitous intracellular endoproteases such as furin [97]. As a consequence, the HA is transported to the cell surface in a preactivated form, and released virions are immediately infectious. In fact, the avian H5N1 viruses that in 1997 caused the death of 6 out of 18 infected humans carried such a type of HA [26,186]. Analyses of human H5N1 isolates in the mouse model showed that the presence of a furin recognition motif is an important factor for virulence and organ tropism in mammalian hosts [71]. It should be noted, however, that histopathological examinations of two fatal cases from the H5N1 outbreak gave no evidence for a systemic viral infection despite the presence of a furin recognition site in the HA [196]. Previously, it had been speculated that the vigorous virulence of the 1918 pandemic strain was due to a multibasic HA processing motif. However, this theory was recently disproved by the sequencing of ampliﬁed viral nucleic acids recovered from samples of 1918 inﬂuenza victims that were preserved by storage in the permafrost or by parafﬁn embedment [191]. Thus, although there is no evidence that further human strains expressing HA with a multibasic

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cleavage site exist or have existed, we should be aware of this possibility in the future. Another mechanism that enhances viral pathogenicity is mediated by speciﬁc inﬂuenza viral NA enzymes such as the one expressed by the A/WSN/33 strain that was generated by multiple passages in mice [63,100]. The WSN NA has acquired the ability to recruit the ubiquitous serum protease precursor plasminogen to cellular and viral membranes through loss of a glycosylation site at Asn130 and the presence of a carboxy-terminal lysine residue. As a consequence, HA can be immediately cleaved after activation of the protease. The sequestration of plasminogen allows the virus to grow in the absence of exogenous protease in vitro and to spread to and replicate in several organs of mice, including the brain. Although neither the inﬂuenza virus strain from 1918 nor any natural isolate have been found to express this type of NA [160], the possibility remains that protease recruitment may occur in future epidemic strains. In conclusion, there is currently no rationale for predicting the virulence of interpandemic inﬂuenza virus strains. Clearly, the disclosure of speciﬁc determinants that inﬂuence the severity of epidemics or pandemics remains an important challenge for future scientiﬁc work.

6 6.1

CLINICAL AND MICROBIOLOGICAL DIAGNOSIS OF INFLUENZA Clinical Diagnosis of Inﬂuenza and Its Complications

Inﬂuenza has a short incubation period, varying from 1 to 4 days. Uncomplicated inﬂuenza in nonvaccinated individuals is associated with both respiratory and systemic symptoms (reviewed in Ref. 14) that are characterized by a sudden onset of fever that often peaks within 24 h at 38–41 8C, dry cough in the absence of underlying lung disease, myalgia, and headache [36,127,136]. In clinical trials during epidemics, the use of a strict clinical deﬁnition of inﬂuenza had an accuracy rate of 63–75% [124]. Other frequent symptoms are chills, malaise, anorexia, sore throat, dizziness, nasal congestion, weakness, and vomiting. The frequency of particular complaints appears to vary with age. For instance, children seem to be more affected by gastrointestinal disturbances, whereas myalgia and sweats are more common in adults [127,136,216]. Findings during physical examination are poor, but fever, toxic facies associated with prostration, and injected eyes are often observed. Auscultatory and radiological ﬁndings are usually normal. Differential diagnosis of uncomplicated inﬂuenza infection is common cold, pharyngitis, and

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tracheobronchitis. During local inﬂuenza outbreaks or if the patient has had close contact with a conﬁrmed case, a presumptive diagnosis of inﬂuenza in adults aged less than 65 years with an acceptable positive predictive value can be made when the patient presents with sudden onset of fever (feverishness and chills) plus any one of the following sudden symptoms: cough, myalgia (aching, stiffness), or malaise (tiredness) [181]. Symptoms of inﬂuenza in children and infants are broader and less suggestive than in adults. In addition to commonly observed fever, key symptoms in children may be less important or even absent. The most frequent sign of inﬂuenza is the common cold, and in previously healthy children, otitis media is the most common cause of complication and may be the only sign of the infection. Aside from Reye’s syndrome, fever or upper respiratory tract infection followed by generalized convulsions must arouse suspicion for an inﬂuenzaassociated encephalopathy [52]. A reverse transcription (RT)-PCR for detecting inﬂuenza RNA performed on cerebrospinal ﬂuid will easily conﬁrm the diagnosis. Regional inﬂuenza outbreaks are always preceded by sporadic cases, essentially in infants and in children. Accurate clinical diagnosis is very difﬁcult during this period, and a microbiological conﬁrmation is always needed. Later, when the number of cases increases and an outbreak is declared, the wide variety of inﬂuenza symptoms requires virological diagnosis for neuraminidase inhibitors to be used appropriately (see Sec. 7). During epidemics, general practitioners are often overloaded, and there is no near-bed rapid test available. The abovementioned key symptoms increase the positive predictive value of a clinical diagnosis during epidemics, and their accuracy is sufﬁcient for the use of neuraminidase inhibitors as a presumptive treatment. This attitude has to be taken very cautiously, especially if there are other cocirculating pathogens such as RSV that can mimic inﬂuenza infection, particularly in young children and in the elderly. This is why clinicians should regularly look at the results of the national surveillance networks, e.g., on www.eiss.org or www.cdc.gov. Adults above 65 years of age, young children less than 4, immunocompromised persons, and individuals with preexisting chronic airway disease or heart disease are at increased risk for serious respiratory or systemic complications and death due to inﬂuenza (reviewed in Ref. 136). If there are no known underlying chronic conditions, it is of ﬁrst importance to determine whether such conditions exist when a ﬂulike syndrome occurs in order to prevent severe complications. During interpandemic periods, the most worrisome complication is pneumonia, which may result either from an early primary viral infection or from a

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subsequent secondary bacterial infection, which occurs predominantly in the elderly. Primary viral pneumonia is characterized by diffuse interstitial inﬁltrates and rapid progress after the appearance of inﬂuenza-like symptoms that may lead to hypoxemia and death within 1–5 days. Combined inﬂuenza virus and bacterial infection is three times as common a complication, with Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus inﬂuenzae being the most involved bacterial species [185]. In the 1957 and 1968 inﬂuenza pandemics, up to threefourths of patients with serious or fatal pneumonia suffered from secondary bacterial infection [103,105]. Biochemical and animal model studies have demonstrated that S. aureus secretes a protease that supports extracellular cleavage activation of the HA protein from human inﬂuenza viruses [169,189]. Because processing of the HA is a pivotal step of viral multiplication, the bacterial coinfection may enhance and/or accelerate virus-induced tissue damage in the lung. Other respiratory complications of inﬂuenza are acute bronchitis, bronchiolitis, and laryngotracheobronchitis (croup) among children and adolescents. Otitis media is the most common complication, but inﬂuenza virus infections can also induce Reye’s syndrome, a rare neurological and metabolic disorder that is associated with a progressive noninﬂammatory encephalopathy and fatty liver degeneration, in particular when patients are given salicylates for symptom relief. Finally, atypical courses of inﬂuenza have been observed during which patients have developed myocarditis, pericarditis, myositis, rhabdomyolysis, or encephalitis. 6.2

Microbiological Diagnosis

Microbiological diagnosis has three main objectives, namely, the identiﬁcation of the inﬂuenza virus for the clinician, the detection of the virus for the network of surveillance for an early warning system, and the identiﬁcation of the viral strain by the national inﬂuenza reference centers, which will provide essential data on circulating strains for the vaccine composition. Nasopharyngeal swabs or, better, washes have to be sent in a viral transport medium, possibly frozen, but might be posted by mail in winter conditions, i.e., at temperatures of less than 4 8C. Recovery of inﬂuenza viruses from diagnostic samples and their identiﬁcation are still performed in embryonated chick eggs, whereas cell culture with MDCK cells is increasingly used. Nevertheless, in egg culture, inﬂuenza isolates are less antigenically homogeneous than the same MDCK-grown isolates [162]. Results can be obtained in a matter of days with either method. Erythrocytes of various species, preferably turkeys, may be used for the detection of agglutinating viruses and

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titration of neutralizing serum antibodies. These methods are widely used for their reliability and the speed of the response (several hours). Subtyping of the strains is performed by a hemagglutination-inhibition technique with subtype-speciﬁc ferret antisera. Near-hand rapid diagnosis of inﬂuenza viruses by immunoﬂuorescence or enzyme immunosorbent assays are commercially available. These kits frequently use monoclonal antibodies that recognize the conserved viral NP or M1 protein and show a sensitivity ranging from 75% to 100% [221]. Radio- and ﬂuoroimmunoassay are also used for rapid detection of viral antigen with a sensitivity of 50–80% and a time to perform of less than 24 hr. Like commercial kits, these latter methods are highly dependent on the quality of the sample. Detection of viral RNA by RT-PCR provides essential information, even in archive material, on the type, subtype, and origin of the RNA segments. With appropriate primers, RT-PCR shows a higher sensitivity than viral culture techniques. Serological diagnosis has no place in clinical practice but is useful for public health retrospective purposes. 7.

AGENTS FOR TREATMENT AND ANTIVIRAL CHEMOPROPHYLAXIS OF INFLUENZA

7.1

Amantadine and Rimantadine

Amantadine (1-amino adamantane hydrochloride) and its derivative rimantadine (a-methyl-1-adamantane methylamine hydrochloride) were among the ﬁrst discovered antiviral substances [37,42,158]. Both have been proven effective in the treatment and prevention of inﬂuenza caused by type A, but not type B, viruses [34,198]. Until the recent development of the neuraminidase inhibitors, amantadine and rimantadine were the only licensed drugs for treatment of and prophylaxis against inﬂuenza virus infections. However, for several reasons, including their inactivity toward inﬂuenza B viruses and their potential to provoke adverse effects and to induce the development of viral drug resistance, the clinical use of adamantanamine compounds has been limited [34]. Nevertheless, these drugs are still recommended by the North American Advisory Committee on Immunization Practices (ACIP) as a cost-effective choice, particularly in inﬂuenza chemoprophylaxis. 7.1.1

Antiviral Mechanism

Amantadine is a colorless substance with an unusual symmetrical cagelike structure (Fig. 3). It inhibits the early stage of infection of most human inﬂuenza A viruses in tissue culture at micromolar concentra-

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FIGURE 3 Structural formulas of antiviral compounds that block the M2 ion channel proteins (A) or inhibit viral neuraminidase activity (B). For a comparison, the structure of N-acetylneuraminic acid (sialic acid) is shown on the left side.

tions that can also be achieved in vivo [72]. Both amantadine and rimantadine target the M2 proteins of all inﬂuenza A virus subtypes at an early step of infection. The 97. amino acid M2 polypeptide is a tetrameric type III integral membrane protein containing an 18-residue transmembrane domain that forms a pH-regulated proton channel in the viral membrane [99]. After internalization of the virus through endocytosis, the adamantane compounds are thought to block the endosomal ﬂow of protons into the interior of the virion by interacting with the M2 transmembrane domain [153,154]. As a consequence, uncoating of the virus in the cytoplasm is inhibited, resulting in a block of viral infection. At concentrations higher than 10 mg/mL, amantadine also has activity against a broader spectrum of RNA viruses, including parainﬂuenza virus, respiratory syncytial virus, and Dengue virus, in tissue culture [34]. However, these drug concentrations cannot be achieved in respiratory secretions or plasma, which rules out a clinical use of adamantane compounds to combat such infections. 7.1.2

Effectiveness

In many countries amantadine, but not rimantadine, has been approved for treatment of uncomplicated inﬂuenza A, although the latter has been

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found to have fewer side effects. Both substances can reduce the duration of fever and system symptoms by approximately 1 day when given within 2 days after onset of disease signs [40,69,193,198,201,213,223]. The clinical improvements correlate with a reduction of the viral titers in respiratory secretions, suggesting that these drugs function by reducing viral replication [69,79,193]. Amantadine and rimantadine also have prophylactic effectiveness in reducing inﬂuenza-associated morbidity and clinical symptoms [1,29,38,41,128,142,197]. A recent review of studies undertaken with healthy adults demonstrated an average effectiveness of 61% for amantadine and 72% for rimantadine in preventing laboratory-conﬁrmed inﬂuenza [40]. During long-term prophylaxis, amantadine was found to cause mild reversible adverse effects in 5–30% of the recipients, which involved central nervous system (CNS) and minor gastrointestinal complaints [41,54,128]. In contrast, CNS side effects during treatment with rimantadine were reported to be only slightly higher than with placebo [40,152]. 7.1.3. Development of Viral Resistance to M2 Blockers and Its Molecular Basis Most natural inﬂuenza A virus strains appear to be sensitive to adamantane compounds, requiring a 50% inhibitory concentration (IC50) of only 0.2–0.4 mg/mL as determined in plaque reduction assays [3,75,171]. However, an early recognized caveat for widespread clinical use of M2 blockers was the rapid emergence of drug-resistant viruses in tissue culture and in animal models [3,8,145,208]. It therefore came to no surprise that resistant viruses were isolated from infected patients as early as on the second day of drug treatment [12,69,74,113,193]. One study found that a total of 27% of children with laboratory-conﬁrmed inﬂuenza shed resistant viruses after 7 days of treatment with rimantadine [69]. Such selected drug-resistant viruses appear to be virulent, because they could transmit to family members and cause subsequent disease even when the contact persons were treated prophylactically with rimantadine [74]. Hay and colleagues [72] were the ﬁrst to demonstrate that resistance to amantadine was conferred by single amino acid changes within the transmembrane domain of the M2 protein. Several follow-up studies of resistant viruses selected during tissue culture passage or isolated from drug-treated patients or animals conﬁrmed that drug sensitivity is associated with mutations at the M2 positions 26, 27, 30, and 31 (reviewed in Ref. 78). These changes presumably reduce the accessibility of the M2 ion channel for the drugs while still allowing a sufﬁciently high ﬂow of protons [154]. Viruses that become insensitive to

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amantadine show complete cross-resistance to rimantadine and vice versa. Escape mutant viruses replicated normally and appear to be genetically stable in the absence of drug [8,145]. These ﬁndings have raised concerns about a possible selection of new amantadine-resistant epidemic strains. However, despite the use of amantadine and rimantadine for decades, a recent survey of more than 2000 virus samples collected in 43 countries identiﬁed as little as 0.8% of isolates to be resistant to adamantanamines [222]. Moreover, no tendency for an increase in the occurrence of resistant viruses was reported. Thus, it appears that the replication or transmission of such viruses is for unknown reasons reduced compared to that of drug-sensitive wild-type strains. 7.1.4

Clinical Use for Prophylaxis and Treatment

Amantadine and rimantadine are available in oral formulations as either tablets or syrup. Therapy of acute inﬂuenza illness with adamantane compounds is recommended by the ACIP (1) for patients at high risk for complications even when they were vaccinated, (2) for those who care for such patients, and (3) for persons with severe inﬂuenza [18]. This also includes individuals who suffer from natural or acquired immunodeﬁciency. To avoid emergence and transmission of drug-resistant viruses, treatment should be continued until disease symptoms disappear. Amantadine and rimantadine are approved at two daily 100 mg doses for treatment of adults and children older than 12 years (Table 2). Due to declining renal function, individuals above 64 years of age should not be administered more than 100 mg/day. The substances should also be used carefully in other patients with impaired renal functions, and halving of TABLE 2

Properties of Approved Anti-inﬂuenza Drugs Amantadine

Active against inﬂuenza virus type(s) Administration Ages approved for treatment Ages approved for prophylaxis

Rimantadine

Relenza

Tamiﬂu

A

A

A and B

A and B

Oral

Oral

Oral

1 year

13 years

Powder inhalation 7 years

1 year

1 year

1 year

Not approved

13 years

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the daily dose is recommended. Only amantadine is licensed for treatment of children between 1 and 9 years of age and should be dosed at 5 mg/kg per day. It is recommended that antiviral prophylaxis with adamantane compounds commence after epidemiological and virological conﬁrmation of inﬂuenza A virus activity in the community, because the drugs are inactive toward inﬂuenza B viruses [18]. Chemoprophylaxis can also be considered for protection among high-risk groups, including children and adults with chronic pulmonary or cardiac disease, immunocompromised persons with a reduced response to vaccines, or in the case of a poor match between an epidemic virus strain and the current vaccine. Because the adamantane compounds do not interfere with the development of neutralizing antibodies [198], they can also be used for the protection of persons at high risk to bridge the time gap between vaccination and the appearance of an efﬁcient immune status. For adults and children older than 9 years, two 100 mg doses of amantadine or rimantadine per day are recommended. Children between 1 and 9 years should receive a maximum of 150 mg per day in two divided doses. 7.2

Neuraminidase (NA) Inhibitors

Two new anti-inﬂuenza drugs that inhibit both inﬂuenza A and B viruses, zanamivir (Relenza2, manufactured by (Glaxo-Smith-Kline) and oseltamivir (Tamiﬂu2, manufactured by Roche Pharmaceuticals), have been approved for general use in the United States, Australia, Europe, and Japan. In contrast to the rather fortuitous identiﬁcation of the antiviral activity of amantadine, the development of zanamivir and oseltamivir, which inhibit the viral neuraminidase, was based upon modern rational drug design. With the arrival of NA inhibitors (NIs), M2 blockers will probably be considered obsolete if the promising results from trials on NIs are conﬁrmed by successful use in general practice. Nevertheless, NIs, in spite of their safety and efﬁcacy, are still expensive. 7.2.1

Mechanism

Sialic acids terminate the sugar oligosaccharide side chains attached to eukaryotic glycoproteins and are essential receptor determinants that are bound by the viral hemagglutinin during attachment of the virus to the cell surface. By the time progeny virus bud, these receptors need to be removed to allow efﬁcient release of the progeny viruses. The viral NA (acylneuraminyl hydrolase, EC 3.2.1.18) is the receptor-destroying enzyme that hydrolyzes glycosidic linkages adjacent to N-acetylneuraminic acid (Neu5Ac, sialic acid), (Fig. 3). It is established that NA

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activity is important for virus propagation, because its blockade by antibodies [31], temperature-sensitive mutation [151], or inhibitory substances [148] results in the aggregation of budding virions at the cell membrane and hence reduction of virus release. Additionally, NA removes the terminal sialic acids attached to the carbohydrate side chains on the viral HA and NA glycoproteins, which contribute to viral clumping and prevent virus spread unless they are cleared away by sialidase activity [151]. In infected animals or humans, NA may also enhance virus penetration through the viscous mucus in the lung airways, which contains sialic acids [98]. Of importance, the NA exhibits its enzymatic activity outside the cell, which means that inhibitory compounds do not need to cross the cell membrane. This avoids the requirement for membrane permeability that is mandatory for other antiviral agents that act on internal viral components. Thus, inhibition of viral NA activity was the rationale of several efforts to identify substances that would reduce viral spread and replication. The sialic acid transition state analog 2-deoxy-2,3-dehydro-Nacetylneuraminic acid (Neu5Ac2en), the ﬁrst ancestor of the current NA inhibitory substances, was characterized by Meindl and Tuppy in 1969 [122]. Neu5Ac2en and derivatives thereof inhibited viral neuraminidase activity and replication in tissue culture in the micromolar range but were inactive in the mouse model of inﬂuenza [121,149,150]. Later, the determination of the three-dimensional structure of NA by the Colman group provided detailed structural information for understanding the interactions between the enzyme and its substrates and inhibitors. It was revealed that the enzymatic site is located in a cavity on the protein surface that is lined by amino acids highly conserved among inﬂuenza A and B virus NA [30,202,203]. Nine conserved amino acids interact directly with sialic acid, and there are 10 more residues that provide structural support. Using computer-assisted drug design, von Itzstein et al. [205] demonstrated that the introduction of positively charged amino or guanidino moieties at position 4 of the Neu5Ac2en ring structure increased NA inhibition by two to four orders of magnitude [205]. The inhibition of NA activity by 4-guanidinoNeu5Ac2en, which is now also called zanamivir (Fig. 3), translated into efﬁcient reduction of viral replication of type A and B inﬂuenza viruses in the nanomolar range in vitro and dose-dependent decrease of viral titers in infected animals [205,215]. Thus, zanamivir is 500–1000-fold as active as the M2 blocker amantadine. Furthermore, cellular neuraminidases are only weakly inhibited by zanamivir, suggesting that it should have few side effects [205]. Zanamivir has low oral bioavailability but shows high antiviral activity in humans or animals when administered

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topically by inhalation of dry-powder aerosol [22]. The second currently approved NA inhibitor compound oseltamivir [3R,4R, 5S-4-acetamido-5amino-3-(1-ethylpropoxyl)-1-cyclohexene-1-carboxylic acid, also termed GS4071/Ro64-0802] (Fig. 3) has similar antiviral activities against type A and B viruses [96]. Oseltamivir emerged from an independent NA structure–based study and is based on a cyclohexene ring structure in which the polar glycerol side chain of the sialic acid analogs is replaced by a lipophilic 3-pentyloxy moiety [95]. It is important to note that oseltamivir has high oral antiviral activity when administered as its methylester prodrug, GS4071/oseltamivir phosphate, which is converted to the active drug by hepatic enzymes [82,101,123]. More neuraminidase inhibitory substances such as the cyclopentane compound RWJ-270201 are in clinical testing and might broaden the spectrum of anti-inﬂuenza drugs in the future [4]. 7.2.2

Effectiveness

Several double-blinded, placebo-controlled studies of communityacquired inﬂuenza A and B conﬁrmed that both zanamivir and oseltamivir have potent antiviral properties and are in general safe to use in healthy adults [16,80,112,124,129]. Initially, two daily doses of inhaled or oral NA inhibitors were found to signiﬁcantly shorten disease duration and to reduce symptoms and viral loads when treatment was initiated within 26 hr postinfection [81,82]. Even when inhalation of zanamivir was begun within 30 hr after onset of symptoms, the time to alleviation of major disease signs (cough, myalgia, fever, headache) was shortened by 1–2 days and patients were able to resume normal activities earlier [80,124,126]. Initiation of therapy later than 30 hr after disease onset still reduced viral loads but was less beneﬁcial for symptom recovery. Side effects, which included diarrhea, nausea, and nasal symptoms, occurred during clinical testing with an incidence of 51.5% but were equally high with placebo [60]. Studies of orally administered oseltamivir concluded that treatment with two 75 mg daily doses for 5 days reduced shedding of virus and the severity and duration of inﬂuenza symptoms by 1–2 days when therapy was begun within 36 hr after onset of disease signs [137,199]. Although no changes in clinical parameters were reported during oseltamivir administration, three to four times as many persons experienced mild upper gastrointestinal side effects in comparison to placebo controls. Both zanamivir and oseltamivir also demonstrated high effectiveness in preventing naturally acquired inﬂuenza [73,77,129,209]. One 10 mg dose of inhaled zanamivir reduced the occurrence of laboratoryconﬁrmed clinical inﬂuenza by 67% and that of febrile inﬂuenza by 84%

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among 1107 healthy adults during a 4 week trial period [129]. Similarly, a postexposure protection study demonstrated that the same dosage had 79% efﬁcacy in preventing transmission of inﬂuenza to family members when the index case was treated with zanamivir as well [77]. However, zanamivir did not affect the generation of neutralizing antibodies after immunization with inactivated inﬂuenza vaccine [207]. Thus, the drug could be used for interim protection until a sufﬁcient immune status is acquired after vaccination. Oseltamivir had a comparably high efﬁcacy in preventing laboratory-conﬁrmed inﬂuenza by 74% and inﬂuenza with fever by 82% with one or two daily doses of 75 mg [73]. Within households, one 75 mg dose of oseltamivir per day was 89% protective against clinical inﬂuenza even when the index cases were not treated [209]. Thus, to prevent the spread of inﬂuenza within household contacts, the NA inhibitors appear to be preferable to the M2 blockers, which can induce the emergence of virulent drug-resistant viruses [74]. It should be noted that most studies so far have been conducted during high inﬂuenza A activity and the efﬁcacy against inﬂuenza B virus infections remains to be validated. 7.2.3

Viral Resistance to NA Inhibitors

During the development of NI for clinical use, it was recognized that inﬂuenza viruses with a reduced drug sensitivity could be selected in tissue culture (summarized in Refs. 119 and 195). Drug resistance can be characterized by various methods including IC50 determination of the viral NA by plaque reduction assays (number and size) and yield reduction assay in tissue culture [195]. It should be stressed that under laboratory conditions several passages are usually required to select such variants, which is different from amantadine-resistant viruses, which can emerge in a single-cycle experiment. However, drug-resistant viruses were also isolated in the meantime from diseased persons treated with NI [67,68]. It is not precisely known at which frequencies these mutants develop in humans. Because NA inhibitors will probably ﬁnd widespread use in the future, it seems important to analyze whether resistant virus strains can become clinically relevant and to evaluate their potential to contribute to future epidemics. Resistance to NI was found to be complex, because it can be associated with mutations in the NA or the HA or synergistically in both genes. NA mutations that confer reduced drug sensitivity were identiﬁed at amino acid positions Glu119, Arg152, Arg 252, and His274 [67,119]. These amino acids are part of or cluster around the conserved catalytic pocket. Consequently, these mutations can decrease the enzymatic activity to below 5%, and some also destabilize the whole enzyme.

Inﬂuenza

65

Structural analysis has demonstrated that these amino acid alterations signiﬁcantly disturb interactions of the inhibitor within the catalytic pocket and decrease the strength of binding [204]. The various NI molecules slightly differ in their interactions with the enzyme. Thus, a given NA mutant enzyme may show a range of sensitivity against different inhibitors [67,119,188]. Interestingly, viruses with a reduced sensitivity to NI were also frequently found to have mutations in the HA that affected the globular head region containing the receptor binding site, the stalk region, and the HA2 subunit. Apparently the HA mutations reduce drug sensitivity by decreasing the afﬁnity for cellular sialic acid receptor molecules and thereby easing the release of budding viruses from the plasma membrane [66,120]. Thus, HA mutations compensate for the lower receptor-destroying activity of the NA induced by the presence of inhibitor or mutation. Ironically, HA mutant viruses have been selected that, owing to very weak receptor binding, depend on the presence of zanamivir for infection [15]. These ﬁndings corroborate the concept that efﬁcient viral replication requires a carefully balanced interplay between the strength of HA binding to sialic acid–conjugated receptor proteins and the NA activity in hydrolyzing these receptor determinants [206]. Recent reports have described escape mutants that emerged in drug-treated patients. Those studies also revealed some limitations of the current cell culture systems that are used to detect such viruses. After prolonged zanamivir therapy an inﬂuenza B virus was isolated from an immunocompromised child whose NA had acquired an Arg152?Lys mutation and was 1000-fold less drug-sensitive than the progenitor [68]. Furthermore, the escape mutant also contained a Thr198?Ile alteration in the HA close to the receptor binding site. This mutation reduced its afﬁnity for a-2,6-linked sialic acid receptor determinants on epithelial cells in the human respiratory tract, thereby decreasing the dependency of the virus on NA activity for release. However, the same mutation did not affect binding to a-2,3-linked sialic acids present on MDCK cells. As a consequence, this mutant virus was fully drug-susceptible in a plaquereduction assay in MDCK cells and would have remained undetected if screening had been based merely on this assay. Conversely, some unselected clinical isolates appear to be remarkably resistant against NA inhibitors in tissue culture assays, although they are fully sensitive in animal models [5,215]. This phenomenon is probably explained by a low dependence of some strains on efﬁcient release in tissue culture, where they can spread directly from cell to cell. These ﬁndings demonstrate that for reliable monitoring of clinical isolates there is a need for improved tissue culture systems that more closely mimic the situation of the human

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epithelium. Drug-resistant viruses with NA mutations Arg292?Lys, Glu119?Val, and His274?Tyr were isolated during clinical trials from 1–2% of volunteers receiving oseltamivir [67,220]. The available data on the pathogenicity of these mutant viruses in animal models suggest that they have reduced replication capability in vivo and may therefore be clinically less relevant in humans. 7.2.4

Clinical Use for Prophylaxis

Currently, Tamiﬂu (oseltamivir) but not Relenza (zanamavir) is licensed for chemoprophylaxis in children older than 12 years and in adults (Table 2). For persons with creatinine clearance of 10–30 mL/min, halving of the usual dosage for therapy or prophylaxis is recommended. Two approaches are possible, a seasonal prophylaxis that provides a 92% reduction of conﬁrmed inﬂuenza infection in a vaccinated population of frail elderly persons [118] and a short-term prophylaxis for controlling institutional outbreaks by breaking the virus circulation. In the summarized indications below, the ﬁrst one is a 7 days postexposure prophylaxis, and the three others are intended to give long-term protection. Summarized indications for chemoprophylaxis with neuraminidase inhibitors (Tamiﬂu for persons older than 13 years of age): Control of starting epidemics in institutions, particularly in nursing homes for the elderly Unvaccinated persons who are in frequent or close contact with one or more high-risk persons HIV-infected persons, especially if CD4 þ is low, and for whom vaccination probably would have given no serological response Unvaccinated persons for whom a contraindication for vaccination does exist and who belong to a high-risk group 7.2.5

Clinical Use for Treatment

The use of zanamivir (Relenza) and oseltamivir (Tamiﬂu) is indicated for the treatment of uncomplicated inﬂuenza caused by type A and B viruses [18]. Therapy with either drug should be initiated within 48 hr after the onset of disease signs and should be continued for 5 days [60,163]. It is important to consider that bacterial superinfections may occur that would not be affected by antiviral agents. Neither substance has been shown to prevent serious complications of inﬂuenza such as pneumonia. Relenza is approved for treatment of inﬂuenza in persons aged 7 years and older. The recommended dosage is two inhalations of 5 mg doses twice a day using the inhalation device provided by the manufacturer. Relenza is not recommended for persons with underlying

Inﬂuenza

67

respiratory conditions such as asthma or chronic obstructive pulmonary disease, because there have been some reports of serious adverse effects in such patients [60]. Tamiﬂu can be used for treatment of patients aged 1 year or older. Depending on the age, the recommended dose for children above the age of 12 years and adults is two 75 mg capsules a day. Two daily doses of 15–30 mg are recommended for children under 15 kg, two doses of 45 mg for children weighing between 15 and 23 kg and two doses of 60 mg for persons weighing >23–40 kg. In principle, NIs should not be used without conﬁrmation of the presence of inﬂuenza virus, because they are ineffective against other pathogens that may induce similar symptoms. For this main reason, presumptive treatment must remain the exception. Summarized indications for treatment with neuraminidase inhibitors (Tamiﬂu from 1 year of age and Relenza from 7 years of age): Proven cases of uncomplicated inﬂuenza A and B infection Unconﬁrmed secondary cases, if postexposure prophylaxis was not used. The use of neuraminidase inhibitors could be envisaged in Primary inﬂuenzal pneumonia and other inﬂuenza-associated complications, excluding superinfections. Presumptive treatment of an infected person with very suggestive symptoms during epidemic activity if no microbiological conﬁrmation is possible. Presumptive treatment of an infected person with suggestive symptoms in case of antigenic mismatch during an inﬂuenza outbreak, regardless of the vaccination status. Immunosuppressed patients who did not receive prior chemoprophylaxis. in this case, diagnostic criteria will be less demanding. In any case, salicylates have to be avoided as supportive treatment in children, and paracetamol should be preferred.

8 8.1

IMMUNIZATION Inactivated Vaccine

Current inﬂuenza vaccines are made from puriﬁed hemagglutinin of inactivated egg-grown viruses. This split or subunit surface protein is derived from the three A/H3N2, A/H1N1, and B circulating strains annually recommended by WHO. Randomized controlled trials have shown 70–90% efﬁcacy [125], i.e., protection against laboratory-con-

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ﬁrmed inﬂuenza infection, when circulating and vaccination strains match well. As expected, postvaccination antibody titers decrease with age [161] but nevertheless induce protecting antibodies in 50% of vaccinated elderly persons [64]. Despite an important mismatch, a very low efﬁcacy (12%) by protecting HI antibodies might be observed in old persons [39]. Twenty cohort studies on vaccine effectiveness were evaluated by metaanalysis that showed prevention rates in the elderly of 56% for respiratory illness, 53% for pneumonia, 50% for hospitalization, and 68% for death [65]. Even if vaccine efﬁcacy is low for protecting elderly individuals against infection, the vaccine remains highly effective in preventing outcomes associated to inﬂuenza in this particular high-risk group. In addition, economic studies have shown that immunization can reduce work absenteeism by 43% [135] and physician visits by 42% [19] in healthy adults. Inﬂuenza vaccination was ranked ﬁrst among 587 lifesaving interventions with the best cost per life gained [192]. The costeffectiveness ratio, in comparison with other interventions in the elderly, was found to be the best one, and the vaccine was even considered costsaving for the elderly [47]. Even though safe and effective, inactivated vaccine has important limitations: Included strains have to be chosen, and possibly changed, by WHO each year on a timely basis. Mismatch between vaccine and circulating strains can occur. During the period 1982–1991, 12% of vaccine compositions were inadequate [146]. In contrast with other vaccines, inﬂuenza vaccine offers partial protection. Vaccine efﬁcacy and vaccine effectiveness are often confounded [28]. Compliance of vaccination is limited, especially in health care workers. Production in eggs increases vaccine production time, and it is a real race against time from the moment the appropriate composition of the vaccine is declared by WHO. 8.2

Persons Recommended for Vaccination

The main objective of inﬂuenza vaccination is to reduce the impact of the disease, and targeted vaccination is essentially oriented to persons who are at high risk for complications. Inﬂuenza immunization policies vary greatly from country to country [138], reﬂecting uncertainties concerning

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the risks of inﬂuenza and beneﬁts of vaccination. For instance, advanced age (>65 years) without any high-risk condition was not recognized by some countries as additional risk, whereas publications have shown that even healthy elderly individuals should be vaccinated [211]. Moreover, American health authorities recently extended their recommendations by lowering the age for vaccination from 65 to 50 [18]. The main reason is that one-third of this age group (50–64 years old) has at least one highrisk medical condition, and the argument for extending immunization policy is that an age-based strategy is more efﬁcient than strategies based on individuals’ medical conditions. Selection of persons to be vaccinated relies on the reduction of complications in high-risk groups, the reduction of morbidity related to inﬂuenza in particular individuals, and breaking the circulation of the virus by immunizing persons who can transmit the diseases to high-risk groups. In this way, it is debated whether all children have to be vaccinated for enhancing herd immunity as suggested by the Japanese experience, where vaccination of schoolchildren was shown to protect and decrease the mortality in the elderly [159]. Individual protection and societal beneﬁts unfortunately often compete for the support of policymakers, and particular attention should be drawn if the use of attenuated vaccine were generalized in the child population. Health care workers (HCWs) are also important groups of people who can transmit the disease to high-risk persons, and publications on the beneﬁts of HCW vaccination for the protection of high-risk persons are convincing [21,157]. That is why current U.S. recommendations about which persons should absolutely be vaccinated have included HCWs, and these seem to be applicable to all countries [18]. Summarized indications for inﬂuenza vaccination: 1. Persons of 65 and more, even healthy ones 2. Institutionalized persons (those in nursing homes and health care facilities) 3. Persons of the age of 6 months and older with at least one underlying chronic condition: a. Lung disease, essentially asthma and COPD b. Cardiovascular diseases such as cardiac failure, valvulopathy, and pulmonary hypertension c. Hepatic disorders such as cirrhosis or chronic viral hepatitis d. Renal diseases, particularly in dialysis or transplant patients

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e.

Metabolic diseases such as diabetes types I and II, cystic ﬁbrosis, or hemoglobinopathies f. Immune disorders, in all situations threatening immunocompetence: therapy with corticoids, therapy for malignant tumors, HIV infection, organ transplants, pregnancy in which the second or third trimester will occur during the winter g. Children aged 6 months to 18 years who are receiving longterm aspirin therapy (risk of Reye’s syndrome) 4.

Persons who can transmit the disease to high-risk groups a.

Medical and administrative staff of health care facilities, nursing homes, or outpatient care settings b. Households of high-risk persons 5.

6. 7.

Travelers who are at high risk and who travel to the southern hemisphere between April and September or to the northern hemisphere between October and March. Normally these persons should have been vaccinated in the autumn, and a second immunization should be given with a vaccine or the last available composition. Persons between 50 and 64 years, with particular attention to smokers, excessive alcohol drinkers, and obese persons. Anyone else who wishes to be protected against the infection.

The only contraindication is allergy to egg protein, and the vaccine should not be given, for psychological reasons, to persons with an upper respiratory infection. Side effects are essentially local (redness, pain) or rarely general (low grade fever) and in both cases are self-limiting. 8.3

Live Attenuated Vaccines

Live cold-adapted inﬂuenza vaccine was ﬁrst developed in the 1960s [111], and this reassortant attenuated vaccine is currently submitted to the Food and Drug Administration. The vaccine reassortant strain contains six genes from attenuated master strains and two genes coding for hemagglutinin and neuraminidase of contemporary wild viruses. Safe and highly immunogenic, this attenuated intranasally administered vaccine has a 92% efﬁcacy rate in preventing laboratory-conﬁrmed inﬂuenza infection [11]. In 1997, when the A/Sydney/5/97(H3N2) strain was not included in its composition, the vaccine was nevertheless 86% efﬁcacious against the wild strain [10]. Effectiveness of the vaccine is also appreciable in that it reduces febrile otitis media by 30% and illness

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necessitating antibiotics by 29% [11]. Rhinorrhea, especially after the ﬁrst dose, is the main side effect.

9

SUMMARY OF APPROACH FOR CONTROLLING AND MANAGING INFLUENZA IN INDIVIDUALS

Control and management of inﬂuenza are intended to decrease the incidence rate, the spread of the virus, the intensity of symptoms, and the severity of associated complications. Vaccination is the most important preventive intervention among persons at high risk or persons who are likely to transmit the disease to high-risk persons. Dosage varies according to age and prior vaccination. Recommendations are as follows: In previously unvaccinated children aged from 6 months to 35 months: two half-doses (0.25 mL) separated by 4 weeks In previously unvaccinated children aged from 36 months to 8 years: two doses (0.50 mL) separated by 4 weeks In previously vaccinated children over 36 months of age, whatever the prior vaccination status: one dose (0.50 mL) The intramuscular route of administration has to be used, in the anterolateral part of the thigh in children less than 2 years of age and in the shoulder in both children over the age of 2 years and adults. Vaccination must be given between mid-October and the end of November in the northern hemisphere. For chemoprophylaxis, neuraminidase inhibitors (NIs) are preferred in place of M2 blockers. Long-term prophylaxis (4 weeks) with oseltamivir can be initiated in persons aged 13 years or older when the risk of exposure to inﬂuenza virus is high, with a dosage of 75 mg/day. In this case, combined chemoprophylaxis and vaccination provide additional protection. Contact chemoprophylaxis with oseltamivir may also be given in a family setting where an index case is discovered. The same dosage is used for 7 days. Treatment of uncomplicated cases of conﬁrmed inﬂuenza can be initiated with Relenza, 5 mg twice daily in persons aged 7 years or more, or Tamiﬂu, 75 mg twice daily in adults. As mentioned above (Sec. 7.2.5), dosage of Tamiﬂu in children aged 1 year and older depends on the weight. Both drugs have to be taken for 5 days. Effectiveness cannot be expected if the treatment is not started within 2 days after the onset of illness. As for chemoprophylaxis, both NIs might be given as treatment in vaccinated persons.

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PANDEMICS OF INFLUENZA Three Major Pandemics of the Twentieth Century

Three major pandemics occurred during the 20th century, namely the Spanish ﬂu in 1918, the Asian ﬂu in 1957, and the Hong Kong ﬂu in 1968. Possible mechanisms of the occurrence of new strains and disappearance of old strains have been described above. The death toll of each pandemic was impressive, with 20–40 million worldwide during the Spanish ﬂu and 100,000 deaths in the United States for both pandemics in 1957 and 1968 [58]. A large proportion of deaths occurred in young adults, suggesting a putative acquisition of protection against the illness in younger persons [176]. This age pattern and the variable severity of the pandemic virus have to be taken into account for preparing contingency plans including scenarios of variable intensity. Lessons could also be learned from the Spanish ﬂu to help plan for the next inﬂuenza pandemic. Issues observed in 1918, such as authoritative measures for wearing masks or closing schools, which were often unpopular and rejected, could arise again. The main objectives of a national plan are to reduce panic-related problems, to ensure a reliable communication strategy, and to ensure equity in access to prevention and treatment measures. 10.2

Preparedness Plans

The World Health Organization issued guidelines in 1998 for helping national and regional authorities prepare a preparedness plan that can be used in the case of an inﬂuenza pandemic [210]. The latter is deﬁned as The emergence of an inﬂuenza A strain with a different hemagglutinin subtype than strains that have been circulating for many years A high proportion of susceptible people in the community, i.e., no or low antibody titers to the novel hemagglutinin High person-to-person transmissibility, with accompanying human disease To assess the risk before proposing ways for managing and controlling a pandemic, it was necessary to rank the different threat levels and the successive phases of a pandemic. This makes it possible to deﬁne a strategy according to the importance of the risk. If the risk is to be assessed by international institutions and teams, then each country is responsible for the management process. It is strongly recommended that a National Pandemic Committee be established in each country or

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that it be improved if it already exists. To prevent inadequate measures in the case of false alarms, preparedness levels have been deﬁned that must exist before an inﬂuenza pandemic is declared: Phase 0, preparedness level 1 corresponds to the detection during the interpandemic period of a novel human strain without evidence of outbreak. In phase 0, preparedness level 2, two or more human infections are observed. Human transmission is conﬁrmed by WHO in preparedness level 3. In phase I, outbreaks occur in at least one country. In phase II, the outbreak is extended to multiple countries. In phase III, outbreak activity has stopped in initially affected regions and epidemics occur elsewhere. After the end of the ﬁrst world wave, additional waves are expected in phase IV. In phase V, WHO will declare the pandemic to be over. When there is a pandemic or a pandemic alert, WHO will collaborate closely with the four collaborating centers in London (UK), Atlanta (USA), Tokyo (Japan), and Melbourne (Australia), essentially for preparing diagnostic agents, characterizing the strain, and preparing a new vaccine. The most delicate actions to be taken if a pandemic occurs will be organizing antiviral agent stockpiles for the ﬁrst wave and obtaining a speciﬁc vaccine before the next wave. Inequity in distribution and social disruption will be the main problems to be addressed by the authorities.

11

CONCLUSIONS

Interpandemic inﬂuenza outbreaks pose an important challenge for both individuals and society. Management and control of the disease rely essentially on surveillance, immunization, and treatment. The reduction of the impact that can be obtained depends on the awareness of the illness by both clinical staff and decision-makers. Strategies for enhancing herd immunity by vaccinating children have to be considered, because immunization of groups at high risk will not be sufﬁcient for controlling the disease. Likewise, the appropriate use of new antiviral agents in both chemoprophylaxis and treatment will help to reduce the impact. Meanwhile, national preparedness for an inﬂuenza pandemic, which is certain to occur, is of major importance.

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Acknowledgments We thank J. Manuguerra (Institute Pasteur, Paris, France) and S. Pleschka (University of Giessen, Germany) for critical comments on the manuscript and D. Heuer (Max Planck Institute of Infection Biology, Berlin, Germany) for help in the preparation of ﬁgures.

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3 Respiratory Syncytial Virus Philip R. Wyde and Pedro A. Piedra Baylor College of Medicine, Houston, Texas, U.S.A.

1 1.1

INTRODUCTION Human and Economic Impact of Respiratory Syncytial Virus Infections

Respiratory syncytial virus (RSV) is ubiquitous, causing epidemics annually worldwide [1]. Community attack rates are usually high (30– 50%), and almost all children have an RSV infection by 3 years of age [2]. The majority of these infections are subclinical or mild. However, if virus descends to the lower respiratory tract, serious manifestations and even death can occur. Respiratory syncytial virus infections impact a number of populations especially hard [3]. In the very young (those less than 2 years of age), RSV is the leading producer of bronchiolitis, pneumonia, and lower respiratory tract infection (LRTI). This virus can also cause signiﬁcant problems in children with underlying chronic health conditions, for example, those with nephritic syndrome, chronic heart disease (CHD), or bronchopulmonary dysplasia (BPD). Children with cystic ﬁbrosis (CF) are also at high risk. In this populations, RSV infections result in reduced lung function and a greater rate of hospitalization (43%) than any other viral infection [4]. Adults with chronic pulmonary and/or cardiac 91

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disease are also at high risk, and in the elderly the impact of RSV appears to be second only to that of nonpandemic inﬂuenza [5]. However, most at risk are immunocompromised individuals [6], especially those receiving chemotherapy for leukemia or undergoing bone marrow transplantation. In these populations, once pneumonia develops, the overall mortality can range from 50–100% without treatment; even with treatment, unless therapy begins before infection of the lower respiratory tract takes place the prognosis is very poor. In toto, the human and economic impact of RSV is staggering. Presently there are approximately 100,000 hospital admissions and 4,500 deaths in the United States annually due to RSV infections [7]. These infections may be responsible for 40–50% of hospitalizations for bronchiolitis and 25% of pediatric hospitalizations for pneumonia [8]. Worse, many RSV infections may have long-lasting consequences because a signiﬁcant number of infected children may develop hyperactive airway disease and diminished pulmonary function later in life [9]. Here, too, the numbers are astounding. Between 40% and 50% of infants hospitalized with RSV bronchiolitis have recurrent episodes of wheezing during early childhood, and many go on to develop asthma or other long-term airway morbidity. There may also be a link between RSV infection in infancy and the development of chronic obstructive pulmonary disease (COPD) in adult life [10]. Indeed, it has been reported that even children who have mild RSV disease can have recurrent wheezing up to 6 or even 10 years after the acute episode [11,12]. However, a causal relationship between RSV infection early in life and the development of asthma later in life has not been proven. Adding to the problem, RSV epidemics often occur in clusters (usually during late fall and winter in the northern hemisphere), and this clustering of cases can cause major problems in both primary and secondary care centers [13]. In terms of dollars, the average hospitalization charge for an RSV infection (weighted in 1998 US dollars) was $7,140 for infants and $6,910 among all children younger than 5 years old [14]. Overall, it is estimated that in the United States alone, RSVrelated illnesses cost between $300 million [7] and $400 million annually [15]. Amazingly, these statistics may be an underestimation. Bronchiolitis-associated hospitalizations among U.S. children is rising [16], and recent data suggest that annual bronchiolitis hospitalizations associated with RSV infection among infants may be greater than previous estimates for RSV bronchiolitis and pneumonia hospitalization combined [17].

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Epidemiology

There are two major subgroups of RSV, designated A and B. Strains are placed into one of these groups primarily on the basis of their reactivity with panels of monoclonal antibodies raised against epitopes on RSV fusion (F) and attachment (G) envelope proteins [18,19]. During an epidemic, a single strain may circulate, or strains belonging to both subtypes can be present [20]. Reinfections with RSV are common [21], possibly due to the antigenic variability that occurs among RSV strains. However, the actual mechanisms involved remain unclear. Regardless, signiﬁcant illness and appreciable morbidity can occur during reinfections, and it does not seem to matter which subtypes the individuals were initially infected with. Although most reinfections do not lead to serious disease, they may be an important source of virus that can lead to infection of more vulnerable populations (e.g., infants or immunoincompetent individuals). Spread of RSV appears to take place primarily through saliva drops and close contact and not by aerosol. Infected persons with symptoms are highly contagious [22]. Moreover, the virus can remain infectious for 6 hr on solid surfaces. Thus, disinfection and hygienic practices are very important in preventing transmission, with hand washing being of paramount importance. Other steps include (1) avoidance of close contact with infants by persons who have symptoms of respiratory tract infections, (2) reducing contact of very young infants (<3 months of age) with other infants and young children, and (3) testing using rapid diagnostic methodology in suspected cases to enable segregation of infected patients [23]. Similar, but stricter, procedures need to be followed with immunocompromised individuals [6]. Nosocomial infections and outbreaks in institutions are common and can be explosive. Virus may be introduced by visitors from the community, but it is also often spread by institutional personnel. Attack rates of 40% or more are not uncommon in these situations [24,25]. As elsewhere, education in infection control, frequent hand washing, and other simple hygienic procedures can reduce the incidence and magnitude of these outbreaks. 2 2.1

REPLICATION RSV Structure and Function

All RSV strains contain a linear, nonsegmented, negative-stranded RNA genome that encodes for 11 proteins [26]. This RNA is surrounded by a

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helical nucleocapsid, which in turn is encompassed by matrix (M) protein and a protective coat composed of three proteins, F (fusion), G (attachment), and SH (small hydrophobic) [26]. These coat proteins are encased in a lipid membrane that is derived from the plasma membrane of the host cell when the virus buds from this cell (Fig. 1). (The lipid membrane is required for infectivity.) Together, the nonsegmented single-stranded RNA, helical nucleocapsid, and lipoprotein envelope (i.e., coat proteins þ lipid membrane) make up the virion. Mature virions are pleomorphic and range from 150 to 300 nm in diameter. Enclosed inside the virion are two nonglycosylated matrix proteins, designated M and M2, and three viral RNA-associated proteins identiﬁed as N (major nucleocapsid protein), L (viral RNA polymerase), and P (phosphoprotein). The M protein may mediate interaction between the nucleocapsid and the envelope, whereas the M2 protein apparently is a

FIGURE 1 Schema of the replication cycle of respiratory syncytial virus. (Modeled after the schematic representation of paramyxoviruses presented in Ref. [244].) Lamb RA and Kolakofsky D. Paramyxoviridae: The Viruses and Their Replication. In: Fields B, Knipe DM, Chanock RM, Hirsch MS, Melnick JL, Monath TP, Roizman B, editors. Fields Virology. New York: Raven Press, 1996: 1191.)

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transcriptional elongation factor that interacts with the N protein and enhances read-through of intergenic junctions during viral transcription [27]. This protein may also have a negative regulatory function [28]. Two nonstructural proteins, NS1 and NS2, are present in the cytoplasm of infected cells but not in virions. NS1 is thought to be involved in transcriptional regulation [29], but the role of NS2 is not clear. An 11th gene codes for a 90-residue protein whose function is unknown but that is thought to be contained as a second open reading frame within the M2 protein [26]. The F and G proteins have paramount importance immunologically, for vaccine development and for virus pathogenesis. The native fusion protein (F0) is cleaved in the Golgi apparatus by a cellular protease into two subunits, F1 and F2 [30]. These two subunits are held together covalently by a disulﬁde bond and are fusion-competent. The hydrophobic N-terminus of the F1 portion is thought to insert into the host cell membrane when fusion occurs. This portion of the F1 subunit is conserved within each RSV subfamily (i.e., subgroup A or B) but not between the two subgroups [26]. Despite this and other heterogeneity, there is identity among the F proteins of all RSV strains at the two positions in the N-terminus of the F1 protein that have been identiﬁed as being essential to F protein structure and fusion activity [31]. Fusion activity is thought to begin after the F1 protein undergoes a conformational change that exposes the active site of the fusion peptide and allows insertion of F1 into the host cell membrane [32]. Presumably, the inactive native conformation exists on free virions after budding from infected cells and undergoes this conformational change when the virus interacts with the host cell receptor. After the viral envelope fuses with target cell plasma membranes, the viral nucleocapsid is released into the host cell cytoplasm, where transcription and replication can occur. The virus does not have to enter cells from without. F protein can also induce fusion of infected cells with uninfected neighboring cells, causing syncytium formation, a hallmark of paramyxovirus infections. This ability to go from cell to cell without leaving the infected host cell may help protect the virus from the host’s immune system. Regardless, the conservation of the amino acid sequences in the active sites of the F protein between RSV strains suggests that this protein is vital to the survival and pathogenesis of the virus. This conservation, the essential role in pathogenesis, and the fact that antibodies to the resulting epitopes in this conserved region of the F protein are virus-neutralizing (i.e., are protective) have made the F protein a major target for both RSV vaccines and chemotherapeutic agents.

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There is no such conservation among the G proteins of different RSV strains. Amino acid sequences >50% have been reported between different group A and B strains for this protein, and even within the same antigenic group, 20% differences in G protein sequences have been determined [33–35]. Indeed, G proteins from different RSV strains have been reported to vary markedly in length and to have differences in molecular weight ranging from 84 to 90 kDa (mature form of the protein). Highly varied glycosylation adds to this heterogeneity [33]. The role of this glycosolation is not clear, but it has been speculated that it may protect epitopes on the G protein from being recognized as a foreign antigen [36]. Regardless, although the G protein is responsible for promoting attachment and entry into host cells and can induce virusneutralizing antibodies, it has not been the target of many RSV vaccines. The signiﬁcant variability just discussed is partially responsible for this. However, a second factor is that several studies have suggested that the G protein is capable of inducing eosinophilia and other adverse immune responses [37]. (This subject is discussed in more detail below.) The third coat protein, SH, is small (only 64 amino acids) and hydrophobic. Although it has been shown to facilitate virus-host cell fusion [26], it is not essential to this process or to infection [38]. However, SH deletion mutants have been shown to be attenuated in mice compared to wild-type RSV strains and may therefore be of some interest in developing attenuated live virus vaccines [38]. The large polymerase protein (L) and phosphoprotein (P) that are part of the polymerase complex and viral nucleocapsid are not well characterized. To date, neither protein has been a major target for either vaccines or chemotherapeutics. Neither have the two matrix proteins, M and M2, or the two nonstructural genes, NS1 and NS2. 2.2

Replication

Respiratory syncytial virus replicates primarily in the ciliated epithelial cells lining the respiratory tract. Infection is initiated by the binding of the linear heparin-binding domain of the virion’s G protein to receptors on the host cell plasma membrane [39]. What exactly the receptor is is not known, but it is thought to be a sulfated peptidoglycan [40]. The virus then penetrates and enters the host cell, where it uncoats, releasing the matrix protein and the nucleocapsid [26]. Transcription of vRNA to positive-stranded messenger RNA (mRNA) then occurs, followed by translation of the viral mRNA into early viral proteins. There is then a switch to the production of positive sense RNA that serves as a template for the synthesis of progeny genomes. Viral proteins accumulate in the

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FIGURE 2 Diagramatic representation of respiratory syncytial virus. Shown are the lipid bilayer (light gray) with the three coat proteins (G, F and SH) of RSV penetrating. Inside the virion are the matrix protein (depicted in black), the matrix 2 protein (black dots), and negative-strand RNA, which is surrounded with nucleocapsid protein (NP). Associated with the NP are the two other proteins associated with the transcriptase complex, the large polymerase (L) and phospho (P) proteins. The two nonstructural proteins (NS1 and NS2) of RSV are found only in the host cell and not in RSV virions. (Modeled after the general representation of a paramyxovirus virion presented in Ref. 244.)

host cell cytoplasm and associate with viral genetic material and polymerase to form the nucleocapsid. This complex is incorporated into the virion, which then buds through the apical membrane, where the virions acquire their lipid envelopes [41].

3

PATHOGENESIS

Respiratory syncytial virus infections usually begin in the nasopharynx, from where they may spread to the lower airways (Fig. 3). In

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FIGURE 3 Comparison of the general kinetics of respiratory syncytial virus shedding and relationship to clinical illness in infants and adults.

immunocompetent individuals, infection of cells other than the respiratory epithelium is unusual, and the virus is usually restricted to the respiratory tract [42,43]. The major exception to this is that the virus often spreads to the middle ear, so the development of otitis media is common in this population [44]. In immunoincompetent persons, extrapulmonary dissemination can occur, and virus may migrate to the kidneys, liver, central nervous system, and heart with some regularity [6,26]. There is a high frequency of virus isolation from the nasopharynx in the early phase of infection, especially in children [45]. After 14 days, virus recovery from this region is less likely. However, in immunosuppressed individuals, excretion of virus may continue for 28 days or longer. In general, the peak viral infection occurs about 5–7 days after onset of illness [3]. Many things can inﬂuence clinical outcome, including poor general condition and poor nutritional status [46,47]. Gender, ethnic group, and

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levels of maternal antibody [48]; age at which the ﬁrst RSV infection takes place [49]; an underlying pulmonary or cardiac condition [3]; reduced lung function; or an immunodeﬁciency condition can also contribute to disease severity. In addition to causing bronchiolitis and pneumonia, RSV has been implicated in the pathogenesis of childhood asthma and reactive airway disease [50,51]. Indeed, although other viruses have been associated with wheezing [52,53], RSV appears to be the single most important agent in children <3 years of age associated with this response [54]. Even children who experience only mild RSV disease and who do not require hospitalization can have recurrent wheezing up to 10 years after the acute episode [11]. Immunoglobulin E induced during RSV infection is thought to initiate the pathogenic processes leading to airway disease by binding to the cells lining the epithelium of the respiratory tract [55]. There, this antibody can interact with eosinophils and mast cells that are also present in the area, causing these granulocytic cells to release the contents of their prominent granules [56,57]. Many of the released molecules are inﬂammatory and can attract still other eosinophils and mast cells, T-cells, and polymorphonuclear neutrophils [58]. In addition, there is upregulation of cellular adhesion molecules, their ligands [e.g., CD11B, intercellular adhesion molecule-1 (ICAM-1) and E-selectin], and HLA class I and II antigen-presenting molecules on different leukocyte populations [54]. These changes occur on the plasma membranes of both epithelial and inﬁltrating cells and facilitate binding of the latter to the former, localizing them and allowing them to produce still more damage. The process can accelerate and spiral out of control as more cells are recruited and more inﬂammatory molecules are released. Interleukin (IL)-1a, IL-4, IL-5, IL-8, IL-11, and IL-12, IFN-g, arachidonic acid metabolites (e.g., leukotrienes), and chemokines such as RANTES, MIP-1, and MCP-1 are just some of the mediators and modulators that have been implicated in this process [54,55,59]. Other mechanisms may also be involved, because increases in IgE and type 2 immune responses are not always seen in children manifesting reactive airway disease or asthma after RSV infection [60,61]. The RSV G coat protein may play an important role in the induction of reactive airway diseases, because it can induce marked eosinophilia and inﬂammatory responses when administered alone or as part of an inactivated vaccine. As described above, this protein is unique in that its amino acid composition has considerable variation among different RSV strains. Moreover, it is also heavily glycosylated and does not resemble the surface glycoproteins of other members of the Paramyxovirinae

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subfamily [62]. No protein-speciﬁc CTL response has been found to the G protein, but it has been shown to elicit a predominantly type 2 immune response [63,64]. In addition, the presence of G protein has been shown to depress natural killer cell and IFN-g responses (both hallmarks of type 1 immune responses) [65]. Interestingly, RSV produces soluble as well as membrane-anchored forms of G protein [66], and the eosinophilic responses to the soluble G protein exceed those to the membraneanchored form [67]. The best evidence for a link between the RSV G protein, inactivated vaccines, and eosinophils comes from mouse studies [67].

4

DIAGNOSIS

Respiratory syncytial virus infections can be misdiagnosed, particularly in adults. One reason for this is that the symptoms of RSV in adults are often very similar to those of many other respiratory viruses, especially inﬂuenza [68]. In addition, they can frequently be masked by the symptoms of accompanying underlying (e.g., cardiopulmonary) disease, especially in the elderly and/or chronically ill. The increasing availability of rapid diagnostic tests that are relatively easy to perform should make misdiagnosis less of a problem, particularly as these tests become more sensitive (Table 1). Diagnostic tests for RSV are usually performed on secretion samples obtained from the nasopharynx of a patient by washing, suctioning, or swabbing. These samples can be cultured for virus in tissue culture cell monolayers or tested directly for antigen presence using ﬂuorescent antibody staining procedures on recovered cells or a rapid diagnostic ELISA test in which a speciﬁc color change indicates the presence of RSV antigens in the samples. Alternatively, vRNA can be detected by genome ampliﬁcation using reverse transcriptase–polymerase chain reaction (RT-PCR) assays (Table 2). The ﬂuorescent antibody, ELISA, and RT-PCR assays are so rapid as to permit treatment the same day [69,70]. In contrast, isolation and identiﬁcation of virus using tissue culture cells can take from days to weeks to complete. In addition, the procedures used for tissue culture–based assays usually require a wellequipped laboratory with highly trained technicians. Regardless, isolating and speciﬁcally identifying the virus can provide important epidemiological information that the more rapid tests generally do not offer. This is particularly true at the beginning of an epidemic when it is important to determine which viruses and strains are circulating [23].

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TABLE 1 Rapid RSV Antigen Detection Tests in the Ofﬁce and Hospital Setting Goal or property Pathogen-speciﬁc diagnosis Decision to start ribavirin therapy Reduce inappropriate use of antibiotics Identify outbreak of RSV in the community Group patients for prevention of RSV transmission Cost of rapid antigen test Sensitivity of rapid antigen test

Ofﬁce/clinic

Emergency room/hospital

Generally not required

Useful

Not applicable (ribavirin given only in hospital setting) Useful

Useful

Useful

Useful

Useful

Not required

Useful

Modest compared to ofﬁce/clinic visit Poor for children, adults, and those with mild illness

Minimal compared to ER/hospital costs Very good in ill infants, poor in adults

TABLE 2 Diagnosis of RSV Infections Diagnostic method

Comments

Rapid antigen test

Simple, 15–30 min, low cost, easy to perform Simple, half-day test, low cost, requires trained technician Complex, 1–2 day test, low to moderate cost, requires highly trained technicians Complex, 2–14 day test, moderate cost, requires highly trained technicians Requires acute and convalescent blood specimens, complex, 2–7 days, moderate cost, requires highly trained technician

Fluorescent antibody test RT-PCR and other genomic ampliﬁcation methods Tissue culture

Antibody assays for detection of antibody rises to RSV

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VACCINATION

5.1 5.1.1

Background History

The roles of the different virus-speciﬁc humoral and cell-mediated immune (CMI) responses induced following RSV infection are not entirely understood. Indeed, some of these responses may be detrimental. This possibility was made most evident in the failed RSV vaccine trials performed in the late 1960s [71–74]. In these trials, children immunized with test vaccine containing formalin-inactivated (FI) whole virus had more severe disease when they became naturally infected with RSV than children who got infected but did not receive the vaccine. This exacerbation of disease occurred despite the fact that the FI-RSV vaccine induced relatively high titers of RSV-speciﬁc antibodies [71,75]. Many theories have been put forth for why exaggerated disease occurred following the administration of FI vaccine in the 1960s ﬁeld trials [75,76]. More recent data derived from studies carried out in mice suggest that an imbalance in immune responses could have been involved [67,77,78]. For example, it has been shown that mice challenged with live virus following vaccination with FI RSV produce predominantly CD4 þ (type 2) T-helper cells and cytokines that are associated with inﬂammation and disease (e.g., IL-4 and IL-10), but only low levels of major histocompatibility complex class I restricted, RSV-speciﬁc cytotoxic T-lymphocyte (CTL) activity, which is thought to be critical for rapid viral clearance and reduced illness [79–81]. Unvaccinated mice inoculated with RSV produce predominantly a CD8 þ (type 1) immune response. 5.1.2

Humoral Immune Responses to RSV

Since the 1960s, much effort has been spent assessing the contributions of humoral and CMI responses in protecting or exacerbating RSV disease. Looking ﬁrst at humoral immunity, there is much evidence that indicates that virus-speciﬁc secretory [83–85], and serum [86–88] antibodies play important roles in protecting the upper and lower respiratory tracts, respectively, from RSV infection. The best evidence that virus-speciﬁc, serum-neutralizing (Nt) antibodies can be protective comes from studies showing that prophylactically administered monoclonal and polyclonal antibodies can prevent or reduce RSV-induced illness in children exposed to this virus (discussed in more detail below) [89–91]. On the other hand, it is equally clear that although signiﬁcant levels of virus-Nt antibodies are produced and are readily detectable in the serum

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following infection, the protection of these antibodies is often incomplete or transient, because reinfection, particularly of the upper respiratory tract, can readily occur [21]. This is most apparent in young infants, who often get reinfected in successive RSV seasons, but also occurs commonly in older children and adults. The frequency of reinfection despite the presence of signiﬁcant levels of circulating Nt antibodies suggests that virus-speciﬁc antibodies produced locally in the respiratory tract have a role in protecting the respiratory mucosa [83]. Maternally derived virusspeciﬁc Nt antibodies can also be protective [49,92,93]. However, because these antibodies are obtained passively, they are not long-lasting, and the newborn infant may become vulnerable after a period of time (e.g., between 6 weeks and 6 months of age) [49,93]. In addition, maternally derived antibodies can interfere with the efﬁcacy of vaccines targeted for very young infants and diminish the ability of infants to mount active immune responses to infection [94]. The class and subclass of the immunoglobulin (Ig) induced may affect disease outcome. For example, both IgE and IgG4 have been associated with RSV-induced pathogenicity [95,96]. 5.1.3

Cell-Mediated Immune Responses to RSV

The role of T-cell-mediated immune (CMI) responses in protection or exacerbation of RSV disease has also been closely examined. Increases in HLA class I restricted CD8þ cytotoxic T-lymphocyte (CTL) responses speciﬁc for RSV antigens have been reported following RSV infection of humans [97,98] and mice [99–101]. The data obtained from these and other studies suggest that virus-speciﬁc CTL responses do not play an important role in preventing infection or reinfection but are critical in clearance of virus from the lungs [102]. Supporting this idea is the ﬁnding that patients with impaired cellular immunity often have recurrent, severe, and/or prolonged disease with virus shedding that can last for weeks to months [103,104]. Paradoxically, T-cells may also contribute to the prominent immunopathology associated with RSV disease, because T-cell-mediated enhanced lung pathology has been well described in RSV rodent models [105,106] and, as discussed above, may also play a signiﬁcant role in RSV-induced wheezing, hyperreactive airway disease, and asthma. Type I cytokines (e.g., IL-2 and IFN-g) are associated with virusspeciﬁc CTL responses, and these have been reported to predominate following natural RSV infection [107] although perhaps not as prominently as occurs following other respiratory virus infections (e.g., inﬂuenza) [108]. In contrast to these ﬁndings, a predominance of type 2 cytokines and immune responses that may contribute to the immune

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pathology seen in young children infected with RSV has also been reported [109,110]. Another problem is that both correlations and lack of correlations of cytokine levels, particularly IFN-g, with disease severity have been found (see Sec. 6.2.8). Some of the variability seen may be due to the fact that different tissues or collection sites were used in the different studies. Regardless, the roles of virus-speciﬁc CTL and CMI responses in RSV disease are not clear. 5.2 5.2.1

RSV Vaccines Live Virus Vaccines

After the disastrous outcome of the 1960s ﬁeld trials, attention turned to trying to produce live attenuated vaccines that could potentially induce balanced immune responses, stimulate mucosal immunity, and be less likely to induce the enhanced disease that was seen with the inactivated preparations administered parenterally. Initially, attention focused on creating cold-passaged (cp) and temperature-sensitive (ts) mutant viruses. In either instance, the viruses picked were more likely to grow relatively well at the lower temperatures found in the upper respiratory tract and less well in the lower respiratory tract where temperatures are higher. Hence, they were less likely to cause serious illnesses. Indeed, this proved to be true of most of the live virus vaccine candidates when they were inoculated into adults. However, in the early trials and even in many of the more recent trials, these viruses proved to be too virulent, too attenuated, or too unstable when tested in children [111]. In more recent efforts to produce live attenuated RSV vaccines, interest has centered on increasing the attenuation of the candidate virus by using repetitive rounds of chemical mutagenesis to increase the number of mutations present in them [111–115]. Others have tried to create viruses with genome deletions that lead to altered growth characteristics and attenuation [116,117]. The development of reverse genetics has revolutionized the live virus vaccine ﬁeld. Mutations can now be systematically created and their effects on attenuation, immunogenicity, and protection methodically studied [118]. Two new candidate RSV strains developed using this technology have already been shown to be immunogenic in seropositive subjects [118], and it is certain that more potential candidates will be developed using these procedures. The technique also allows other possibilities. For example, recently a recombinant RSV virus that expresses IFN-g during infection of mice was created [119]. This expression did not affect the attenuation of the virus but did appear to enhance its immunogenicity.

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No live virus vaccine is likely to be licensed in the near future for all targeted populations (e.g., infants and immunoincompetent individuals). However, some may prove suitable for selected subpopulations (e.g., immunocompetent adults and the elderly). Moreover, there has been a positive side to the work done so far. Enhanced disease has not been seen in any of the trials testing these candidate vaccines, and in one study the test virus was able to induce signiﬁcant levels of virus-neutralizing antibodies in animals given passive antibodies [113]. Thus, it appears that at least some live virus vaccines can induce antibodies in the presence of maternally derived antibodies. 5.2.2

Recombinant Vaccines

Bacteria (e.g., Escherichia coli and Staphylococcus carnosus) and viruses (e.g., adenovirus, vaccinia virus, and baculovirus) have been used to develop recombinant vaccines that produce RSV proteins with protective epitopes following their administration. For example, resistance to RSV challenge has been seen in animals inoculated with recombinant vaccinia viruses expressing the F, G, M2, or N proteins of RSV. However, the protection induced by the M2 and N proteins was relatively short-lived [120]. In another study, genetically engineered chimeric protein composed of the external domains of the F protein of RSV and the hemagglutininneuraminidase (HN) protein of PIV3 were produced in insect cells using a baculovirus expression system [121]. Bovine RSV and PIV3 have also been used to produce recombinant viruses that express human RSV and PIV3 viral proteins [122]. Plant viruses have also been used to engineer recombinant RSV vaccines that can be used to infect embryonic plants. This can lead to the production of antigen-laden plant products that can induce protective antibodies when they are eaten [123–125]. 5.2.3

Subunit and Peptide Vaccines

Much effort has also been spent on developing subunit vaccines for use against RSV. Because portions of the F, but not the G, protein are conserved among both RSV A and B subtypes, most of these preparations contain RSV F protein either alone or mixed with the G glycoproteins. The two subunit vaccines that are furthest along in development are the puriﬁed fusion protein (PFP) vaccine being manufactured by Wyeth Ayerst and BBG2Na produced by the Centre d’Immunologie Pierre Fabre. There are actually three generations of the Wyeth Ayerst PFP vaccine. The ﬁrst preparation, PFP-1, was composed of immunoafﬁnity puriﬁed, alum-adsorbed F glycoprotein. PFP-2 is a more puriﬁed

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preparation and contains >99.9% F glycoprotein. It has been shown to be safe and immunogenic in healthy young, seropositive children [126], in seropositive children with BPD [127], in seropositive children with CF [128], in ambulatory adults over age 60 [129], and in the elderly [130]. As with PFP-1, no signiﬁcant adverse reactions were associated with PFP-2 during testing. Moreover, no enhanced RSV disease was seen in any of the trials in vaccinated children who subsequently became infected with RSV. Unfortunately, neither PFP preparation has been studied in young infants, particularly those with underlying risk factors. A third generation of PFP, PFP-3, is also being developed. This material is currently in phase II clinical trials. A systematic overview of the PFP trials was recently performed to assess whether these vaccine preparations are efﬁcacious in preventing RSV-induced LRTI [131]. It was concluded that they do reduce the overall incidence of all RSV infections but that the clinically important outcome of RSV LRTI is not reduced. It was recommended that, because of concerns about the pooling of data that was done in different clinical trials, these vaccines be tested in large ﬁeld trials. BBG2Na is a synthetic polypeptide produced in Escherichia coli. It is composed of residues 130–230 of RSV-A G protein (G2Na) fused to BB, an albumin-binding domain of streptococcal protein G. (This fusion potentiates the immunogenicity of the polypeptide.) Within this domain are 12 amino acids that are present in both A and B RSV subtypes. BBG2Na has been shown to be immunogenic and protective in mice following administration by different inoculation routes including intramuscular [132] and intranasal [133]. Equivalent protection was seen against both subgroup A and B RSV strains [134]. In other animal studies, BBG2Na administered intranasally with cholera toxin B or zwittergent 3–14 generated both mucosal and systemic antibody responses that protected the test animals against RSV challenge and did not induce lung immunopathology upon subsequent RSV challenge [135]. This ﬁnding is important because early studies showed that BBG2Na induced a predominant type 2 T-cell response upon immunization. In recent testing in adults, BBG2Na appeared to be both safe and immunogenic [136]. Less favorable results may have occurred in other trials since testing of this product has been discontinued. Synthetic RSV peptides have also been tested as potential vaccine candidates. Mucosal delivery of one of these, when combined with enterotoxin-based adjuvants, elicited CD8þ T-cell responses in mice [137]. However, this response appeared to be both protective and immunopathogenic. All of these peptide vaccine candidates are still experimental and in preclinical study.

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Vaccines based on RSV subunits or synthetic peptides may be particularly useful in older children, adults, and the elderly. These are populations that have already experienced RSV infections and are thus immunologically primed. For this reason, a peptide or subunit vaccine may be able to induce protective secondary immune responses in these populations that it does not induce in infants. These vaccines may also be useful to boost levels of protective antibodies in pregnant women. (These women would be immunologically primed and thus should respond relatively well to subunit or peptide vaccines.) In addition to standard testing, subunit RSV vaccines have been tested in combination with recombinant virus preparations [138]. Other investigators have been investigating the effects of various adjuvants on the immunogenicity of subunit vaccines (see, e.g., Ref. 139). One caution should be emphasized. Increased pulmonary cellularity has been seen in cotton rats infected with RSV after immunization with one of the chimeric FG preparations [140]. 5.2.4

DNA Vaccines

Several RSV DNA vaccines aimed at inducing expression of proteins with protective epitopes have been developed [141–144]. Such vaccines have the potential to be highly immunogenic and capable of inducing strong protective humoral and CMI responses. However, a major concern with DNA vaccines is their safety, especially in the long term and in infants who have relatively high endogenous DNA synthesis and replication. For this reason, DNA vaccines may be more suitable in an older population, because there is less cellular replication taking place in these individuals.

6 6.1

ANTIVIRAL AGENTS Passive Antibodies

At the present time, two agents, RSV immunoglobulin (RSVIG) (RespiGamTM, MedImmune, Inc., Gaithersburg, MD) and palivizumab (SynergisTM, MedImmune, Inc., Gaithersburg, MD), are currently licensed for prophylactic use against RSV. The former material contains polyclonal antibody to RSV and is prepared from serum that has been shown by screening tests to have high Nt antibody titers to this virus. For this reason RSVIG is more efﬁcient against RSV than standard Ig preparations. RSVIG is generally administered intravenously once a month during the RSV season and has been shown to prevent or reduce

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RSV disease severity in infants and young children who are exposed to this virus after its administration [145–148]. RSVIG was licensed in the United States in 1996 [167]. However, it is not recommended for use in infants with cyanotic CHD, because it was associated with an excess of adverse events in this population during testing. In addition, it is generally not used outside the United States because of the difﬁculty in administrating it (i.e., i.v.) and because of the large ﬂuid volumes (15 mL/kg) and high protein load (750 mg/kg) involved in its use. Because passively administered antibodies can interfere with vaccine efﬁcacy, administration of live virus vaccines (e.g., MMR and varicella) is often postponed after use of RSVIG. Another problem associated with RSVIG is its high cost [149,150]. Palivizumab was developed because it was desirable to ﬁnd a material that did not require intravenous infusion, administration of relatively large volumes, multiple visits, and a hospital setting for proper administration [146]. This humanized monoclonal IgG1 antibody has virus-Nt activity against antigenic site A on the F protein of RSV and thus potentially can prevent infection of both A and B subtypes of RSV [151]. Although it contains a low amount (5%) of murine amino acid sequences, no adverse effects have been seen with this product. Palivizumab has been shown to prevent or reduce RSV disease severity in preterm infants and infants with chronic lung disease who are exposed to this virus after its administration [90,151–153]. Currently palivizumab is usually given intramuscularly at a dose of 15 mg/kg prior to the beginning of the RSV season, with subsequent doses given monthly throughout the RSV season. Speciﬁc prophylaxis usually starts when circulation of RSV in the community is veriﬁed. The Food and Drug Administration approved palivizumab for clinical use in 1999, and guidelines have been put forth by the American Academy of Pediatrics Committee on Infectious Diseases [154]. However, it is approved only for prophylactic use because it has not been shown to prevent RSV replication following therapeutic administration. Interestingly, unlike RSVIG, it has not been shown to reduce development of otitis media [155]. Regardless, palivizumab is the ﬁrst monoclonal antibody ever licensed for use against any infectious disease. Like RSVIG, palivizumab is expensive, and this has caused heated debate about its cost-effectiveness [156,157]. The debate is in ﬂux because of differences in analysis, the increasing cost of an average hospital stay, and the ethics of withholding a treatment that has been shown to be effective and safe [10]. Because of the expense, it has been suggested that patients selected for immunoprophylaxis with palivizumab be selected carefully [158]. The American Academy of Pediatrics [154] has issued

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guidelines recommending that the decision to use palivizumab should be made by a specialist and that it be used in children at greatest risk of complication to RSV (e.g., babies with chronic lung disease severe BPD and very preterm babies discharged from the neonatal unit shortly before or during the RSV season). Palivizumab is currently not recommended for use in some high-risk groups such as children with congenital heart malformations, cystic ﬁbrosis, malignancies, immune deﬁciencies, or neuromuscular diseases, because it has not been studied in these groups.

6.2 6.2.1

Chemotherapeutic Agents Antiviral Targets

Most antiviral agents are designed to prevent virus replication by interfering with one or more of the replicative steps essential for virus replication. In the case of RSV this would be virus attachment to and penetration of the host cell, transcription of negative-stranded vRNA into mRNAs and more vRNA, translation of mRNAs into functional (e.g., P and L polymerase enzymes), structural (e.g., M, SH, F, and G), and nonstructural (e.g., NS1 and NS2) proteins, and/or viral assembly and/ or egress (budding) of the virus out of the host cell. Interference at any of these steps can reduce or truncate infection and result in decreased infection and disease. These agents can come in many forms, some of which are discussed below. In the case of RSV, some host cell enzymes that have been targeted are inosine monophosphate dehydrogenase (IMPDH), S-adenosylhomocysteine hydrolase (SAH), L-aspartic acid transcarbamoylase, ornithine monophosphate decarboxylase (OMP decarboxylase), and cytosine triphosphate synthetase (CTP synthetase; see Ref. 159 for a detailed review). Selectivity apparently comes about in this situation because in most nonrapidly dividing host cells these enzymes are functioning at a signiﬁcantly lower level than occurs in virus-infected cells. Regardless, these compounds generally have lower therapeutic indices (i.e., generally are less selective toward virus-infected cells) and can be cytotoxic to the host. Many nucleoside analogs, the next subject in this chapter, fall in this category. The only one that will be discussed in detail is ribavirin. That is because this nucleoside analog is currently the only one that is licensed for use against RSV, and it is, of course, a prototype for this class of antiviral agents. Its successes and problems are probably true, to a greater or lesser extent, of many nucleoside analogs, particularly those designed to inhibit negativestranded RNA viruses.

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Ribavirin

Ribavirin (1-b-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) (Virazole, ICN Pharmaceuticals) is a synthetic nucleoside analog of guanosine. It was approved in 1986 to treat RSV infections when administered as an aerosol [160] following demonstration that it could inhibit RSV replication in tissue culture [161], protect cotton rats from pulmonary RSV infection [162], and in clinical trials provide medical beneﬁt (i.e., signiﬁcantly improve overall clinical scores, decrease lower respiratory tract–associated symptoms, and improve the arterial saturated air (SaO2) in treated infected infants [163,164], even in those with underlying CHD or BPD) [165]. Mechanism studies have shown that ribavirin must be phosphorylated to manifest its antiviral activity. Initial phosphorylation to its monophosphate form occurs as the drug is transported into the host cell. It is then serially phosphorylated to a triphosphate, which is believed to be the active form of the drug that inhibits IMPDH in host cells. Inhibition of this enzyme is thought to lead to a depletion of intracellular pools of guanosine triphosphate, which in turn interferes with vRNA synthesis [166]. Support for this mechanism is provided by the fact that the addition of guanosine can reverse the inhibitory activity of ribavirin in cell culture [167]. Ribavirin triphosphate may also interfere with virusspeciﬁc RNA polymerase initiation and elongation steps required for the synthesis of essential viral proteins, because it is known to do this during inﬂuenza virus replication [168,169]. The half-life of ribavirin in lung tissues is approximately 2 hr. In contrast, its T1/2 in serum is 300 hr. The major catabolite of ribavirin is triazole carboxamide, which is inactive and is excreted in the urine [170]. It is important that ribavirin treatment be started as soon as possible after the onset of symptoms because RSV pulmonary titers peak shortly after the onset of symptoms [23]. This point deserves emphasis, because it may be responsible for much of the variable ﬁndings and controversy associated with this compound. It is also important to emphasize that although it is highly desirable to deliver ribavirin early in infection, it is also desirable that the RSV diagnosis be veriﬁed, e.g., by antigen detection. Ribavirin alone or combined with RSVIG is recommended for use with transplant patients with mild or moderate pneumonia (inﬁltration with or without mild or moderate hypoxia) [171]. However, transplant patients with serious pneumonia requiring ventilator treatment presently have a mortality rate of 100%, irrespective of antiviral therapy, and therefore such therapy is not recommended for these patients [172,173]. It

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is highly recommended that transplant patients exhibiting symptoms of respiratory infection be tested frequently for RSV and if this virus is present, that they be treated before it descends to the lower respiratory tract. Despite its long use and licensing, ribavirin’s favor has been declining steadily. There are a number of reasons for this. First, there are potential safety problems, not only to patients receiving the drug but also to health care workers working in the vicinity of where ribavirin is being aerosolized [174]. The primary concern is that ribavirin is known to have teratogenic, carcinogenic, and mutagenic side-effects in animal models [175,176]. Thus, it is important that pregnant women (e.g., female nurses and doctors) not be exposed to the drug. It is recommended that all individuals concerned be informed about possible exposure risks during ribavirin aerosolizations and that adequate ventilation be ensured during the procedure. Another factor contributing to the declining acceptance of ribavirin has been the failure of many more recent studies to demonstrate any signiﬁcant beneﬁcial clinical effect of treatment with ribavirin. Indeed, many of the early studies have now been criticized with respect to their methodology and their use of subjective endpoints such as clinical score rather than major endpoints such as mortality, SaO2 , and mechanical ventilation. Furthermore, aerosolized water was used in some of these studies as the placebo, and this may have induced bronchospasms in the placebo groups [177]. These problems and the high cost of ribavirin ($3,300 per case [178]) led the American Academy of Pediatrics in 1996 to alter their recommendations for the use of ribavirin from ‘‘should be used’’ to ‘‘may be considered.’’ In addition, their new guidelines indicate that treatment with ribavirin should be limited to use in high-risk children (i.e., those with CHD, BPD, or premature infants or those aged <6 weeks) or immunodeﬁcient or severely ill children, with or without mechanical ventilation. 6.2.3

Other Nucleoside Analogs with Activity Against RSV

Inosine monophosphate dehydrogenase (IMPDH) has been the target of numerous other compounds, several of which are analogs of ribavirin that inhibit RSV replication better than the parent compound. One of these, EICAR (5-ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide) appears to have the same mechanisms of action as ribavirin but is approximately 30-fold as potent as the licensed compound in cell culture assays [167] and is also more active against RSV than ribavirin in cotton [179]. Two other analogs, mizoribine (4-carbamoyl-1-b-D-ribofuranosylimidazolium-5-olate) and 3-deazaguanine, also appear to be more potent than ribavirin in tissue culture testing [180,181]. However, neither of

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these compounds has been tested in animals. LY-253963 and VX-497 are two compounds unrelated to ribavirin. The former is a prodrug of an inhibitor of IMPDH that requires sequential glycosylation and phosphorylation in order to express its biological activity. This compound exhibited signiﬁcant RSV inhibitory activity in vitro and in vivo [182] but did not make it into clinical studies. VX-497 is an IMPDH inhibitor that has come into the fore rather recently. This material reversibly inhibits IMPDH catalysis of inosine monophosphate (IMP) to xanthine monophosphate and has a 20-fold higher activity against RSV than ribavirin on a molar basis [183]. However, its selective index (ratio of median cytotoxic concentration to median efﬁcacious concentration) is nearly threefold lower than that of the licensed compound. No in vivo studies have been reported. Two compounds that have been shown to strongly inhibit SAH and RSV replication are 60 -ðRÞ-60 -C-methylneplanocin A [184] and carbocyclic 3-deazaadenosine [185]. However, both of these compounds also had cytotoxic manifestations [185,186] and did not advance to clinical trials. Pyrazofurin [3-(b-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide] inhibits OMP decarboxylase and RSV in tissue culture assays at low concentrations (e.g., 70 ng/mL) [180]. However, in cotton rats, cytotoxic effects were noted in the test animals at relatively low doses, and a therapeutic index of <10 was obtained [187]. This ﬁnding makes evident a common occurrence seen in antiviral testing. Many potential antiviral agents that exhibit signiﬁcant selective antiviral activity in tissue culture assays do not do so in vivo where there are more complex and sensitive tissues, organs, and systems. N-(phosphonoacetyl)-L-aspartate (PALA) is an example of a compound that is a potent inhibitor of L-aspartic acid transcarbamoylase. In in vitro testing, PALA exhibited marked cytotoxicity to proliferating cells but much less toxicity to cell cultures containing conﬂuent monolayers (i.e., stationary cells). In cotton rats, signiﬁcant reductions in pulmonary titers (0.8–1.4 log10/g lung), compared to pulmonary viral titers in placebo-treated control animals, were consistently seen in cotton rats given 510 mg PALA kg1 day1 twice daily i.p. on days 1–3 postinfection with either subtype A or B RSV. No toxic effects were noted even in animals given 100 mg of PALA kg1 day1 for seven consecutive days [188]. This compound did not advance to clinical trials. Cyclopentenylcytosine is a broad-spectrum antiviral agent that has been shown to be active against several paramyxoviruses [189]. The putative target is cytodine triphosphate synthetase, an enzyme that converts uridine triphosphate to cytodine triphosphate (CTP). Supporting this hypothesis is the ﬁnding that the antiviral and cytocidal effects of

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cyclopentenylcytosine are both readily reversed by the addition of cytosine and, to a lesser extent, uridine but not by other nucleosides such as thymidine. Although this material does not appear to be suitable as an antiviral agent itself, it has been recommended that further work be done testing it in combination with inhibitors of other enzymes essential for virus replication (e.g., OMP decarboxylase and inhibitors of this enzyme such as pyrazofurin or 6-azauridine) [189]. 50 -Noraristeromycin, 3-deaza-5’-noraristeromycin, and 7-deaza-5’noraristeromycin are inhibitors of SAH. They are worthy of mention primarily because their enantiomeric forms exhibit markedly different antiviral activity [190,191]. Two carbocyclic nucleoside analogs with modiﬁed purine bases, 9238X [192] and 20 -deoxy-20 -ﬂuororibonucleoside [193], also inhibit RSV in vitro assays and in 1996 were patented by the Wellcome Foundation [194]. The status of these compounds with respect to whether they are moving forward is not known. None of the nucleoside analogs just described, with the exception of ribavirin, has advanced out of preclinical testing. Part of the problem is that many of these agents have already been shown to be toxic to proliferating cells (e.g., pyrazofurin [187] and neplanocin [186]) and are not good risks to take into a clinical setting, particularly for use against a virus that primarily affects infants, immunocompromised individuals, or those already greatly weakened by age and/or inﬁrmities.

6.2.4

Antisense Inhibitors

Another, rather unique, approach to controlling RSV infections that has been evolving during the past decade is the development of antisense oligonucleotides designed to speciﬁcally inhibit RSV replication. These molecules are synthesized strands of nucleic acids that are complementary and of opposite polarity to the portion of the viral genome that is being targeted. To be effective, these molecules must be able to get to targeted (RSV-infected) cells, enter them, and reach the cellular compartment that contains viral nucleic acids. There they must bind, inhibit, or inactivate viral nucleic acid transcription or translation, all before they are removed and/or degraded. Various RSV genes have been targeted. However, those complementary to the M2 and L genes have exhibited the most signiﬁcant antiviral activity in tissue culture–based assays [195,196]. Still to be overcome is the problem of how to deliver these molecules in vivo. For respiratory viruses, it may be possible to reduce some aspects of this problem by direct delivery of these molecules to the respiratory tract by small-particle aerosolization [197].

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Non-Antibody Inhibitors of RSV Attachment to Host Cells

The host cell receptor for RSV is thought to be a highly sulfated, heparinlike glycosaminoglycan located on the cytoplasmic membranes of host epithelial cells [39,198]. The receptors for A and B RSV subgroups have also been well studied [39]. Using this knowledge, disruption of infection through competitive binding of either the G protein or the cell receptor should be possible. Indeed, many compounds have been shown to interfere with this interaction, including heparin [199], heparin-like modiﬁed biopolymers [200], derivatized dextrans [201], and polysulfonates and sulfated polysaccharides from marine microalga [202]. Among the most interesting of the compounds that block RSV attachment to host cells are the negatively charged polyoxometalates (POM). This class of compounds consists of complexes of cationic alkaline earth metals containing oxygen and various transition metal ions. Several POMs have been tested and found to inhibit laboratory and clinical strains of RSV in cell culture [203]. In these studies it was determined that those containing germanium, niobium, tin, and zirconium were the most potent and selective for RSV replication in Hep-2 cells. EC50 values ranging from 0.1 to 10 mM were reported. Thus, in vitro, these compounds appear to be up to 100 times as active as ribavirin. The inhibition seen is apparently due to interference with virus adsorption and syncytium formation [204]. Virtually all of the inhibitors of virus attachment and penetration suffer the same weakness. They must be present in relatively high concentrations at, or near, the time of virus infection, making them mostly impractical for clinical use. However, their extraordinary antiviral activity and low cytotoxicity in tissue culture assays (i.e., many have selective indices >1000) make them of continuing interest. 6.2.6

F Protein Inhibitors

The F protein is thought to play a role in RSV attachment and penetration of the host cell as well as being involved in cell-to-cell spread of the virus (i.e., syncytium formation) [205]. With this in mind, a computer-based searching strategy designated C.A.S.T. was used to identify domains of heptad repeats of the F protein of RSV that are thought to be important in the later stages of fusion of the RSV envelope and host cell membrane during attachment and penetration of RSV into the host cell. Three such domains were identiﬁed, two in the F1 portion of the F protein designated HR1 and HR2, and one in the F2 portion, designated HR3 [206]. An amino acid peptide with overlapping sequences of RSV-HR2

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(amino acids 486–521) was found to have the most potent anti-RSV activity in vitro (EC50 ¼ 0.05 mM), and this inhibitory activity was virusspeciﬁc. Reports of the activity of this material in animals have not been made. However, like other peptides that inhibit RSV attachment, for this peptide to be effective in vivo a way will have to be found to maintain protective levels of it at the site of infection. VP-14637 is the lead compound in a series of low molecular weight inhibitors that are under preclinical investigation by ViroPharma for potential use against RSV infection. In tissue culture assays, VP-14637 inhibited RSV strains in very low concentration (6 nM compared to 40 mM for ribavirin) [207]. Time of addition studies suggested that this compound acts on an early event in the virus replication cycle, most probably fusion of the virus and host cell membrane. Virus yield assays indicated that RSV production is completely inhibited during multiple rounds of viral replication. Moreover, the compound is effective against both RSV A and B subtypes. Resistance to VP-14637 was observed and mapped to mutations in the virus F protein, providing more evidence that VP-14637 is an inhibitor of viral fusion. Cotton rats experimentally inoculated with RSV and exposed to doses of 7.2 mg VP-14637 per day by small-particle aerosol had reduced lung virus titers ranging from 1.3 to 42:8 log10 =g lung, compared to the mean pulmonary virus titers seen in the control animals. Researchers at Wyeth Ayerst using a high-throughput screening program to ﬁnd an effective inhibitor of RSV elucidated a series of biphenyltriazine anionic compounds that possess speciﬁc anti-RSV activity [208]. The most active compound identiﬁed was given the designation RFI-641. Mutations in RSV strains resistant to RFI-641 map to the F protein [209], and the compound inhibits an RSV strain that is devoid of both the G and SH surface proteins. These data strongly suggest that the target of this compound is the F protein. In tissue culture, RFI-641 was found to be active against both A and B groups of RSV [209]. In these tests, the compound appeared to be 32-fold more active than ribavirin (median EC50 value against ﬁve RSV strains ¼ 143 nM), and selectivity indices ranged from >417 to >2500. It was not effective in cotton rats when administered parenterally, but it was when given topically [210]. RFI-641 has also been shown to have antiviral activity in African green monkeys [211]. In the latter model, prophylactic administration of RFI-641 signiﬁcantly reduced viral titers in nasal washes (1.7 log10 measured at the peak of virus infection). The fact that this compound can signiﬁcantly inhibit RSV in animals following topical administration is exciting. These are really the ﬁrst studies to provide evidence that

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molecules that can inhibit virus attachment and/or penetration into host cells can be practically administered and still be active. Development of this compound is ongoing [211]. 6.2.7

Miscellaneous Compounds and Materials

Human eosinophil–derived neurotoxin (EDN) has been shown to mediate the ribonucleolytic destruction of extracellular RSV [212]. Cetirizine (2-[2-[4-(4-chlorophenyl)phenyl-methyl]-1-piperazinyl] ethoxyacetic acid) is an antihistamine that has been reported in a patent application to inhibit RSV replication [213]. RD3-0028 [1,4-dihydro-2,3bonzodithiin] is an interesting compound that inhibits RSV infection both in tissue culture and in mice. In tissue culture, this compound inhibited laboratory and clinical isolates of both RSV A and B subgroups. The EC50 obtained was *4 mM, and its median toxic concentration to cells was 271 mM [214]. NMSO3 is a sulfated sialyl lipid (molecular weight 1,478.8) that appears to inhibit RSV replication by several mechanisms: (1) binding directly to the F protein and inhibiting fusion, (2) inhibiting penetration (seen by shifting temperature during the period of contact between the virus and cells), and (3) inhibiting syncytia formation [215]. A peptide comprising amino acids 77–95 of Rho A (a member of the Ras superfamily of small GTP-binding proteins) has been shown to block RSV syncytia formation and RSV entry into host cells [216]. In tissue culture assays, an EC50 of 0.54 mg peptide/mL was obtained. When administered intranasally to mice at a dose of 500 mg/animal, a 200-fold reduction in pulmonary RSV titer was seen if the Rho A was administered at the same time as virus challenge, and a 20-fold reduction was observed in this model if the Rho A was given 4 days postinfection (1 day prior to killing) [217]. Interestingly, mice given the Rho A simultaneously with virus had no apparent illness or weight loss, whereas those treated 4 days after infection exhibited the same degree of illness and weight loss as infected control animals given placebo. In a related study, mice were treated with lovastatin, a drug that inhibits prenylation pathways in cells by directly inhibiting hydroxymethylglutaryl coenzyme A reductase [218]. Giving this material to mice up to 24 hr after RSV inoculation caused a signiﬁcant reduction in mean pulmonary virus titer, weight loss, and illness compared to control animals that did not get this treatment. Because lovastatin also reduces syncytia formation in cell culture and eliminates RSV replication in HEp2 cells, it is more likely that this compound inhibited RhoA membrane localization and virus fusion. Because lovastatin is already approved for use, it has been recommended that this compound should be considered

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for evaluation as a preventive antiviral therapy for selected groups of patients at high risk for severe RSV disease such as the institutionalized elderly and bone marrow or lung transplant recipients [218]. A number of plant extracts have been shown to inhibit RSV replication. Some examples reported recently include an extract from Eleutherococcus senticosus roots [219], iridoid glycosides isolated from Barleria prionitis [220], ferulic and isoferulic acid extracted from Cimicifuga beracleifolja rhizomes [221], and a 10,425 Da protein puriﬁed from the edible mushroom Rozites caperata [222]. Several extracts from the medicinal plant Barleria prionitis found throughout Africa, India, Sri Lanka, and tropical Asia [220] and extracts of three species of medicinal plants obtained from Argentina (i.e., Polygonum punctatum, Lithraea molleoides, and Myrcianthes cisplatenis) [223] have also been reported to inhibit RSV. None of these plant extracts has been evaluated in vivo. Other miscellaneous compounds that have been reported to have RSV-inhibiting activity are pyridobenzazoles [224], a synthetic derivative of 1,5-dideoxy-1,5-imino-D-glucitol [225], pyridobenzazol [226], benz[de]anthracen-7-one [227], and 2,20 ,4’-methylidynetriphenol [228]. The latter is of particular interest because it has been reported by Viropharma Inc. to have a selective index of >150,000 against RSV. 6.2.8

Interferons

In mice, interferon gamma (IFN-g) clearly appears to have the potential to limit RSV replication and inﬂammatory responses, because animals to which the gene for IFN-g has been transferred are protected against infection with this virus [229]. Moreover, knockout mice that cannot produce this cytokine innately or following inoculation with antibodies speciﬁc for IFN-g develop more extensive inﬂammation of the airways and disease than control mice after RSV infection [230]. However, the IFN-defective mice also develop less obstruction of their airways, suggesting that this cytokine may have a pathogenic as well as protective role in these models. Also of interest is the fact that IFN-g production in mice does not appear to reduce pulmonary eosinophilia. The latter can occur following experimental infection with RSV despite abundant IFN-g production by local T-cells [231]. In humans, IFN-g is often detected following RSV infection and has been detected in the lower respiratory tracts of infected babies [232]. It has also been shown that signiﬁcant levels of IFN-g are produced following infection of human leukocyte cultures with RSV [233]. A protective effect for IFN-g is indicated by the fact that children with severe RSV LRTI that requires mechanical ventilation is associated with low IFN-g production [234]. In addition, peripheral blood mononuclear

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cells obtained from infants with severe RSV disease express less IFN-g than peripheral blood mononuclear cells obtained from other test groups [233]. However, there are also data suggesting that IFN-g may contribute to some RSV-induced disease. For example, higher levels of this cytokine have been reported in children with virus-induced wheezing than in nonwheezers [230]. Thus, the roles of IFN-a and IFN-b in RSV disease remain unclear. The effects of exogenously administered IFN-a on the course of RSV infection have been studied following administration intramuscularly [235–237], intranasally, or as a small-particle aerosol [238]. No clinical efﬁcacy or cytotoxicity was seen in these studies, and IFN therapy for RSV infections has not been pursued. 6.2.9

Bronchodilators and Corticosteroids

Although not true antiviral agents, bronchodilators and corticosteroids have commonly been used to try to mitigate the inﬂammatory effects that take place in the lower respiratory tract of the host in response to RSV infection and that appear to play an important role in the bronchiolitis and other respiratory problems initiated by this virus. However, highly variable results have been obtained following the use of these agents, and their effectiveness in ameliorating RSV disease is unclear and a subject of much debate (see reviews in Refs. 239–241). 6.2.10

Oxygen

There seems to be a consensus that oxygen is useful for the treatment of acute bronchiolitis by functioning to maintain oxygen saturation (SaO2) levels [10]. 6.2.11

Vitamin A

Because vitamin A concentrations in infants with RSV disease are often inversely proportional to disease severity, it was thought that this vitamin might be a useful adjunctive therapy for treating severe RSV infections [242–243]. However, although proven safe, oral administration of vitamin A has not proved to be effective in decreasing morbidity in children with acute RSV infection [242]. 7

CONCLUSIONS

An ever-increasing appreciation of the prevalence of RSV and its medical impact in multiple populations has led to a burgeoning interest in this virus as a target for prophylactic and therapeutic agents. The success of Synagis as a prophylactic agent and the signiﬁcant deﬁciencies

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associated with ribavirin as a therapeutic agent have further heightened interest in this area. Live attenuated, recombinant, subunit, peptide, and DNA RSV vaccines are being developed and evaluated. However, licensing of any of these vaccines may take years. Moreover, if any is approved, it will not likely be approved for, or efﬁcacious in, all target populations (especially immunoincompetent individuals and very young infants). Use of a combination of separate strategies, e.g., maternal immunization followed by immunization of the infant with a subunit vaccine, may be necessary to increase the utility and protection of any approved vaccine(s) [82]. Regardless, even following the licensing of one or more RSV vaccines, there will still likely be a need for effective chemotherapeutic or biological antiviral agents to be used against infections caused by this virus. This is certainly true in the absence of an approved RSV vaccine. Similarly, when new safe and efﬁcacious RSV antiviral agents are approved, they may not be effective alone and may have to be given in combination or coadministered with an antiinﬂammatory agent in order to more effectively alleviate disease symptoms. Clearly it is desirable to have available both safe and effective vaccines and antiviral agents to reduce the medical impact of RSV. Based on current efforts, there is good reason to be optimistic that this state will come about in the not too distant future.

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4 Rhinovirus Ronald B. Turner and Frederick G. Hayden University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A.

1

INTRODUCTION

The isolation of a rhinovirus in cell culture in 1956 was promptly followed by the recognition that these viruses were a major cause of the common cold. Rhinoviruses are now recognized as the most frequent cause of viral upper respiratory tract infections [1–3]. Epidemiological studies based on isolation of virus in cell culture indicate that both adults and children experience a rhinovirus infection every 1–2 years [4,5]. The relative insensitivity of cell culture isolation suggests that these attack rates are underestimates, but systematic epidemiological surveys using more sensitive polymerase chain reaction methods have not been done. The risk of rhinovirus infection is highest in young infants and gradually declines with increasing age. These infections are frequently brought into the home by young children, and there is a slight increase in the incidence of infection in young adult parents. The rhinoviruses cause infection yearround but are associated with seasonal epidemics in the fall and spring. The onset of common cold symptoms associated with rhinovirus infection typically occurs 1–2 days after infection. The time to peak symptoms is generally 2–4 days after infection [6]. Nasal obstruction, 139

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rhinorrhea, and sneezing are present early in the course of the illness, but sore or ‘‘scratchy’’ throat is frequently reported as the most bothersome symptom on the ﬁrst day of symptoms [1,6–8]. The sore throat usually resolved quickly, and by the second and third day of illness the nasal symptoms predominate. Cough is associated with approximately 30% of colds and typically does not become the most bothersome symptom until later in the illness when the nasal symptoms decrease in severity [6,7]. The usual cold lasts about a week, although 25% last 2 weeks [7]. Virus shedding persists after the resolution of symptoms, and virus may be cultured from 10–20% of subjects for 2–3 weeks after infection [9]. The common cold is generally associated with little morbidity, although the complications of these illnesses have a substantial medical impact (see Table 1). The most important complications of common colds are otitis media in children and exacerbations of reactive airways disease. Bacterial sinusitis is also a recognized complication of viral upper respiratory infection, although recent evidence indicates that sinus involvement is a part of the common cold syndrome and it is difﬁcult to differentiate this viral sinusitis from bacterial superinfection. In spite

TABLE 1 Illnesses Associated with Rhinovirus Infection

Illness Common cold Otitis media

Sinusitis Exacerbation of asthma Exacerbation of cystic ﬁbrosis Exacerbation of chronic obstructive pulmonary disease

Population at risk All ages Primary children, although ~70% of colds are complicated by abnormal middle ear pressure in all age groups All ages All ages

Proportion of illnesses associated with rhinovirus 50% 30%

All ages

6–50% 60–70% in children, 19% in adults 16%

Adult

23%

Source: Refs. 9a, 9b, 9c, 9d, 9e, 9f, 62.

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of the medical signiﬁcance of the common cold and its complications, attempts to develop effective treatments have been relatively limited and unsuccessful. Another important consequence of the common cold is the inappropriate use of antibiotics for these illnesses and the associated contribution to the problem of increasing antibiotic resistance in pathogenic respiratory bacteria. In the United States in 1998, there were an estimated 84 million ofﬁce visits for acute respiratory illnesses (ARIs), including 25 million primary care ofﬁce visits for colds [10]. Antibiotics were prescribed for over one-half of all ARIs and 30% of colds, despite lack of evidence to indicate clinical beneﬁt [11,12]. Effective prevention or treatment of rhinovirus infection would potentially have a beneﬁcial impact on this problem. 2 2.1

PATHOGENESIS Transmission

The pathogenesis of the common cold presents several potential targets for interrupting rhinovirus transmission, infection, and rhinovirusassociated illness. During a rhinovirus cold, the virus is present in nasal secretions at titers of 102–103 TCID50/mL of nasal lavage ﬂuid [13,14]. This virus is readily transmitted to the hands of the infected individual and to objects in the environment. Virus can be recovered from the hands of approximately 50% of infected individuals and approximately 10% of objects in the environment of these individuals. Once in the environment, rhinovirus can survive hours to days [13–15]. Rhinovirus may be transmitted either by direct contact of a susceptible individual with virus on the hands of the infected person or on objects in the environment, followed by self-inoculation into the nose or eye, or by large-particle aerosols generated by the infected individual [15,16]. The role of direct contact in the transmission of rhinovirus infection suggests the possibility that transmission could be prevented by the use of virucidal agents directed at removing rhinovirus from the hands. 2.2

Replication

Rhinovirus replication is initiated by attachment to a receptor on the cell surface. The cellular receptor for most rhinovirus serotypes (major receptor group) is intercellular adhesion molecule-1 (ICAM-1) [17–19]. The rhinovirus capsid has an icosahedral symmetry formed of 60 identical protomers consisting of four proteins designated VP1–VP4. Crystallographic techniques have identiﬁed a depression in the capsid

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surface, the so-called ‘‘canyon,’’ surrounding each of the 12 ﬁvefold axes of symmetry [20]. This canyon is the binding site for the ICAM-1 receptor molecule [21]. At the bottom of the viral canyon is a hydrophobic pocket. The current working hypothesis for the mechanism of viral uncoating suggests that this pocket contains a lipid moiety or ‘‘pocket factor’’ that is expelled when ICAM-1 binding occurs (reviewed in Ref. 22). The loss of the lipid moiety destabilizes the viral capsid and results in a change in conformation that allows release of the viral RNA. The hydrophobic pocket is also the site of binding for the capsid-binding agents that are currently under investigation as antiviral agents for treatment of rhinovirus infections [23]. Binding of these agents in the capsid pocket appears to inhibit attachment to ICAM-1 and stabilize the conformation of the viral capsid, thus inhibiting viral uncoating [24–27]. The relative importance of these mechanisms of action appears to vary depending on the viral serotype. A second aspect of viral replication that has been targeted by antiviral agents is the post-translational modiﬁcation of viral proteins. The rhinovirus genome encodes a single large polyprotein that is cleaved to produce the individual structural and enzymatic proteins of the virus (reviewed in Ref. 28). Most of these cleavage reactions are catalyzed by a protease designated the 3C protease. This protease has a structure similar to that of trypsin, a serine protease, but the active site of the protease is a cysteine sulfhydryl [29]. The active site sequences are highly conserved in the different rhinovirus serotypes, and inhibitors of 3C protease have potent anti-rhinovirus activity for a broad spectrum of rhinovirus serotypes [30]. As discussed below, one of these agents is currently under study as treatments for rhinovirus colds. 2.3

Pathogenesis of Symptoms

The role of viral replication in the initiation of rhinovirus illness is clear, but efforts to treat rhinovirus colds by inhibition of virus replication have been generally disappointing. Recent research efforts have been directed at understanding the pathogenesis of rhinovirus-induced illness with the expectation that this effort might lead to more effective treatments for common cold symptoms. Only a small proportion of nasal epithelial cells are infected during a rhinovirus cold, and there is little evidence of direct viral damage to the nasal epithelium [31–35]. The observation that a polymorphonuclear leukocyte response was present during symptomatic rhinovirus infection but was absent in infected volunteers who were asymptomatic suggested that the host response to the virus might contribute to the symptom complex [36]. Subsequent studies identiﬁed

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interleukin-8 as one potential mediator of this host response [37,38]. Increased concentrations of IL-8 are present in the nasal secretions of subjects with symptomatic rhinovirus infection [38–40], and in experimental rhinovirus infection there is a modest correlation between the severity of common cold symptoms and the concentrations of IL-8 in the nasal secretions [40]. The observation that intranasal challenge of normal subjects with IL-8 produces symptoms that in some respects mimic the common cold also provides support for the hypothesis that IL-8 may contribute to common cold symptoms [41]. Other proinﬂammatory mediators have also been associated with rhinovirus cold symptoms. The kinins, bradykinin and lysylbradykinin, have been found in the nasal secretions of volunteers with rhinovirus colds, both experimentally induced and naturally acquired [36,42]. The concentration and time course of the production of kinins were roughly correlated with the severity and time course of symptoms in these subjects. Subjects who were infected with rhinovirus but who did not develop symptoms did not have an increase in nasal secretion kinin concentration. Intranasal challenge of uninfected volunteers with increasing concentrations of bradykinin resulted in symptoms of nasal obstruction, rhinorrhea, and sore throat [43]. The role of kinins in the pathogenesis of common cold symptoms is less clear, however, in light of the failure of a bradykinin antagonist to moderate common cold symptoms [44]. Similarly, in a more recent study, steroid therapy signiﬁcantly reduced the concentration of kinins in nasal washes but had no effect on symptoms [45]. The interleukins IL-1 and IL-6 have also been reported in the nasal secretions of symptomatic subjects with experimental rhinovirus colds [46,47]. As with IL-8 and the kinins, the concentration of these proteins increases and then decreases as symptoms wax and wane. The concentration of Il-6 in nasal secretions appears to be directly correlated with the severity of the common cold symptoms [47]. In spite of these data demonstrating an association between common cold symptoms and various inﬂammatory mediators, the role of these mediators in pathogenesis will not be clear until speciﬁc inhibitors are available for use in human studies. If these proinﬂammatory mediators are involved in the symptomatic response to rhinovirus infection, then treatments directed at inhibiting the elaboration or action of these mediators might be beneﬁcial. It remains to be determined, however, whether the nonspeciﬁc proinﬂammatory response to rhinovirus infection is an important contributor to the adaptive host responses that are required for natural recovery from, and development of immunity to, the rhinovirus infection.

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DIAGNOSIS OF RHINOVIRUS INFECTIONS

There are no rapid diagnostic methods currently available for the detection of rhinovirus infection that will be useful for guiding appropriate antiviral therapy. In the absence of effective antiviral treatments, a speciﬁc diagnosis of rhinovirus infection is not useful for the management of the common cold. Thus efforts to develop point-ofcare diagnostics for rhinovirus have been limited. Initial efforts to develop an immunoassay by using antibodies directed at the rhinovirus 3C protease have recently been described [48]. This assay, which uses thin-ﬁlm biosensor technology that has been successfully adapted for rapid point-of-care diagnosis of other human pathogens was capable of detecting a broad spectrum of different rhinovirus serotypes although it remains to be determined whether the assay will be sufﬁciently sensitive for clinical use. The only generally available method for the laboratory diagnosis of rhinovirus infection is isolation of the virus in cell culture. Polymerase chain reaction (PCR) has been used in the research setting and appears to be the most sensitive method for detection of rhinovirus infection (reviewed in Ref. 49). Under ideal conditions, cell culture will detect approximately 75% of the infections documented by PCR. Neither of these currently available diagnostic methods is useful for guiding clinical decisions in patients with the common cold syndrome. In the absence of clinically useful laboratory methods, the diagnosis of rhinovirus infection relies on clinical criteria. Recent evidence suggests that 60–80% of patients with a common cold syndrome (afebrile, prominent nasal symptoms, and minimal systemic symptoms) that occurs between late August and early November will have a rhinovirus infection. The sensitivity of clinical diagnosis of rhinovirus infection is not known. 4 4.1

GENERAL ISSUES IN USE OF ANTI-RHINOVIRAL AGENTS Assessment of Antiviral Action

The virologic course of experimentally induced and naturally occurring rhinovirus colds has usually been characterized by determining the yields of infectious virus in sequentially collected nasal secretion samples. During the ﬁrst several days of infection the titers of infectious virus peak at relatively low levels [approximately 103–104 50% tissue culture infectious doses (TCID50) per milliliter] and generally correlate with symptom severity (reviewed in Ref. 50). Recent studies indicate that viral RNA levels in nasal secretions are higher but also correlate broadly

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with symptom scores. However, clear relationships between pharmacokinetic (PK) properties and pharmacodynamic (PD) effects in terms of antiviral or clinical measures have not been deﬁned for the investigational anti-rhinoviral agents currently available or previously studied. For example, it is uncertain what magnitudes of antiviral effect with respect to reductions in upper respiratory tract viral titers are needed to prevent or treat rhinovirus illness. One limitation to deﬁning PK–PD relationships is the absence of a practical animal model of human rhinovirus infection. These viruses can successfully infect non-human primates, speciﬁcally chimpanzees and gibbons, but experimentally induced infections in susceptible human volunteers have provided the most informative in vivo model for assessing candidate anti-rhinoviral agents. For example, experimental prophylaxis studies with intranasally administered interferons have shown that dose-related protection can be achieved against rhinovirus infection and illness, such that high doses can prevent both infection, deﬁned by virus recovery and/or virus-speciﬁc serologic responses, and common cold illness (reviewed in Ref. 51). Somewhat lower doses allow laboratory-documented infection but protect against illness, whereas even lower doses do not prevent illness. Substantial evidence indicates that the speciﬁc mechanism of antiviral action and pharmacokinetic properties of candidate antirhinovirus compounds are important factors in determining route and frequency of administration as well as probable clinical utility. Agents that interact directly with intact virions (e.g., receptor decoys, capsidbinding agents), and possibly those that bind to speciﬁc host cell receptors (e.g., interferons, anti-receptor antibodies), need to achieve adequate concentrations in the extracellular ﬂuids lining the respiratory epithelium, whereas agents that inhibit an intracellular event in viral replication (e.g., viral protease or transcriptase action) need to reach adequate intracellular concentrations in the mucosa. These characteristics; the anatomy and physiology of the upper respiratory tract, including the effects of mucociliary clearance; and the pathogenesis of infection heavily inﬂuence the potential antiviral and clinical activities of antiviral agents for prevention and treatment of rhinovirus infections. 4.2

Prophylaxis Versus Treatment

In general, prevention of illness is easier to achieve than treatment of established rhinovirus colds. Several investigational agents (for example, intranasal interferons or pirodavir) are effective for chemoprophylaxis but not treatment of experimental or natural colds (reviewed in Ref. 51).

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Although these agents exert signiﬁcant but incomplete antiviral effects when used for treatment, no symptom beneﬁt has been recognized in treating colds. This lack of clinical efﬁcacy is probably due to the pattern of viral replication during rhinovirus colds, with early peaking and rapid declines in nasal viral titers, and to the fact that host inﬂammatory responses and neurogenic reﬂexes play important roles in the symptom pathogenesis (reviewed in Ref. 52). In addition, the magnitude of the observed antiviral effects with these agents was likely insufﬁcient. More recent studies with orally administered pleconaril establish that treatment of rhinovirus colds with selective inhibitors can reduce symptom duration and severity [53]. These ﬁndings indicate that ongoing, that is, continuing, viral replication is important in symptom pathogenesis and offer the possibility that prompt termination of replication might provide even greater clinical effects. Although symptom pathogenesis is incompletely understood, combinations of antiviral and host response–modifying agents would likely provide the greatest therapeutic beneﬁt. Several current challenges are to identify key host inﬂammatory responses and selective, welltolerated inhibitors of these responses. For example, studies of systemic or intranasally applied corticosteroids have found evidence for both lack of consistent symptom beneﬁt and upregulation of rhinovirus replication [45,54]. One treatment trial in young children with rhinovirus colds found that intranasal ﬂuticasone not only failed to provide clinical beneﬁt but also appeared to increase the likelihood of developing acute otitis media compared to placebo [55]. Such experiences indicate that host immune modulators need to be carefully assessed with regard to adverse effects on the virologic course of infection and will likely need to be used in combination with antiviral therapy. 4.3

Intranasal Administration

Available data indicate that delivery of antiviral agent to the nasal mucosa alone would be adequate for chemoprophylaxis of most rhinovirus colds. In particular, intranasal deposition of small quantities of infectious virus is sufﬁcient to initiate infection in almost all persons lacking serotype-speciﬁc humoral immunity. Most infections appear to start after virus reaches the posterior nasopharynx, which is rich in ICAM-1-expressing cells overlying the adenoidal tissue [56]. Furthermore, studies of intranasal interferon established that intranasal delivery of antiviral agents to the nasopharyngeal mucosa by coarse sprays or drops is protective against both experimentally induced and naturally occurring rhinovirus infection and illness (reviewed in Ref. 51).

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The method used for intranasal administration may have implications for clinical effectiveness. Several studies employing radiolabeled tracers found broader, more uniform distribution but more rapid clearance after administration by nasal drops in the supine position than by nasal sprays in the sitting position [57,58]. Although drops appear more active under experimental infection conditions, in which the virus inoculum is also given as drops, a deﬁnitive study comparing the efﬁcacy of antiviral sprays and drops has not been reported for natural rhinovirus colds. Although some rhinovirus infections may result from deposition of the virus in the lower respiratory tract, the clinical observations to date indicate that intranasal drug delivery is sufﬁcient for prevention of most rhinovirus colds. Rapid mucociliary clearance of topically applied agents that do not interact with speciﬁc cellular receptors (e.g., unlike interferons, antiICAM-1 antibodies) or reach inhibitory intracellular levels would likely necessitate frequent intranasal dosing. For example, pirodavir is a capsid-binding agent that presumably interacts with extracellular virus to inhibit early replication events. Intranasal sprays of pirodavir were protective against experimental rhinovirus when given six times daily but not when given three times daily [59]. A topically applied agent such as soluble ICAM-1 (tremacamra) that serves as a reversible receptor decoy would also probably require frequent intranasal dosing. In contrast, a topically applied agent that inhibits an intracellular replication event (e.g., the 3C protease inhibitor ruprintrivir or AG7088) might be effective on a relatively infrequent dosing basis if taken up and retained by cells in an antivirally active form. Although mucociliary clearance is reduced, often dramatically, during established colds, it is unclear what implications this pathophysiological event has for the distribution, retention, and activity of topically administered antiviral agents. In addition, the increased respiratory secretions due to vascular leak, goblet cells, and glandular activity during colds would likely diminish the local antiviral effects of topically applied agents. 4.4

Systemic Administration

Once symptoms have developed, broader antiviral distribution within the respiratory tract appears to be desirable for treatment of established illness. The extent of viral replication, as reﬂected by peak viral titers in nasal secretions and by in situ hybridization studies of nasal mucosal biopsies [31], appears limited in rhinovirus colds. However, rhinovirus colds cause radiologically documented, self-limited sinusitis in most cold sufferers [60] and are frequently associated with otitis media in children

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[61,62], as well as otological abnormalities indicative of Eustachian tube dysfunction in adults [63]. In situ hybridization studies indicate that virus replication can occur in the tracheobronchial epithelium [64] and in the maxillary sinus mucosa [65], and rhinoviral RNA is frequently detected in the middle ear ﬂuid of children with acute otitis media. Such observations suggest that replication commonly occurs at sites beyond the nasopharyngealmucosa and that inhibition of virus replication at such sites would be important to providing therapeutic beneﬁt and reducing the risk of complications. For example, drug delivery to the tracheobronchial tree might be needed to reduce the likelihood of exacerbations of asthma or chronic obstructive pulmonary disease. This would require an inhaled route of delivery for agents that lack oral bioavailability. Conversely, an orally administered agent that distributes widely within the respiratory tract has a greater likelihood of inhibiting viral replication at extranasal sites (i.e., sinus, Eustachian tube, middle ear, tracheobronchial tree) and reducing associated complications. However, adequate drug distribution to the respiratory tract from the blood compartment following oral administration was a problem for several earlier capsid-binding agents, which achieved reasonable concentrations in the blood but ineffective concentrations in nasal secretions [66]. Furthermore, systemic administration might increase the risk of unacceptable drug-related toxicities.

4.5

Combination Therapies

The combined use of anti-rhinoviral agents may offer enhanced antiviral action, reduced toxicity, and reduced likelihood of the emergence of resistance. However, the number of active agents available is very limited, and combined use of antiviral agents has received little study. Additive or synergistic antiviral activity has been found with combinations of certain anti-rhinoviral agents (e.g., interferon-alfa and enviroxime), but one clinical trial found no greater protection from the combination than with interferon alone [67]. More effort has focused on using combinations of antiviral agents and host response modiﬁers to exert greater therapeutic beneﬁts. Intranasal interferon-alfa 2b combined with an oral nonsteroidal antiinﬂammatory drug (naproxen) and a topical anticholinergic drug (ipratropium) exerted greater therapeutic activity than individual agents in experimental rhinovirus infections [68], and further studies of this combination antiviral–antimediator approach are under way for treating naturally occurring colds. Progress in this approach will depend on an

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improved understanding of the pathogenesis of rhinovirus illness and the identiﬁcation of selective inhibitors of key host inﬂammatory responses. 5

ANTI-RHINOVIRUS AGENTS OF INVESTIGATIVE INTEREST

A substantial number of agents have been identiﬁed that have antirhinoviral activity in vitro (reviewed in Ref. 51), and candidate agents from several different mechanistic classes have progressed to clinical testing (Table 2). However, most have been abandoned because of lack of efﬁcacy, limited antiviral spectrum, poor tolerance or pharmacokinetic properties, or high cost of production. Several investigational agents that have been recently studied or remain under active clinical development are discussed below. 5.1

Pleconaril

Pleconaril (3-[3,5-dimethyl-4-[[3-(3-methyl-5-isoxazolyl)propyl]oxy]phenyl]-5-(triﬂuoromethyl)-1,2,4-oxadiazole; VP-63843) is a novel, orally bioavailable, small-molecule inhibitor of picornaviruses that interacts with the virus capsid. It has progressed the furthest in clinical development of anti-rhinovirus agents to date. 5.1.1

Spectrum of Activity

Pleconaril has broad activity in vitro against rhinoviruses and enteroviruses, including polioviruses, coxsackieviruses, echoviruses, and TABLE 2 Antiviral Targets for Rhinovirus and Representative Agents That Have Been Studied in Clinical Trials Antiviral mechanism/ viral target

Antiviral class

Alteration of cell susceptibility Attachment

Interferons

Uncoating/capsid function Nucleic acid synthesis

Capsid binders

Post-translational protein modiﬁcation

Receptor blockers

Transcriptase complex inhibitors 3C Protease inhibitors

Representative agents Recombinant IFN-a 2a/b, leukocyte IFN-a Anti-ICAM-1 antibody, soluble ICAM-1 Pleconaril, pirodavir Enviroxime Ruprintrivir (AG7088)

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human enterovirus. In cell culture, concentrations of 40.18 mM inhibit 90% of enterovirus clinical isolates [69]. The median EC50 value for rhinovirus clinical isolates is 0.04 mg/mL, but the range (<0.01 to >1.0 mg/mL) is broad [70]. Approximately 10% of the numbered rhinovirus serotypes and 10% of clinical isolates are not inhibited by pleconaril concentrations of 1.0 mg/mL or more. Plasma concentrations in adults after a single 400 mg dose of pleconaril exceed the IC90 values for approximately 95% of enterovirus and 90% of rhinovirus serotypes. Oral pleconaril is active in experimental murine models of enteroviral central nervous system infection and in experimental human coxsackie A21 virus infection of the respiratory tract [71]. 5.1.2

Mechanism of Action

The capsid of picornaviruses plays essential roles in binding of the virus to and entry into the host cell. The crystallographic structure of picornaviruses shows a relatively conserved hydrophobic pocket within the capsid protein VP1 for both enteroviruses and rhinoviruses. Pleconaril was developed to bind into this hydrophobic pocket, and pleconaril binding induces conformational changes in the viral capsid that lead to altered attachment and/or viral uncoating. For major receptor group rhinoviruses, which use ICAM-1 as their cellular receptor, pleconaril alters the conformation of the canyon ﬂoor and inhibits the receptor binding interaction. For these and other picornaviruses, pleconaril ﬁlling the pocket stabilizes the capsid and inhibits the intracellular uncoating and release of viral RNA. This is indicated in part by increased thermostability of the virions. 5.1.3

Pharmacology

Pleconaril is orally absorbed with an estimated bioavailability that approaches 70% when it is administered with food. Pharmacokinetic studies using both a hard gelatin capsule and oral solution show ﬁrstorder absorption and dose-proportional blood concentrations [72–74]. Coadministration of pleconaril with food results in two- to four-fold enhanced bioavailability. Following multiple doses of 400 mg given with food, peak plasma concentrations occur at 2–3 hr and average 2.2 mg/mL on day 1 and 3.4 mg/mL on day 7 in healthy adults [75]. The elimination half-life of pleconaril is best characterized by a two-compartment model with a shorter initial phase (T1/2 of 2–3 hr) and a prolonged terminal elimination phase (T1/2 of approximately 180 hr). Studies in children suggest decreased maximal concentrations and smaller area under the curve compared to adults [73]. In neonates, oral bioavailability of a liquid formulation is variable with normalized Cmax averaging 29 ng/mL/mg

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dose [76]. Animal studies have shown that pleconaril distributes to the liver, nasal epithelium, and brain within 2 hr of the administration of oral solution; concentrations of drug in the central nervous system and nasal secretions exceed those of plasma. Pleconaril has a large volume of distribution consistent with signiﬁcant tissue distribution [77]. It undergoes slow but extensive metabolism, with over 30 metabolites that apparently lack antiviral activity. The major route appears to be reductive cleavage of the triﬂuoromethyl oxadiazole ring. Pleconaril and its metabolites are excreted primarily in the feces, with approximately 80% of an orally administered dose excreted in this way within 48 hr of administration; less than 1% of pleconaril is excreted unchanged in the urine. 5.1.4

Adverse Effects

Pleconaril appears to be safe and generally well tolerated in adults and children. In placebo-controlled studies of the use of pleconaril for rhinoviral illness in adults, the only signiﬁcant differences in adverse events between pleconaril and placebo recipients have been in gastrointestinal complaints (nausea, emesis) and menstrual irregularities. In a review of 32 patients treated with compassionate release pleconaril for severe enterovirus disease, 13 experienced no adverse effects, and the most common adverse events among the remaining patients were nausea and vomiting. No important effects on hepatic levels of CYP450 isoenzymes in rat or dog microsomes were observed following in vivo dosing (100 mg/kg) for 26 weeks [78]. One human study found modest (<20%) effects of pleconaril on theophylline pharmacokinetics in healthy adults, consistent with inhibition of CYP1A2. However, recent studies have found that signiﬁcant induction of CYP3A4 occurs in pleconaril recipients, so that drug interactions are likely with agents metabolized by this isoenzyme. 5.1.5

Resistance

Rhinoviruses selected in vitro for resistance to pleconaril show mutations affecting capsid proteins outside the drug-binding pocket or within the hydrophobic binding pocket of VP1. Mutations on the surface of the virus appear to allow for greater binding of the virus to its receptor, whereas those within the hydrophobic pocket either block drug entry (pocket-exclusion mutants) or decrease afﬁnity of the pocket for pleconaril. Analysis of coxsackievirus type B variants resistant to pleconaril shows a single amino acid change within the drug-binding hydrophobic pocket of VP1 [79]. Of note, these variants and several clinical isolates of enteroviruses with reduced susceptibility due to point

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mutations in VP1 appeared signiﬁcantly less virulent than wild-type viruses in animal models of infection. In studies of pleconaril treatment of rhinovirus colds, post-treatment isolates with reduced drug susceptibility have been found in about 10% of pleconaril recipients. 5.1.6

Clinical Efﬁcacy

Pleconaril is investigational at present (March 2002) in the United States, although it is available for use on a compassionate basis for the treatment of potentially life-threatening enterovirus infections. In placebo-controlled studies of adult and pediatric patients with moderate to severe enteroviral meningitis, reductions in morbidity were seen, but phase III studies involving adults with viral meningitis showed no statistically signiﬁcant beneﬁts. When used to treat life-threatening enterovirus infections, open-label pleconaril has been associated with favorable responses to treatment in a majority of patients [79a]. In an experimental coxsackie A21 virus infection, in which virus was inoculated intranasally, oral pleconaril starting before challenge reduced viral titers and illness measures compared to placebo [71]. Retrospective analysis of two phase II treatment studies of rhinovirus colds in adults found that pleconaril recipients (400 mg t.i.d. for 7 days) had a 1.5 day reduction in the time to alleviation of their symptoms (deﬁned as absence of rhinorrhea and other symptoms to mild or absent) compared to placebo [97]. No signiﬁcant effects were seen in those without picornavirus illness. Two controlled phase III studies involving over 2000 persons, approximately two-thirds of whom had proven picornavirus illness, found that early treatment (within 24 hr of symptom onset) of picornavirus colds was associated with an overall average 1 day reduction in illness duration and signiﬁcantly accelerated reduction in viral loads and symptom scores as early as the second day compared to placebo [53]. Pleconaril is currently under study for chemoprophylaxis and for pediatric treatment. 5.2

Tremacamra

Tremacamra [recombinant soluble intercellular adhesion molecule-1 (ICAM-1); BIRR 4] is a truncated soluble form of ICAM-1, the receptor for the major group of rhinoviruses, and is composed of the ﬁve extracellular immunoglobulin-like domains of the molecule. 5.2.1

Spectrum of Activity

Cellular ICAM-1 acts as the cell surface receptor for the major receptor group of human rhinoviruses, which account for approximately 90% of

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numbered serotypes. In cell culture the 50% effective concentrations (EC50) of tremacamra against the major receptor group human rhinovirus serotypes average about 4 mg/mL but range from 0.1 to 41 mg/mL [80]. Activity has also been conﬁrmed for most clinical rhinovirus isolates [81]. 5.2.2

Mechanism of Action

The major group of human rhinoviruses uses ICAM-1 as its cellular receptor [18,19]. Tremacamra appears to act as a receptor decoy and selectively inhibits receptor binding of this group of rhinoviruses in a competitive, reversible, concentration-dependent fashion [82]. In addition, it may cause extracellular uncoating of virions to render them noninfectious. 5.2.3

Pharmacokinetics

Tremacamra has been studied as a nasally inhaled powder. Two formulations have been tested: a phosphate-buffered saline spray and a carboxymethyl cellulose-mannitol–based powder selected to increase intranasal duration of tremacamra. They appear equally active, but the CMC is less well tolerated [83]. Tremacamra does not appear to be absorbed across the nasal mucosa. 5.2.4

Adverse Effects

Intranasal tremacamra is generally well tolerated, but nasal irritation was associated with use of the CMC powder. The most common adverse events reported in a single volunteer study of experimental colds were headache and nasal irritation [83]. 5.2.5

Resistance

Rhinovirus variants with decreased susceptibility to s-ICAM-1 were selected by in vitro passage in the presence of the molecule [84]. These variants show 10–40-fold reductions in susceptibility in vitro. The mechanism of reduced susceptibility remains to be determined, and no data are available on resistance to tremacamra in clinical studies. In addition, prolonged in vitro passage of one major receptor group rhinovirus in marginally permissive cells eventually selected virus variants capable of growth independent of the ICAM-1 receptor [85]. 5.2.6

Clinical Efﬁcacy

Preclinical studies found that intranasal s-ICAM-1 was effective in preventing rhinovirus infection in chimpanzees [86]. A recent two-center study of tremacamra for the treatment of experimental rhinovirus infections in adult volunteers showed that intranasal tremacamra (1.6 mg

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sprays ﬁve times daily for 5 days) begun either 4 hr before or at 12 hr after virus inoculation signiﬁcantly decreased cold frequency by 23%, symptom scores by 45%, nasal mucus production by 56%, and, when used postinoculation, viral titers and IL-8 levels in nasal lavage specimens [83]. 5.3

Ruprintrivir

Ruprintrivir (formerly AG7088) is a potent peptidomimetic inhibitor of rhinovirus 3C proteases that was developed through structure-based drug design [87]. Picornavirus 3C proteases are small (*20 kDa) monomeric molecules in which the substrate binding site is highly conserved across viral serotypes. Although a cysteine protease, the 3C protease is related mechanistically to trypsin-like serine proteases. 5.3.1

Spectrum of Activity

In cell culture ruprintrivir inhibits the replication of all studied rhinovirus serotypes with a mean EC50 of 0.02 mM and a relatively narrow 27-fold range of 0.003–0.081 mM [30]. Cytotoxic concentrations exceed 1000 mM. Ruprintrivir is also active against clinical rhinovirus isolates and representative enteroviruses at similar concentrations [70]. Proteins present in nasal secretions, mucin, and alpha 1-acid glycoprotein do not negate its activity in vitro [30]. In cultures of BEAS-2B human bronchial epithelial cells, ruprintrivir is associated with concentrationdependent inhibition of both viral replication and cytokine elaboration with reductions in IL-6 and IL-8 levels at 0.1 mM concentrations [88]. 5.3.2

Mechanism of Action

The rhinovirus 3C protease mediates cleavage of viral precursor polyproteins into structural and enzymatic ones and is essential for viral replication. Ruprintrivir binds irreversibly to the protease active site through formation of covalent bonds and potently inhibits enzyme activity; this leads to the accumulation of viral precursor polyproteins in infected cells [30]. Timing of addition studies show that ruprintrivir inhibits single rounds of rhinovirus replication when added as late as 6 hr after viral inoculation, whereas a representative capsid-binding agent was active only within the ﬁrst hour [30]. Picornavirus 3C proteases are also involved in proteolytic degradation of certain cellular proteins and in direct binding to viral RNA as part of the replication complex [87]; whether ruprintrivir interferes with the latter action has not been reported.

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Pharmacokinetics

Ruprintrivir has minimal oral bioavailability (<10%) and has been tested as an intranasal spray in studies to date. Most studies have used a 2% suspension in carboxymethyl cellulose. When given intranasally to uninfected persons at doses of 8 mg ﬁve or six times daily for 7 days, a minority have detectable plasma levels (>0.2 ng/mL). Peak plasma concentrations of the acid metabolite (AG7185) average 2.2 ng/mL after multiple doses and decline with T1/2 of about 3–6 hr [89]. Nasal lavage concentrations after an 8 mg dose are much higher and average 3.1 mg/ mL at 4 hr after dosing. 5.3.4

Adverse Effects

Intranasal ruprintrivir is generally well tolerated, but nasal irritation may occur with repeated intranasal dosing and be associated with bloodtinged nasal mucus. 5.3.5

Resistance

Rhinovirus variants with modestly decreased susceptibility to ruprintrivir have been selected by in vitro passage (A. Patick, personal communication), but no data are available on resistance in clinical studies. 5.3.6

Clinical Efﬁcacy

One multicenter study of ruprintrivir found that nasal sprays given twice or ﬁve times daily starting before beginning experimental rhinovirus inoculation in adult volunteers did not reduce infection frequency but did reduce viral replication markers and moderated the severity of colds [90]. When administered ﬁve times daily, treatment starting at 24 hr after inoculation signiﬁcantly reduced viral titers, symptom scores, and nasal mucus production compared to placebo. A treatment study in naturally occurring colds was confounded by a low rate of rhinovirus positivity, but a retrospective subset analysis of rhinovirus-infected subjects who received treatment within 24 hr of symptom onset suggested clinical beneﬁt [91]. Studies of other intranasal formulations of ruprintrivir and of orally bioavailable 3C protease inhibitors are in progress. 5.4

Interferons

Interferons are potent, generally species-speciﬁc cytokines that have antiviral, antiproliferative, and immunomodulatory effects that contribute to reduced host cell susceptibility to picornavirus infections. Only low concentrations of endogenously produced interferon are detectable

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in the nasal secretions of rhinovirus-infected persons [92]. Extensive clinical testing of intranasal leukocyte-derived and recombinant DNA– produced monospeciﬁc interferons was conducted during the 1980s (reviewed in Ref. 51), and limited work continues at present. 5.4.1

Spectrum of Activity

Interferons of various types are active against the full spectrum of rhinoand enteroviruses, although inhibitory concentrations are highly dependent on the test system. 5.4.2

Mechanisms of Action

The antiviral effects of interferons are mediated through speciﬁc host cell receptors and signal transduction pathways that, in conjunction with double-stranded viral RNA, lead to the expression of multiple cellular proteins (for example, 20 ,50 -adenylate synthetase, protein kinase, Mx protein) that mediate antiviral actions. An isoform of the 20 ,50 -adenylate synthetase system has been shown to inhibit picornavirus replication in vitro [93]. The immunomodulatory actions of interferons, including activation of natural killer cells and macrophages, may also contribute to control of replication. 5.4.3

Pharmacokinetics

The intranasal pharmacology of different interferons has not been well characterized. Consistent with the prolonged duration of the antiviral state induced in exposed cells, once-daily intranasal interferon administration is protective against both experimental and natural rhinovirus infections. 5.4.4

Adverse Effects

Intranasal interferons cause dose- and duration-related nasal mucosal irritation manifested by stufﬁness, dryness, local discomfort, bloodtinged mucus, mucosal erosions, and ulcerations. Symptoms usually begin after the ﬁrst week of prophylactic use, but histopathological abnormalities including inﬂux of T-lymphocytes, related to the immunomodulatory effects of interferons, are detectable within 4 days of initiating administration [94]. 5.4.5

Resistance

Resistance among picornaviruses to interferon antiviral actions has not been described.

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Clinical Efﬁcacy

Intranasal a- and b-interferons of several types are effective for prophylaxis of experimental rhinovirus colds (reviewed in Ref. 51), and once daily intranasal interferon-alfa 2b has been proven effective for prevention of rhinovirus illness when used for preexposure prophylaxis in adults (minimal effective doses of approximately 3 MU daily) or for postexposure prophylaxis (doses of 5 MU daily) in families. However, protection is dose-related, and lower doses (2.5 MU daily) were not effective for postexposure prophylaxis in one trial. When initiated 28 hr after experimental rhinovirus infection, intranasal interferon–alfa 2b moderately reduced peak viral titers but did not provide overall symptom beneﬁt [95]. Similarly, high doses (up to 20 MU t.i.d. for 7 days) reduced virus replication but not symptom resolution, in part due to local side effects [96]. When started 24 hr after infection, intranasal interferon-a2b 6MU dosed 3 times over 36 hr added to the effectiveness of chlorpheniramine and ibuprofen in experimental rhinovirus colds [98]. This strategy warrants study in natural colds.

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5 Herpes Simplex Virus Kimberly A. Yeung-Yue, Gisela Torres, Mathijs H. Brentjens, Patricia C. Lee, and Stephen K. Tyring University of Texas Medical Branch, Galveston, Texas, U.S.A.

1

INTRODUCTION

Herpes simplex virus (HSV) infections manifest in many forms. Mucocutaneous lesions in the orolabial (Fig. 1) and anogenital (Fig. 2) areas are the most common. Typically, HSV type 1 (HSV-1) infects the oral mucosa, whereas HSV type 2 (HSV-2) affects the genital mucosa. Genital herpes caused by HSV-1 [1–4] and orolabial herpes due to HSV-2 [5], however, are increasingly more prevalent. Primary oral infection with HSV often manifests as gingivostomatitis. Other locations on the body may be affected by herpetic infection as well, such as the ﬁngers, which can be infected with herpetic whitlow (Fig. 3). Inoculation of the ﬁngers with HSV most commonly occurs in association with primary oral or genital infection [6]. Among health care workers in direct contact with bodily secretions, the incidence of herpetic whitlow has decreased since implementation of the routine use of gloves. HSV keratitis caused by infection of the cornea may lead to neovascularization and corneal clouding [7]. Herpetic sycosis, or follicullitis of the beard area caused by HSV, has also been found to occur after implementation of unsuccessful antibacterial and antifungal therapies [8]. Herpes gladiatorum is most commonly associated with wrestlers, as 165

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Orolabial herpes simplex virus is most often caused by HSV-1.

the name suggests. This cutaneous HSV infection, however, can occur with any direct skin-to-skin contact [9]. Kaposi’s varicelliform eruption, or eczema herpeticum, is a complication of preexisting dermatoses, such as atopic dermatitis, resulting in an extensive vesicular eruption that is most commonly caused by HSV [10]. Erythema multiforme (Fig. 4), characterized by erythematous papules that progress to target lesions, often occurs in genetically susceptible individuals following a recurrent infection with HSV [11]. Polymerase chain reaction has even detected HSV DNA in most erythema multiforme lesions in individuals without a prior history of HSV infection [12,13]. Therefore, it is possible that the lesions seen in erythema multiforme are caused by an immune-mediated response against HSV-speciﬁc antigens in the skin [12]. Neonatal herpes simplex virus infections range in severity and extent. The infection may be localized to the skin, eyes, or mouth (SEM); may cause encephalitis with or without SEM involvement; or may disseminate to multiple locations, including the central nervous system, lungs, liver, or adrenal glands [14,15]. Death or neurological sequelae such as seizures, psychomotor retardation, spasticity, blindness, or learning disabilities may result [14].

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FIGURE 2 Primary genital herpes, as shown here, is usually more extensive than recurrent genital infections. HSV-2 is the most common etiological agent, but the incidence of HSV-1 genital herpes is increasing.

Infection with HSV-1 or HSV-2 in individuals without preexisting antibodies to either HSV-1 or HSV-2 is termed the primary infection. The seroprevalence of HSV-1 is 40–60% in the industrialized nations; HSV-2 prevalence is currently about 20% but has shown an increase during the last twenty years. Recurrent infections result from reactivation of HSV that has been transported to the sensory ganglia during the primary infection and has established a latent infection there. Individuals who are seropositive for one of the two types of HSV can develop a nonprimary infection with the virus of the other type. Exogenous reinfections may also occur if a person is infected with a different strain of either HSV type, although this is rare in immunocompetent hosts [16,17].

2

DIAGNOSIS

The classic morphology of HSV lesions is clustered vesicles on an erythematous base that progress to erosions, then crust. The lesions may be preceded by a prodrome consisting of constitutional symptoms or local symptoms such as pain, burning, paresthesia, itching, or tingling

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FIGURE 3 The incidence of herpetic whitlow is decreasing in medical professionals who routinely use gloves.

[18]. Alternatively, patients may experience prodromal symptoms without any noticeable skin lesions. The primary infection is usually the most severe. The virus establishes latency in sensory ganglia [15,18,19], and recurrent infections often develop with the proper stimuli at varying frequencies [18,20,21]. Obtaining a diagnosis of HSV infection from this clinical picture is occasionally sufﬁcient; however, along with the diagnosis comes a signiﬁcant responsibility of the physician to properly counsel and educate the patient about the disease. The psychosocial impact on the patient’s self-esteem and relationships is great, so it is important to be reasonably certain of the diagnosis. The majority of people who are seropositive for HSV type 2 (HSV-2) never report a history of genital herpes [22,23] suggesting that they are unaware of their potential to transmit the virus. In addition, viral shedding on the surface of the skin may occur in the absence of clinical signs or symptoms on approximately one third of the total number of days that HSV reactivation occurs [23–26]. This asymptomatic viral shedding is responsible for up to 70% of genital herpes transmission cases [27] and can result in life-threatening infections in neonates [28]. Atypical presentations of herpetic lesions such as pustules, ﬁssures, and edematous areas [29,30] may cause frustration when misdiagnosed and

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FIGURE 4 Erythema multiforme, characterized by ‘‘target lesions,’’ may occur after oral or genital infections with HSV.

may place unknowing sexual partners at risk for acquisition of the virus. For these reasons, diagnostic conﬁrmation is recommended. 2.1

Viral Isolation

Currently, viral culture is the gold standard for diagnosis in clinical practice. The virus may be isolated from skin vesicles, cerebral spinal ﬂuid, the throat, nasopharynx, conjunctiva, stool, cervix, or urine. When obtaining samples from the lesions, a Dacron (not cotton) swab should be rubbed vigorously over the base of the unroofed blister or erosion. Viral culture is 100% speciﬁc for HSV. However, sensitivity is low and declines with recurrent infection and with lesion healing [31]. Viral DNA detection using polymerase chain reaction (PCR) is highly speciﬁc for HSV infection, similar to viral culture but of greater sensitivity [23,25]. Samples may be taken from older lesions, and results are more rapid than viral culture. Patients would beneﬁt from a rapid diagnosis via PCR despite increased lab costs and labor because antiviral therapy could be initiated early; however, this technique is not commercially available except for the diagnosis of HSV encephalitis [32].

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Direct Examination from Clinical Specimens

The Tzanck smear technique can be rapid and useful in a clinical setting. The suspicious vesicles should be unroofed, and the bases of the lesions should be scraped, then examined under the microscope. Visualization of multinucleated giant cells is suggestive of HSV infection but is not diagnostic due to limited speciﬁcity. Multinucleated keratinocytes, intranuclear inclusions, and ballooning of cells seen in biopsy specimens have similar limitations. Distinguishing between HSV-1, HSV-2, or varicella-zoster virus (VZV) is not possible by any of these methods alone [33,34]. Direct immunoﬂuorescence will detect HSV in clinical specimens and can distinguish between these three viruses. 2.3

Serological Diagnosis

Serological testing for type-speciﬁc antibodies against HSV may beneﬁt patients with questionable, unrecognized, or subclinical infection. Couples that include one partner with known genital herpes disease may request testing in the other partner to determine his or her risk of acquisition of the virus and allow them to take precautions to prevent it. Pregnant women of unknown HSV status may especially beneﬁt from serological testing in both themselves and their partners. Identifying seronegative pregnant women with seropositive partners would alert the physician to counsel them regarding transmission of the virus and the potential consequences of neonatal herpes. The neonate’s risk of neurological and developmental sequelae from HSV infection increases if the mother develops a primary episode of genital herpes late in the pregnancy. A pregnant woman already seropositive for HSV during labor has a lower risk of transmission to the neonate, and knowledge of her serostatus will help differentiate between primary and recurrent disease if lesions are present during labor [15,28]. A study in the United Kingdom, however, determined that widespread screening of pregnant women and their partners would not be cost-effective [35]. Finally, knowing the serostatus of individuals infected with human immunodeﬁciency virus (HIV) or at high risk of acquiring HIV can prompt physicians to counsel patients regarding the increased risk of transmission or acquisition of HIV. People who are coinfected with HSV and HIV are more likely to transmit HIV to others, and people who are seropositive for HSV are at greater risk of acquiring HIV [36]. The sensitivity and speciﬁcity of serological testing is high and does not require active disease at the time of testing. The usefulness of serological testing, however, is limited during primary infection due to the delay in antibody production after exposure. The presence of

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antibodies cannot give information regarding when the infection was acquired unless signiﬁcant levels of IgM antibodies are present (implying recent infection, within about 1 month). Physicians and patients should be aware that a positive IgG serological test, therefore, does not necessarily determine with certainty if a recent or a distant partner was the source of infection. This type of diagnostic testing also fails to identify the location of the infection, because either HSV-1 or HSV-2 may cause initial or recurrent disease in the same areas (e.g., mouth, ﬁngers, genitals). Similarly, testing for only HSV-2 may miss the diagnosis of genital herpes if that particular case is caused by HSV-1. It is important to correlate diagnostic tests with the clinical picture. HSV-1 and HSV-2 have type-speciﬁc surface glycoproteins. Serological tests detect antibodies against these glycoproteins, such as gG1 and gG2. The U.S. Food and Drug Administration (FDA) has approved six commercially available serological tests, but only four of them are in use. The HSV-1 ELISA and HSV-2 ELISA by Meridian Bioscience (Cincinnati, OH) have been discontinued. Focus Technologies (Cypress, CA) manufactures three laboratory-based tests: an HSV-1 ELISA, and HSV-2 ELISA, and an immunoblot test for antibodies to both HSV-1 and HSV-2 [37]. Only one point-of-care serological test is currently available. The POCkit HSV-2 Rapid Test manufactured by Diagnology (Belfast, Northern Ireland) is performed in 6–10 min using a serum sample or capillary blood from a ﬁngerstick and the reagent in the kit. The test is able to detect antibodies to HSV-2 with high sensitivity and speciﬁcity compared to the gold standard serological test (Western blot) [37,38]. Visual interpretation of the results, however, has varied 5–10% of the time [39]. As mentioned earlier, testing for only HSV-2 can miss the diagnosis of genital herpes that is caused by HSV-1. Performance of serological testing has not been evaluated in children under the age of 14, so testing should be used with caution in this population. 2.3.1

Pregnancy and Neonatal Herpes

Neonatal herpes occurs at a rate of 1600 births/yr in the United States, should always be considered in newborns about 1 week old with fever, irritability, lethargy, or poor feeding [14]. Of note, fewer than 50% of infected newborns with encephalitis or disseminated disease develop cutaneous lesions [14]. Physicians should not rely on a negative maternal history of genital herpes, because only about 5% of pregnant women report a history of genital HSV infection [40]. In contrast, approximately 20% of the population in the United States is seropositive for HSV-2 [41]. Herpes simplex virus has been detected by viral culture during delivery about 1% of the time in women with symptomatic or asymptomatic

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recurrences [40]. Detection of HSV DNA by poly-merase chain reaction, however, has proven to be more sensitive than culture. Polymerase chain reaction detects viral shedding during delivery 9% [43] to 20% [28] of the time. Most neonatal HSV infections are caused by HSV-2 due to the higher prevalence of genital disease caused by HSV-2; however, HSV-1 is transmitted to the newborn more effectively [14,28]. The risk of transmitting HSV from mother to child depends on many factors. Seroconversion that occurs prior to the onset of labor does not appear to affect the outcome of the pregnancy [42]. Primary genital HSV infections in pregnant women who are active during delivery have the highest rate of transmission to newborns, approximately 40% [40]. Recurrent infections pose a much smaller risk of transmission, close to 3% [40]. In practice, it is difﬁcult to distinguish between primary or initial infection versus recurrent infection at the time of delivery, because many women are unaware that they have genital herpes.

2.3.2

Human Immunodeﬁciency Virus and Herpes Simplex Virus

Herpes simplex virus (HSV) and human immunodeﬁciency virus (HIV) appear to inﬂuence each other with regard to disease progression and transmission [44]. HSV is the most common opportunistic infection in HIV-1-infected patients [44]. Clinical manifestations of genital herpes tend to be more severe and persistent in HIV-infected individuals than in HIV-negative persons [45]. In addition, HSV reactivation (clinical and asymptomatic) occurs more frequently in coinfected individuals, which is directly correlated with declining CD4-positive cell counts [46–49]. This may translate to increased transmission of HSV (and HIV). Prior genital infection with HSV has been noted as an important risk factor for acquisition of HIV infection [50–53], likely due to the disrupted epithelial barrier caused by HSV reactivation and the recruitment of activated CD4-positive cells to the site, which are target cells for HIV [36,54,55]. Coinfection with HSV and HIV is believed to potentiate transmission of HIV as well. HIV virions capable of infection and replication have been detected in genital lesions caused by HSV [56]. Finally, there are conﬂicting reports regarding the effect of HSV reactivation on the course of HIV disease progression. Some authors have noted increased levels of HIV RNA in the plasma [57,58] or within cells [54,59] upon reactivation of HSV, whereas others have noted no such ﬂuctuation [60].

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VACCINES

Herpes simplex virus infects its host indeﬁnitely. Current treatment modalities may help minimize or shorten recurrent outbreaks, but no cure is available. Three types of vaccines, however, are under investigation for the prevention and treatment of genital herpes: adjuvant subunit vaccines, a replication-incompetent viral mutant vaccine [disabled infection single cycle (DISC)], and DNA vaccines. Vaccine efﬁcacy may vary, depending on how the goal of the vaccine is deﬁned [61]. Preventing primary infection and the establishment of viral latency may be an unrealistic standard; however, decreasing the severity or frequency of symptomatic recurrences may be a reasonable therapeutic beneﬁt. Pertussis and inﬂuenza vaccines similarly do not prevent the initial infection but do reduce clinical symptoms [61]. Whether a vaccine can reduce asymptomatic viral shedding and subsequent transmission to sexual partners and neonates would be important questions to explore. Cell-mediated immunity rather than circulating antibodies appears to play a larger role in preventing infection with HSV. A helper T-cell type 1 (Th1) response is believed to be more important that a type 2 (Th2) response [61,62]. Circulating antibodies usually prevent autoinnoculation with the same virus type, such as a new genital infection after touching a lesion in the mouth [63–65]. The most common time for a superinfection like this to occur is during the primary infection while antibody titers are relatively low. Antibody titers do not seem to prevent the primary infection, however, possibly due to direct cell-to-cell viral spread rather than occult viremia [62,66]. 3.1

Adjuvant Subunit Vaccine

Adjuvant subunit vaccines contain viral proteins that usually originate from the HSV envelope plus an additional adjuvant to improve immunogenicity. In general, subunit vaccines are less immunogenic than live virus vaccines, and adjuvants that increase immunogenicity run the risk of increasing reactogenicity [61,67]. One recombinant HSV subunit vaccine combined HSV-2 glycoproteins gD2 and gB2 with the adjuvant MF59. Overall, the vaccine lacked efﬁcacy in phase III trials despite its ability to induce neutralizing antibodies, offering women (but not men) only modest and transient protection against infection with HSV-2 [68]. Another recombinant HSV subunit vaccine containing glycoprotein gD2 and an experimental adjuvant monophosphoryl lipid A immunostimulant (MPL) did, however, show statistically signiﬁcant efﬁcacy in preventing symptomatic genital herpes disease in phase III

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trials. The vaccine afforded protection in women who were seronegative for both HSV-1 and HSV-2 and demonstrated a trend toward preventing HSV-2 antibody seroconversion in these women but was not effective for men [69]. The local immune response in cervical secretions that bathe genital mucosal surfaces is speculated to be the reason for the genderspeciﬁc immunity [69]. Further trials are under development to further evaluate efﬁcacy and mechanism of action.

3.2

Replication-Incompetent Mutant Vaccine

A replication-incompetent mutant vaccine consists of live virus that maintains the ability to infect cells but is incapable of spreading between cells. The virus lacks the glycoprotein H gene, which codes for a protein that is required for cell entry [61,67]. In preclinical testing the vaccine was administered at the mucosal surfaces to stimulate the local immunity and was immunogenic and well-tolerated in phase I trials [70,71]. The DISCbased therapeutic vaccine was well tolerated in Phase I trials but the Phase II trial did not meet its clinical endpoint. Studies with the prophylactic DISC vaccine are continuing.

3.3

DNA Vaccine

DNA vaccines contain plasmids that code for select viral antigens such as glycoproteins gB or gD. These gene products can be designed to target various locations in the cell (e.g., cytoplasm, plasma membrane, or extracellular space) to induce different combinations of humoral and cellmediated responses [62,66]. The goal is to engineer a vaccine that will produce a balanced immune response that optimally prevents or treats HSV infections. DNA vaccines currently are in phase I trials and preclinical development [61]. Most HSV vaccine trials are centered around the treatment and prevention of genital herpes but will probably be examined for their usefulness in controlling non-genital HSV infections in the future. The incidence of neonatal herpes is estimated to decrease by 80–90% if women are vaccinated prior to delivery due to either a decrease in viral shedding in the woman’s genital tract or the transfer of maternal antibodies to the infant [61]. Other HSV infections that may be controlled include oral-facial herpes, eczema herpeticum, herpetic keratitis, herpetic encephalitis, herpetic whitlow, herpes gladiatorum, and erythema multiforme.

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VIRAL REPLICATION STRATEGY AND TARGETS FOR THERAPY

Herpes simplex viruses are transmitted through direct contact with saliva, semen, cervical ﬂuid, or vesicle ﬂuid from active lesions. The virus replicates at the portal of entry, such as abraded skin or mucosal surfaces, to initiate infection [72]. Neuronal retrograde axonal ﬂow then transports the capsid to the sensory ganglia, where the virus replicates a second time to establish latency [19]. HSV-1 most commonly replicates in the oropharyngeal mucosa and establishes latency in the trigeminal ganglia, whereas HSV-2 usually initiates infection in the genital mucosa and establishes latency in the sacral ganglia [73]. The virus will persist for the life of the host and has the ability to escape detection by the host immune system. The virus can descend down the sensory nerve spontaneously or with the proper stimulus despite a functional humoral and cell-mediated immunity, resulting in vesicles or ulcers at mucocutaneous sites. Commonly associated stimuli include physical and emotional stress, fever, ultraviolet light exposure, nerve or tissue damage, immunosuppression, heat, cold, menses, infection, and fatigue [18,20]. 4.1

Nucleoside Analogs: Acyclovir, Valacyclovir, and Famciclovir

Antiviral medications that are currently available for the treatment of herpesviruses are nucleoside analogs that prevent viral DNA chain elongation. Acyclovir, valacyclovir, and famciclovir all require activation by a functional viral thymidine kinase and are the main treatment modalities for herpetic infections. Drug-resistant forms of HSV, however, are most commonly thymidine kinase–deﬁcient and therefore require treatments that do not rely on thymidine kinase for activation. Foscarnet or cidofovir can be used in this situation. Acyclovir is an acylic purine nucleoside analog that initially requires phosphorylation by viral thymidine kinase, then further phosphorylation by host cellular enzymes [74]. The active form of the drug, acyclovir triphosphate, competitively binds and inhibits viral DNA polymerase. Termination of chain elongation results in the inhibition of viral replication [22]. Penciclovir is another nucleoside analog with a similar mechanism of action. Valacyclovir and famciclovir are the prodrugs of acyclovir and penciclovir, respectively. These prodrugs have greater oral bioavailabilities than their predecessors and allow for more convenient dosing schedules. Acyclovir, valacyclovir, and famciclovir have similar therapeutic effects against HSV and adverse event

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proﬁles. The nucleoside analogs do not prevent the death of cells already infected with HSV but help minimize spread to other cells [75]. 4.2

Foscarnet and Cidofovir for Drug-Resistant Strains of HSV

Acyclovir-resistant strains of HSV occur in the general population at a rate of 0.4% [76–78]. The rate of resistant strains found in immunocompromised patients is signiﬁcantly higher at 4–11% [76,78–80]. Most cases of resistance involve a mutation in the genes coding for viral thymidine kinase, rendering the enzyme inactive, but will occasionally lead to a variant that does not recognize acyclovir triphosphate or will involve a mutation in the genes for DNA polymerase [76,79,81]. Drug resistance usually develops after long-term exposure to a drug; however, acyclovir-resistant strains of HSV seem to appear when replication is heightened in the setting of decreased immunity. Exposure to acyclovir is not correlated with an increase in acyclovir-resistant strains. Resistant strains of HSV have been detected in specimens collected from patients prior to the introduction of acyclovir and from patients who have never been exposed to acyclovir [76,81–83]. Long-term suppressive therapy with acyclovir appears to decrease the incidence of resistant forms of HSV by limiting replication and thereby decreasing the probability of mutation [82,84]. Viral cultures are heterogeneous; they often contain some acyclovir-resistant virions even if the culture is sensitive to acyclovir [81]. Foscarnet is the only treatment that is approved by the FDA for the therapy of acyclovir-resistant HSV. Unlike the other available antiviral medications, foscarnet is an analog of pyrophosphate. Foscarnet can competitively bind viral DNA polymerase and prevent chain elongation without requiring activation by thymidine kinase [79,85,86]. Strains of acylovir- and foscarnet-resistant HSV have been reported. In these cases, topical cidofovir may be useful [79,87,88]. Cidofovir is already in a monophosphate form and is phosphorylated only by cellular enzymes to its active form. The prevention of chain elongation, therefore, can occur in the absence of a functional viral thymidine kinase. Cidofovir is not approved by the FDA but is recommended by the Centers for Disease Control and Prevention as an alternative therapy for acyclovir-resistant herpes infections. 4.3

Immune System Modiﬁers

New therapeutic options that are unrelated to the nucleoside analogs are under investigation. Immune system modiﬁers such as vaccines for

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decreasing the symptoms of genital herpes were discussed earlier. A topical immune modiﬁer, resiquimod 0.01% gel, is currently in phase III clinical trials and appears very promising. Resiquimod does not have direct antiviral effects. In the presence of HSV antigen, resiquimod functions as an adjuvant by stimulating the local Th-1 response, inducing the secretion of cytokines such as interferon-a, interleukin-12, interleukin-6, and tumor necrosis factor-a. Resiquimod is 100 times more potent at inducing a T helper cell (Th)-1 cytokine response than imiquimod, its ﬁrst generation analog. The combination of antigen and adjuvant essentially mimics the effects of a therapeutic vaccine [69,89,90]. 4.4

Other Classes of Agents for HSV Infections

Two new classes of drugs, the aminothiazolylphenyl-containing drugs and thiazole urea derivatives, are in preclinical development. These nonnucleosidic drugs act by inhibiting the HSV helicase–primase complex, thereby preventing DNA synthesis. The agents appear to have therapeutic efﬁcacy superior to that of acyclovir and other nucleoside analogs in animal models. Treatment is effective even when it is delayed. If proven effective in clinical trials, these drugs could have a signiﬁcant impact on patients infected with acyclovir-resistant strains of HSV, which occurs more commonly in the immunocompromised. Resistance to helicase–primase inhibitors also seems to occur less frequently than resistance to acyclovir. Both compounds were administered orally and were effective against HSV-1 and HSV-2 but not varicella-zoster virus or cytomegalovirus [91–94]. The compounds are also active in topical and parenteral formulations and will cross the blood-brain barrier, increasing their potential usefulness for a variety of herpetic infection [94]. 5

TREATMENT AND MONITORING OF TREATMENT SUCCESS

Determining treatment success in community practice should rely on physician judgment and patient comfort. After the initial diagnosis of HSV infection, speciﬁc laboratory tests are not necessary. Endpoints used in clinical trials for antiviral therapies should be used for patient monitoring: lesion healing time, time to the next recurrence, number of outbreaks per year, severity of lesion symptoms, and frequency of aborted lesions when treatment is started during the prodrome. The success of a speciﬁc oral antiviral regimen depends primarily on patient compliance and severity of disease; therefore, a speciﬁc regimen should be suitable for and tailored to the individual patient. People with mild

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symptoms and infrequent recurrences would probably fare well with episodic therapy, taking a short course of medication at the ﬁrst sign of a recurrence. Other people with frequent, bothersome outbreaks or discordant couples who want to minimize the risk of HSV transmission to the seronegative partner may opt for daily suppressive doses of antiviral medication. The patient should be thoroughly counseled regarding the transmission of HSV so that the partners are aware of the persistent nature of the virus, the likelihood of asymptomatic shedding and possibility of transmission, and the limitations of available antiviral therapies. In general, herpes labialis does not respond to antiviral therapy in the same manner as herpes genitalis. Spruance [95] proposed that the pathogenesis of herpes labialis is slightly different, based on his observations of two types of herpetic lesions that appear after ultraviolet (UV) radiation exposure of the area. The group of ‘‘delayed’’ lesions that appear 3–7 days after UV radiation exposure behave as typical recurrent infections that originate from the sensory ganglia and respond to antiviral therapy. The other set of ‘‘immediate’’ lesions that rapidly surface within 48 h of exposure to the UV radiation, however, probably do not originate from the sensory ganglia. Unlike the ‘‘delayed’’ lesions, the ‘‘immediate’’ lesions are resistant to antiviral therapy. Their quick appearance may be explained by the presence of HSV in the epithelium at the time of irradiation. 5.1

Nucleoside Analogs: Acyclovir, Valacyclovir, Famciclovir, and Penciclovir

The nucleosidic drugs acyclovir, valacyclovir, and famciclovir are the standard treatments for HSV infections. Acyclovir is available in topical, oral, and intravenous preparations. Topical acyclovir showed limited efﬁcacy for the treatment of orolabial HSV infection [95]. The only FDAapproved indication for acyclovir 5% ointment is the treatment of mucocutaneous herpetic infections in immunocompromised patients. Intravenous acyclovir is usually reserved for severe infections because its administration requires hospitalization. Oral acyclovir is the most commonly used form of the drug, but its low oral bioavailability (15– 20%), short plasma half-life (2.5–3hr), and short intracellular half-life (1 hr) warrant frequent dosing [30]. A modiﬁed form of acyclovir, an oral microparticle-based controlled release formulation, is currently under development to improve the dosing schedule [96]. Less frequent dosing with valacyclovir and famciclovir, the prodrugs of acyclovir and penciclovir, respectively, appears to improve patient compliance. The

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oral bioavailability of valacyclovir is three to ﬁve times greater than that of acyclovir [97]. Valacyclovir and famciclovir are available only in oral form. First episode genital herpes infections can be treated with acyclovir 400 mg three times a day or valacyclovir 1 g twice a day for 10 days, although most countries have approved 500 mg twice a day as the accepted dose for valacyclovir. Famciclovir 250 mg three times a day for 10 days, although not FDA-approved for this indication, may also be used. Recurrent genital herpes may be treated with either acyclovir 400 mg three times a day or famciclovir 125 mg twice a day for 5 days. Valacyclovir 500 mg twice daily, however, is now FDA-approved for a shorter 3-day treatment of recurrent genital herpes in place of the previously recommended 5 days [98]. Oral acyclovir has been shown to be effective when used for only two days of treatment in a small trial [99]. Current opinion is moving towards earlier and more common usage of suppressive antiviral therapy in the management of recurrent genital herpes. Acyclovir and famciclovir are commonly used (400 mg and 250 mg p.o., respectively) in twice daily regimens and valacyclovir is effective as a suppressive therapy at a dose of 500 mg per day in patients with fewer than 10 episodes per year prior to therapy. It is important to note that whatever the dose, the half-life of acyclovir means that there are several hours of sub-optimal concentrations in the plasma during a once daily regimen. Clearly compliance is an important factor in once-daily regimen. Clearly compliance is an important factor in once-daily regimens because missed tablets could result in virus breakthrough. Suppressive therapy with acyclovir has already been shown to decrease viral shedding by approximately 90% [26,100], and evaluation of valacyclovir’s effect on viral shedding is currently under way. Intuitively, suppressive therapy that decreases the frequency of recurrences and viral shedding should translate to decreased transmission of HSV between discordant couples. This is currently under investigation. Adverse events reported during use of acyclovir [101,102], valacyclovir [103,104], and famciclovir [105] are usually mild and do not differ signiﬁcantly from placebo or each other. Reduced doses of nucleosidic drugs, however, should be given to patients with impaired renal function. Intravenous acyclovir has been associated with a transient but reversible nephropathy caused by drug crystallization in the renal tubules and collecting ducts [106–108]. The risk of nephropathy is minimized with adequate hydration.

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Penciclovir has very poor oral bioavailability and is therefore available only in a topical form. Penciclovir 1% cream applied every 2 h for 4 days is the only FDA-approved treatment for herpes labialis [109,110]. Other approved treatments are topical docosanl, topical acyclovir-modiﬁed cream, and oral valacyclovir high-dose single-day therapy. Acyclovir 400 mg ﬁve times a day [95,111] and famciclovir 250 mg three times a day [75] for 5 days have shown some beneﬁt for herpes labialis and famciclovir 500 mg three times daily was minimally more effective. However, neither acyclovir nor famciclovir has been recommended for herpes labialis. Valacyclovir 2 g twice a day for only 1 day has recently been shown to be efﬁcacious in the treatment of herpes labialis as well [112]. Acyclovir 400 mg twice a day for suppression of herpes labialis has also been beneﬁcial for patients with frequent recurrences (six or more outbreaks per year) [113].

5.2

Treatment During Pregnancy

Pregnant women with genital herpes may consider taking suppressive doses of acyclovir 400 mg three times daily late in their pregnancy to reduce viral shedding and frequency of recurrence. Suppressive acyclovir has been shown to be cost-effective in pregnant women with a history of genital herpes [114]. This indication is not FDA-approved but has been shown to decrease the need for cesarean delivery in women experiencing a primary episode of genital herpes during pregnancy and is speculated to decrease the risk of neonatal herpes [115–117]. Prophylactic acyclovir use in pregnant women with a history of recurrent genital herpes is less clear, because the risk of transmission to the newborn is signiﬁcantly less compared to an episode of primary genital herpes [40,118]. A registry of women exposed to acyclovir during their pregnancy suggests that the drug is probably safe for use in pregnant women, without any increased frequency of congenital malformations [28,119,120]. However, potential problems associated with acyclovir use during pregnancy include nephrotoxicity in the fetus with decreased renal function, the potential for a delay in presentation of neonatal herpes infection, possible compromised interpretation of viral culture results, or even increased risk of disseminated infection in the neonate [118]. All primary or initial genital HSV infections near term or during labor should be treated with antiviral therapy [14]. Any active lesions during delivery warrant delivery via cesarean section in addition to antiviral therapy [14].

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Treatment of Neonatal Herpes

A high index of suspicion and prompt treatment are essential in preventing or minimizing mortality and neurological sequelae [121]. The currently recommended treatment of neonatal herpes is high-dose intravenous acyclovir 60 mg/kg per day in three divided doses for 14 days in disease of the skin, eye, or mouth [122] or 21 days for involvement of the central nervous system or disseminated disease [123]. This regimen has been demonstrated to decrease neonatal morbidity and mortality more than the standard FDA-approved dose of 30 mg/kg per day for 10 days. Neutrophil count and hydration status should be monitored closely in neonates, because neutropenia and nephrotoxicity due to acyclovir have been reported [123].

5.4

Treatment of Immunocompromised Patients

Immunocompromised patients often suffer from more frequent or more severe HSV infection than individuals with intact immune systems. The approved treatment regimens also vary. Acyclovir 5% ointment applied topically every 3 h for 7 days is FDA-approved for mucocutaneous herpetic infections in the immunocompromised. Oral or intravenous acyclovir, however, is usually more effective [79,124]. The efﬁcacy of intravenous penciclovir 5 mg/kg every 12 hr was demonstrated to be similar to that of intravenous acyclovir 5 mg/kg every 8 hr for 7 days in immunocompromised patients, but with less frequent dosing [125]. The FDA has approved famciclovir 500 mg twice daily for 7 days to treat recurrent orolabial or genital herpes in patients infected with HIV [126], which was as effective as acyclovir 400 mg ﬁve times a day [127]. Episodic treatment with valacyclovir 1 g twice a day was shown to be as safe and effective as acyclovir 200 mg ﬁve times daily for 5 days [128]. Famciclovir 500 mg twice daily can also be used as a suppressive dose in HIV-infected patients, although it is not FDA-approved for this indication [47]. Transplant patients have beneﬁted from suppressive doses of intravenous acyclovir followed by oral administration [129], and suppressive therapy may increase survival in HIV-positive patients [130– 132]. Valacyclovir 500 mg twice a day has recently been shown to be superior to acyclovir 400 mg twice daily for suppression of genital herpes in HIV-infected patients, and both regimens were superior to valacyclovir 1 g once daily [128]. Optimal doses of the various antiviral medications still need to be established.

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Treatment of Drug-Resistant Strains of HSV

Foscarnet, which is a pyrophosphate analog, has poor oral bioavailability and must therefore be administered intravenously when treating acyclovir-resistant HSV infections. The recommended dose of foscarnet is 40 mg/kg every 8 h for at least 10 days, initiated within 7–10 days of treatment failure with acyclovir [83]. Adequate hydration and close monitoring of serum creatinine are essential, because foscarnet may be nephrotoxic. Electrolyte imbalances, nausea, vomiting, diarrhea, headache, fever, anemia, central nervous system disturbances, and penile ulceration (a contact dermatitis from the urine) may also complicate treatment [133]. Foscarnet 1% cream applied ﬁve times a day has also been examined in AIDS patients with some success, but it was not compared to placebo [134]. Occasionally, foscarnet-resistant strains of HSV are encountered [135,136]. Topical cidofovir 1% gel or cream (HPMPC) applied daily for 3 or more days has demonstrated improved lesion healing, decreased viral shedding, decreased lesion size, and reduced pain in a small, randomized, double-blind, placebo-controlled trial [137] and various case reports [87,88]. Of note, isolates from recurrences following cidofovir treatment were often sensitive to acyclovir. After treatment with acyclovir, isolates later became resistant to acyclovir again. Topical cidofovir is not available commercially but may be compounded in the pharmacy using intravenous cidofovir. Systemic side effects are not signiﬁcant with the topical formulation, but signiﬁcant ulcerative reactions have been reported at the site of application [138]. Cidofovir delivered intravenously has nephrotoxic and neutropenic side effects, limiting its use [138,139].

5.6

Immune System Modiﬁers

Topical treatments for HSV infections, if effective, help minimize toxic side effects and are often preferred by patients who are sensitive to oral medications. Resiquimod 0.01% gel, applied directly to active lesions twice a week for 3 weeks, has reduced the frequency of genital herpes episodes in patients with frequently recurring disease. In a pilot study, the median time to ﬁrst recurrence was 57 days and 159 days in the placebo and resiquimod groups, respectively [89]. Several studies to reproduce these results, examine its mechanism of action, and evaluate its impact on viral shedding are currently under way.

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CONCLUSION

The treatment of herpes simplex virus infections continues to rely heavily on nucleoside analog drugs. These treatment modalities can be effective for acute infections and for chronic suppression, thereby minimizing patient discomfort, but they are not curative. Vaccines and resiquimod are being explored for the prevention of and/or treatment of HSV infection although the recent suspension of clinical trials of resiquimod has raised some questions regarding the true effectiveness of the compound. The efﬁcacy of vaccination with the glycoprotein D recombinant vaccine needs to be improved and the mechanisms behind the gender-speciﬁc responses have to be understood before further progress can be made [140]. Anatomical differences might lie behind these gender-related differences in efﬁcacy so further trials are required before the utility of this vaccine can be completely assessed [69]. In addition, new antiviral mechanisms are being explored for the development of novel drug treatments for HSV, such as the helicase-primase inhibitors, one of which is BAY 57-1293. These new drugs may offer a safe and tolerable means of treating strains of HSV that are resistant to the nucleoside analogs and also have the potential to improve treatment efﬁcacy in general. Antiviral therapies might help control infection in the host, and their effect on transmission continues to be studied. Since asymptomatic disease is the source for most cases of transmission, suppression therapy with a compound of high potency and an acceptable tolerability might lead to a reduction in transmission between discordant couples. It should also not be forgotten that patient education and safe sexual practices continue to be important means of disease prevention.

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6 Varicella-Zoster Virus Jashin Joaquin Wu Baylor College of Medicine, Houston, Texas, U.S.A.

Kimberly A. Yeung-Yue, Mathijs H. Brentjens, and Stephen K. Tyring University of Texas Medical Branch, Galveston, Texas, U.S.A.

1

INTRODUCTION

Two different clinical syndromes, varicella (chickenpox) and herpes zoster (shingles), are caused by varicella-zoster virus (VZV), a unique virus with a worldwide distribution. Typically a self-limited disease of childhood, primary infection with varicella is characterized by a pruritic rash. Predominantly a disease affecting adults, herpes zoster is caused by reactivation of the latent virus. The pain associated with active zoster infection and following zoster infection may lead to signiﬁcant impairment in affected persons. Varicella and zoster in immunocompromised patients can often be severe and signiﬁcant conditions.

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HISTORY

Varicella was frequently confused with smallpox infection up to the nineteenth century. Heberden ﬁrst clearly distinguished the two illnesses in 1767 [1]. However, in his book on clinical medicine written in 1892, Osler still saw the need to emphasize the distinction between the two diseases [2]. Although not completely certain, the origin of the name chickenpox may come from the French word ‘‘chiche-pois’’ used to describe the chickpea-size varicella vesicles [3]. Alternatively, chickenpox may have been derived from the Old English word ‘‘gican,’’ which means ‘‘to itch’’ [4]. Herpes zoster has been recognized as a separate clinical disease since ancient times [5]. The word herpes is derived from the Greek word meaning ‘‘to creep.’’ The word zoster is derived from the Greek and Latin words meaning ‘‘girdle’’ or ‘‘belt’’ [6]. Von Bokay [7] ﬁrst postulated in 1888 the association between the varicella and zoster diseases; he noted that varicella often developed in susceptible children following exposure to a patient with herpes zoster infection. Later, Kundratiz (1922) and Bruusgaard (1932) [8] showed that the same agent was the cause of both diseases by successfully inoculating children with vesicle ﬂuid from patients with zoster. These experiments produced localized varicella lesions at the site of inoculation in some patients and a generalized varicella-like exanthem in others. Weller and coworkers [9,10] later proved, through isolation and propagation of the viruses in vitro, that the etiologies of varicella and herpes zoster were identical.

3

INCIDENCE

Varicella causes infection worldwide, being more transmittable in temperate climates than in tropical environments. This results in unique epidemiological differences among regions [11]. In temperate climates varicella is usually a disease of childhood, whereas in tropical regions the infection more commonly occurs in susceptible adults. Herpes zoster primarily affects adults older than 50 years of age, but the condition may occur at any age. Patients with a history of primary varicella infection have a 20% lifetime chance of later developing herpes zoster. The duration and incidence of zoster increases signiﬁcantly with age [12]. The annual incidence is 2.5 per 1,000 persons for ages 20–50 years and increases to 5 per 1,000 persons for ages 51–79. The incidence doubles again for patients older than 80 years of age (10 per 1,000). Zoster rarely occurs in childhood, but it is more frequent in

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children who had primary varicella infection in the ﬁrst year of life. Within one year after transplantation, as many as 35% of bone marrow transplant recipients develop zoster [13].

4

PATHOGENESIS

Varicella disease is one of the most contagious infections, and 80–90% of susceptible household contacts develop clinical infection after exposure [14]. The virus is transmitted by respiratory droplets from the nasopharynx or by contact with infected skin lesions. Patients with varicella are contagious from 2 days prior to the onset of the rash to 5 days after, but dry crusted scabs are not infectious. Patients with zoster are contagious from the skin lesions and less contagious from the nasopharynx secretions. Susceptible contacts may develop primary varicella infection. There is no evidence that herpes zoster illness can be contracted directly from contact with patients with varicella or zoster infection [12,15,16]. Primary varicella initially colonizes the conjunctiva or the mucosa of the upper respiratory tract after exposure to the virus. Two to four days after exposure, the ﬁrst cycle of viral replication occurs in the regional lymph nodes, followed by primary viremia, which develops 4–6 days after exposure. A second cycle of viral replication occurs in the liver, spleen, and other organs after the ﬁrst viremia. A secondary viremia then develops that spreads viral particles throughout the entire body. These virions invade the capillary endothelial cells, then the capillaries, and ﬁnally the epidermis 14–16 days following exposure [17]. The virus travels from the skin and mucosal lesions into the sensory nerve endings during the course of primary varicella infection. The varicella-zoster virus then spreads centripetally along nerve ﬁbers, establishing a permanent latent state in the dorsal ganglion cells. The exact pathogenesis and reactivation of herpes zoster disease is not completely known. A decline in virus-speciﬁc cell-mediated immunity, especially a decrease in T-cell proliferation to the varicella-zoster virus antigen, may be part of the process [18]. A decrease in cell-mediated immunity is typically seen in elderly persons or those with immunocompromised conditions, such as organ transplant, HIV infection, or treatment with radiotherapy or chemotherapy or long-term corticosteroids [19,20]. In the ﬁrst year following transplantation, 20–40% of bone marrow transplant recipients develop herpes zoster [17,21–23]. Likewise, 20–50% of patients with Hodgkin’s disease develop herpes zoster [24– 31], usually within 1 month of induction chemotherapy or within

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7 months of radiotherapy [32]. Herpes zoster may also be the ﬁrst presentation of HIV infection [33,34], and this patient population may present with atypical or recurrent varicella [35–41]. After reactivation of the virus, it undergoes an initial replication cycle in the affected sensory ganglion, which produces active ganglionitis. Severe neuralgia is secondary to the inﬂammatory response and the neuronal necrosis that occurs. When the virus spreads down the sensory nerve, the pain intensiﬁes and produces radiculoneuritis. 5

CLINICAL MANIFESTATIONS OF VARICELLA

Primary varicella is typically a mild and self-limited disease in immunocompetent children, but can be seen in adults (Figs. 1 and 2). Signiﬁcant morbidity may occur, with 11,000 hospitalizations each year in the United States due to complications of varicella infection in children, who are often otherwise healthy. Susceptible adults usually develop more frequent complications, more profuse skin lesions, and more prominent constitutional symptoms, such as prolonged fever (see Tables 1 and 2). Primary varicella infection can result in multiple complications. The most common complication is bacterial superinfection, usually by streptococci or staphylococci. The superinfection may manifest as cellulitis, erysipelas, furuncles, impetigo, or bullous lesions due to the bacterial production of staphylococcal exfoliative toxin [42]. Bacterial infection frequently results in scarring but seldom leads to septicemia [43]. Central nervous system complications occur in less than 1 in 1,000 cases and may include acute cerebellar ataxia, encephalitis, meningoencephalitis, myelitis, polyradiculitis, Reye’s syndrome, and Guillain-Barre´ syndrome [44]. Because aspirin is no longer recommended for children with varicella or other infections, Reye’s syndrome has now become rare. In adults, varicella pneumonia can be a complication. In a study of healthy military recruits with varicella, radiographic evidence of pneumonia was found in 16%. However, only 4% of subjects had clinical evidence of pulmonary involvement [45]. Varicella pneumonia usually develops within 6 days after the onset of rash. The mortality rate for adults with this complication is high, with death occurring in 10% of immunocompetent and 30% of immunocompromised individuals [45]. Other complications of varicella that may rarely occur include appendicitis, arthritis, glomerulonephritis, hepatitis, myocarditis, pancreatitis, orchitis, Henoch-Scho¨nlein vasculitis, optic neuritis, keratitis, iritis [46], and varicella gangrenosum [47]. Varicella infection during pregnancy may produce several fetal complications, ranging from asymptomatic latency to severe congenital

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FIGURE 1

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Primary varicella in a child.

defects [48]. When maternal infection presents in the ﬁrst trimester, congenital developmental malformations most commonly occur. These defects may include cicatricial skin lesions, cortical atrophy, hypoplastic limbs, ocular abnormalities, psychomotor retardation, and low birth weight. In the ﬁrst 20 weeks of pregnancy, the absolute risk of embryopathy after primary maternal VZV infection is about 2% [49]. Maternal varicella infection that occurs after the ﬁrst 20 weeks of pregnancy is associated with a much lower risk of congenital malformation. The risk of congenital defects after maternal herpes zoster at any time during pregnancy is much less than with primary VZV infection. The exposure of a pregnant woman without a history of chickenpox to a

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FIGURE 2

Primary varicella in an adult.

person with a clinically active VZV infection is always of concern. However, about 85% of adults who report that they have not had chickenpox are actually seropositive for antibodies to VZV [49]. Varicella infection may be associated with signiﬁcant morbidity and mortality in immunocompromised children or adults. The lack of immunity in these populations allows for continued virus replication and dissemination, leading to persistent viremia, prolonged fever, a more extensive rash (often with purpuric and/or hemorrhagic lesions), and involvement of other organs of the body such as the liver, lungs, or central nervous system [50,51]. Table 3 highlights these potential complications of varicella. 6

CLINICAL MANIFESTATIONS OF ZOSTER

More than 90% of patients with zoster have a prodrome of intense pain in the involved dermatome preceding the zoster rash. Because the pain is present before cutaneous manifestations, it often leads to a wide array of misdiagnoses, such as appendicitis, cholecystitis, myocardial infarction, pleurisy, peptic ulcer, ovarian cyst, prolapsed intervertebral disk,

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TABLE 1 Time after exposure

Clinical Manifestations of Varicella Clinical manifestations

14–15 days

Occasional prodrome symptoms lasting 2–3 days: low-grade fever, chills, headache, malaise, backache, anorexia, myalgia, nausea, or vomiting.

16–17 days

Rash with crops of small red macules, beginning on the face and scalp and spreading rapidly to the trunk. Relative sparing of the distal upper and lower extremities. Rash progresses over 12–14 hr to 1–3 mm papules, vesicles, then pustules.

16–17 days

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17–19 days

Crust formation

23–27 days

Healing

Miscellaneous observations

Laboratory analysis

Older children and adults may have prodrome symptoms. Younger children usually have an abrupt onset of rash with mild fever and malaise. Pruritus typically occurs with the rash.

Tzanck smear of lesion scraping, but doesn’t differentiate from varicella or herpes simplex.

Viral culture, serology, and direct immunoﬂuorescence.

Vesicles are classically described as ‘‘dew drops on a rose petal’’ (clear serous ﬂuid surrounded by a small red halo). Most children have 250–500 lesions, mostly vesicular. Scarring is rare in uncomplicated cases.

thrombophlebitis, or renal colic. The pain may be constant or intermittent and may be accompanied by hyperesthesia, pruritus, tingling, or tenderness. It is possible for a patient with dermatomal pain and serological or virological evidence of zoster infection never to develop cutaneous manifestations of herpes zoster. This condition is known as zoster sine herpete (zoster without rash) [52,53] and is occasionally suspected but rarely conﬁrmed. However, laboratory conﬁrmation is possible with paired serology (i.e., acute and chronic) or with the polymerase chain reaction (i.e., to detect VZV DNA). Further, patients

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TABLE 2

Wu et al. Differential Diagnoses of Varicella

Contact dermatitis: Pustules typically do not form and fever does not occur; lesions are less likely on the trunk than on the extremities. Drug eruption: Fever does not occur; simultaneous appearance of multiple types of lesions is rare. Hand-foot-and-mouth disease: Lesions are more prominent on the mucous membranes of the mouth and on the distal extremities with hand-footand-mouth disease. Herpes simplex virus: Disseminated disease may sometimes mimic varicella, but herpes simplex typically has a predominance of localized vesicle clusters at the primary site of infection. Insect bites: Insect bites typically occur on the extremities and have an underlying wheal. Scabies: More chronic development of the lesions, which tend to appear in body folds and often have a linear distribution. Smallpox: Historically, smallpox has been an important differential diagnosis, but no longer occurs (although vaccinations have resumed in some populations).

with shingles may rarely have skin changes that are zosteriform (e.g., livedo reticularis) but never develop the classic lesions.

TABLE 3 Complications of Varicella Arthritis Bacterial superinfection of lungs, bones, or skin, rarely with septicemia Myocarditis Neurological complications: aseptic meningitis, Guillain-Barre´ syndrome, meningoencephalitis, Reye’s syndrome, transient cerebella ataxia, transverse myelitis Neutropenia Optic neuritis, keratitis, or iritis Orchitis Pericarditis Pancreatitis Pneumonia Renal complications: glomerulonephritis, nephrotic syndrome, hemolyticuremic syndrome Scarring from skin lesions Thrombocytopenia Vasculitis

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The rash of herpes zoster is commonly localized to the skin area innervated by a single sensory ganglion (dermatome) and does not usually cross the midline of the body (Figs. 3 and 4). Nevertheless, bilateral zoster has been observed in both immunocompetent and immunocompromised persons. The dermatomal rash typically occurs at the site that was most severely affected during primary varicella infection [14]. Any dermatome can be affected, but the most common regions of involvement are the ophthalmic (V1) and midthoracic to upper lumbar (T3–L2) dermatomes [12,54,55]. Herpes zoster in immunocompetent children and young adults tends to evolve rapidly, with few complications and resolution of the neuralgia as the lesion crusts fall off. In immunocompromised and elderly individuals, the rash and pain of zoster are usually more severe and complications occur more frequently (Fig. 5). Postherpetic neuralgia is a common complication, but it has varying deﬁnitions in the medical community. It is most commonly referred to as pain that persists after a certain time period or after all crusts have fallen off. Postherpetic neuralgia occurs in 10–15% of zoster cases [56,57] but infrequently affects persons under 40 years of age. On the other hand, more than one-third of affected

FIGURE 3

Herpes zoster in a thoracic dermatome in an adult.

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FIGURE 4 Herpes zoster of the ﬁrst branch of the trigeminal nerve. Vesicles on the nose are termed ‘‘Hutchinson’s sign.’’

FIGURE 5

Herpes zoster in a lymphoma patient.

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individuals 60 years or older develop postherpetic neuralgia [55–57]. The pain of postherpetic neuralgia usually remits or lessens signiﬁcantly within 1–6 months, although the duration is variable [58]. After crusting of the lesions, other abnormal sensations may also occur in patients, such as anesthesia, dysesthesia, paresthesia, or pruritus. Table 4 lists more clinical manifestations, and Table 5 includes differential diagnoses. Table 6 summarizes complications from herpes zoster. Scarring with hypopigmentation or hyperpigmentation is seen occasionally, especially if the patient did not receive appropriate antiviral therapy. The ophthalmic division of the trigeminal nerve (V1) is involved in 7% of cases and is associated with a high complication rate [45]. Of these zoster cases, 20–70% develop associated ocular disease [57], particularly those cases involving the nasociliary division of the ophthalmic nerve. The Hutchinson’s sign indicates nasociliary branch involvement, presenting as vesicles on the side and top of the nose [59] (Fig. 4). Ocular complications may include acute epithelial keratitis, chorioretinitis, cicatricial lid retraction, scleritis, uveitis, oculomotor palsies, paralytic ptosis, glaucoma, optic neuritis, or panophthalmitis (due to secondary bacterial infection). All of these complications can potentially cause visual impairment or blindness. Corneal sensation is frequently impaired with ophthalmic zoster and may result in neurotrophic keratitis or corneal ulceration. Ophthalmic zoster is also more commonly associated with postherpetic neuralgia [60]. Herpes zoster less frequently involves the second and third divisions of the trigeminal nerve or other cranial nerves [61–64]. Involvement of these nerves may result in lesions of the ears, larynx, mouth, or pharynx. The Ramsay Hunt syndrome is due to involvement of the facial or auditory nerves and consists of ipsilateral facial palsy in addition to zoster lesions of the anterior two-thirds of the tongue, external ear, or tympanic membrane. Involvement of these nerves may also result in deafness, loss of taste, otalgia, tinnitus, or vertigo [61]. Other nervous system complications may rarely occur with herpes zoster. Myelitis and meningoencephalitis have been reported in 0.2–0.5% of patients. These conditions are associated with altered mentation, fever, headache, meningeal irritation, photophobia, vomiting, or nerve palsies [44,65–69]. Herpes zoster may involve the vagus nerve or its ganglia, resulting in cardiac irregularities, dysphagia, gastric upset, nausea, or vomiting. Some patients may develop motor paralysis due to direct extension of the virus from the sensory ganglion to the anterior horn cells. This occurs in 1–5% of all zoster cases [70–73] and usually develops in the ﬁrst 2–3 weeks after rash onset. The muscles associated with the involved dermatome are usually affected, and the prognosis for recovery

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TABLE 4 Clinical Manifestations of Herpes Zoster Time after initial symptoms

Miscellaneous observations

Laboratory analysis

Reactivation of the virus may occur up to several decades after initial varicella infection.

5% of patients (particularly children) will have fever, malaise, or headache during the prodrome period.

0 days

Prodrome of localized pain

Pain is not normally signiﬁcant in children.

Tzanck smear of lesion scraping, but doesn’t differentiate from varicella or herpes simplex. Viral culture, serology, and direct immunoﬂuorescence.

3–7 days or more

Regional lymphadenopathy is usually present.

4–8 days

Unilateral dermatomal rash with erythematous macules and papules Vesicles

6–10 days

Pustules

10–14 days

Crusts

2 weeks, up to 4–6 weeks

Complete healing

Clinical manifestations

Skin lesions resemble varicella but are more conﬂuent. Scarring may occur with healing, particularly in dark-skinned individuals. Localized hypersensitivity or postherpetic neuralgia may persist for months or years after healing.

is generally good [74]. Although mild motor paralysis of the trunk is often unnoticed, the incidence of motor deﬁciencies in zoster cases of the facial nerve or extremities is 10–20% [44]. Granulomatous cerebral angiitis is a delayed central nervous system complication of zoster that develops weeks to months after the zoster rash. This condition predominantly occurs in the elderly and

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TABLE 5

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Differential Diagnoses of Herpes Zoster

Herpes simplex virus: Herpes simplex virus may occasionally simulate zoster, but it is associated with recurrent symptoms and lesions at the same site. Localized bacterial skin infection (e.g., bullous impetigo): Tzanck smear is negative. Localized contact dermatitis: Dermatitis is not typically associated with pain, and Tzanck smear is negative.

results in signs and symptoms that resemble those of a cerebrovascular thrombosis or hemorrhage [75]. Patients may present with manifestations of transient ischemic attacks, stroke-in-evolution, or isolated or multiple cerebral infarctions. The most frequent central nervous system ﬁnding is asymptomatic cerebrospinal ﬂuid abnormalities, such as elevated levels of lymphocytes and protein. The mortality rate with

TABLE 6

Complications of Herpes Zoster

Arthritis Bacterial superinfection Cutaneous dissemination of lesions Ocular complications Cicatricial lid retraction Oculomotor palsies Optic neuritis Chorioretinitis Acute epithelial keratitis Panophthalmitis Scleritis Visual impairment Uveitis Paralytic ptosis Glaucoma Blindness Neurological complications Postherpetic neuralgia Cranial nerve palsies Peripheral nerve palsies Deafness Sensory loss Meningoencephalitis Granulomatous angiitis Motor paralysis Transverse myelitis Scarring of skin lesions Visceral complications Pneumonitis Gastritis Hepatitis Esophagitis Pericarditis

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this complication is 15%, and autopsy examination reveals vascular inﬂammation with microinfarcts and thrombosis [76]. Herpes zoster has a high rate of dissemination (up to 40%) in immunosuppressed patients, but this complication is rare in immunocompetent persons [45]. Of immunocompetent patients 17–35% have a small number of vesicles located remotely from the primary dermatomal eruption, likely due to hematogenous spread of the virus. Cutaneous dissemination is deﬁned as more than 20 vesicles outside the area of the primary and adjacent dermatomes. This is followed by visceral dissemination (i.e., lungs, liver, brain) in 10% of immunocompromised patients. HIV-infected patients with zoster have been observed to have an increased rate of neurological complications (e.g., aseptic meningitis, myelitis, or radiculitis) [77] and ophthalmic complications (particularly progressive outer retinal necrosis) [77–79]. Other manifestations of herpes zoster in immunosuppressed patients include (1) multidermatomal zoster, (2) chronic verrucous nodules, and (3) one very unusual case of postherpetic hyperhidrosis [80]. 7

DERMATOPATHOLOGY

The Tzanck smear is often the ﬁrst test of choice, performed by scraping the base of an early lesion and then staining it with hematoxylin-andeosin, Papanicolaou, Giemsa, Wright’s, or toluidine blue stains. A smear of varicella or zoster lesions will have epithelial cells and multinucleated giant cells containing acidophilic intranuclear inclusions. However, herpes simplex infections have identical ﬁndings, and a Tzanck smear cannot differentiate the two infections. Histopathological ﬁndings from lesion biopsies also cannot differentiate the two. Varicella, zoster, and herpes simplex have ﬁndings of intranuclear inclusion bodies, ballooning degeneration, and multinucleated giant cells. The fusion of adjacent infected cells creates the multinucleated cells [81]. This allows direct cellto-cell spread of the virus and protects the virus from extracellular neutralization by host antibodies [82]. 8

LABORATORY FINDINGS

Most straightforward cases of varicella and herpes zoster are typically diagnosed on the basis of history and clinical ﬁndings [69,83,84]. Varicella zoster virus can be differentiated from herpes simplex virus by several laboratory tests such as direct immunoﬂuorescence, serology, molecular techniques, and viral culture. The most speciﬁc test is isolation of the virus by culture, but it is not always sensitive because the varicella-

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zoster virus is extremely labile [85–87]. Serological testing for the virus (e.g., complement ﬁxation) can retrospectively diagnose varicella-zoster infection [88]. The most useful diagnostic test for varicella-zoster infection is currently direct immunoﬂuorescence of cellular material from skin lesions. Molecular techniques with high sensitivity, such as polymerase chain reaction and dot-blot hybridization, have more recently been used to detect the virus in skin lesions, peripheral blood mononuclear cells, and other tissues of infected patients [86,89,90]. In the future, these tests may become the preferred diagnostic methods. 9

TREATMENT OF VARICELLA

Normal children with varicella have usually been treated symptomatically, because varicella infection in this population is usually self-limited and benign. With the development of antiviral drugs, acyclovir has been shown to reduce the duration and severity of varicella in this population if begun within 24 hr of the rash onset [91], although some beneﬁts are still seen if acyclovir is initiated within 24–72 hr after rash onset (Table 7). Acyclovir has been approved for the treatment of varicella in children aged 2 years and older as well as in adults. However, several factors have forestalled the wide acceptance of this therapy for children: the high cost of treatment, difﬁculty in rapid institution of therapy, and concern of possible development of acyclovir resistance. Therapy allows the child to resume school or play activities 1–2 days earlier, which is considered cost-effective because it allows the caretaker to return to work earlier. Because of the increased risk of severe varicella infection and complications in adults, systemic antiviral treatment is clearly indicated. Oral acyclovir has signiﬁcantly reduced the extent of disease and duration of manifestations in clinical study [92]. Antiviral treatment of varicella is required in immunocompromised patients [93,94], and intravenous acyclovir continues to be the drug of choice in this population. Although not FDA-approved for primary varicella and not studied in controlled trials, valacyclovir and famciclovir are known to be effective against the virus through herpes zoster studies. Interferon alfa and vidarabine have also been proven to be effective in the treatment of varicella in immunocompromised persons, but signiﬁcant toxicity with these drugs has limited their use [95,96]. 10

PREVENTION OF VARICELLA

Emphasis remains on prevention of the infection because the available treatment for varicella is not optimal. Varicella-zoster immune globulin

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TABLE 7 Treatment of Varicella Symptom Pruritus

Fever

Treatment Oral antihistamines, calamine lotion, and tepid baths with baking soda (1/4 cup per bath). Trimming of nails to discourage scratching. Antipyretics (although aspirin should be avoided in children, due to its association with Reye’s syndrome) Systemic antiviral treatment

Immunocompetent adults (and children over 40 kg): Oral acyclovir 800 mg ﬁve times a day for 7 days; valacyclovir 1 g tid or famciclovir 500 tid for 7 days are commonly used but not speciﬁcally FDA-approved for primary varicella in adults. Immunocompromised persons: Intravenous acyclovir: 10 mg/kg (500 mg/ m2) every 8 hr for 7–10 days Otherwise healthy children: Oral acyclovir (20 mg/kg) four times a day for 5 days

(VZIG) is available for immunocompromised patients who have had recent substantial exposure to varicella. Prophylaxis is also indicated for several other exposure situations, such as susceptible pregnant women and neonates whose mothers became infected shortly before delivery. It should be given within 96 hr of varicella exposure, and protection lasts at least 3 weeks [97]. Unfortunately, one-third to one-half of patients still develop clinical infection after VZIG administration [98]. The live attenuated varicella-zoster vaccine (Oka strain) has been shown to be highly effective, with a 96% seroconversion rate in healthy children [99]. Studies determined that the vaccine is 71–91% effective in preventing all disease and 95–100% effective in preventing severe disease. The vaccine is also extremely safe, with only mild side effects such as pain, fever, and slight varicelliform rash at the injection site [100– 102]. Approximately 1–4% of immunized children develop this mild varicella-like syndrome. Some pregnant women have inadvertently been given the vaccine, but no congenital varicella syndromes are known to have resulted from these vaccinations. In addition, the incidence of zoster in vaccinated children is less than that seen in naturally infected children [103]. After vaccination, the decreased incidence of zoster also holds true for leukemic children [104]. In a clinical study of vaccine efﬁcacy in a childcare center, 14% of vaccinated children versus 88% of

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unvaccinated children developed the infection after exposure [105]. The vaccinated children who did become infected had milder disease with fewer lesions and fewer days of absence from school than their unvaccinated counterparts. Another clinical trial showed that the vaccine was 100% efﬁcacious in preventing varicella [106] (Table 8). Children are required by most states to receive the varicella vaccine to attend school (or documentation of previous wild-type chickenpox infection). However, national vaccination coverage for one dose of varicella vaccine among U.S. children aged 19–35 months was only 59% in 1999 [107]. Before the VZV vaccine was licensed in 1995, there were approximately 4 million cases of chickenpox annually in the United States. About 100 persons died of primary varicella, 50% children and 50% adults, each year. Further, there were approximately 11,000 hospitalizations annually from complications of varicella, such as secondary bacterial infections [107–109]. Thus, universal coverage with the VZV vaccine has immense potential beneﬁts in terms of reduced morbidity and mortality. Moreover, an estimated annual societal savings of $384 million would result from the otherwise lost income of parents staying home with sick children, physician ofﬁce visits, and hospitalizations as well as the lifetime income of those patients dying of varicella [110]. However, this estimate does not include the potential reduction in morbidity due to reduced incidence and severity of herpes zoster later in life. Much remains to be discovered about the VZV vaccine, including the question of persistence of immunity. More than 20 years after the ﬁrst use of the vaccine in Japan, there is little evidence that the protection imparted by the vaccine may wane as the individual ages. 11

TREATMENT OF ZOSTER

Several antiviral agents are used for the treatment of herpes zoster, although no medication has been shown to completely prevent

TABLE 8

Prophylaxis of Varicella

Live attenuated varicella vaccine (Oka strain)

Varicella-zoster immune globulin (VZIG)

Routine vaccination at 12–18 months of age. Susceptible persons aged 12 months to 12 years require one vaccine dose; ages 13 and above require two doses, 4–8 weeks apart. 125 U/10 kg (up to 625 U) within 96 hr of exposure for immunosuppressed patients with substantial varicella exposure.

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postherpetic neuralgia (PHN). However, early therapy with antiviral agents has demonstrated a decrease in the duration of PHN. The ﬁrst agent to be evaluated for the treatment of herpes zoster was topical idoxuridine, but its use is not recommended because of the high potential for toxicity and the lack of effect on PHN [111,112]. Vidarabine is a systemic antiviral agent given intravenously that has been shown to reduce the duration of viral shedding, time to cessation of pain and new vesicle formation, healing time, cutaneous dissemination, and complications [49,113]. However, this agent was found later to be no more effective than acyclovir [114], and the signiﬁcant difﬁculty of administration and side effects from vidarabine have limited its use (Table 9). Oral and intravenous acyclovir have an important role in the treatment of herpes zoster. Studies have demonstrated that intravenous acyclovir given to immunocompromised and immunocompetent patients with herpes zoster reduced acute pain and the time to cutaneous healing [115–118]. Intravenous administration is indicated for the treatment of signiﬁcant complications in immunocompetent patients and the treatment of zoster in immunosuppressed patients. Oral acyclovir is indicated for therapy in immunocompetent patients and has been shown to lead to accelerated rash healing and reduction in acute pain [119–124]. However, acyclovir has not been demonstrated to reduce the incidence of PHN [115–117]. Adverse effects with acyclovir are rare and include diarrhea, headache, nausea, and renal toxicity. Central nervous system toxicity is also rare and may result in delirium, disorientation, seizures, slurred speech, or tremor [125]. The treatment of PHN is summarized in Table 10.

TABLE 9 Treatment of Zoster Symptom Local pain and pruritus

Treatment Analgesics, oral antipruritics, calamine lotion, cool compresses Systemic antiviral treatment

Immunocompromised patients: Intravenous acyclovir: 10 mg/kg (500 mg/ m2) every 8 hr for 7 days Immunocompetent patients (oral therapy) Acyclovir 800 mg 5 times daily for 7 days or Valacyclovir 1 g, three times a day for 7 days or Famciclovir 500 mg three times daily for 7 days

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TABLE 10

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Treatment of Postherpetic Neuralgia

Symptom Anticonvulsants

Capsaicin cream

Narcotic analgesics Non-narcotic analgesics Steroids Sympathetic nerve blockade Topical lidocaine gel or patch Transcutaneous electrical stimulation Tricyclic antidepressants (amitriptyline, maprotiline, and desipramine)

Treatment May be effective, especially gabapentin (recently FDAapproved for PHN). Applied topically every 4 hr for pain relief, but usually with local burning sensation. Temporary beneﬁt. Frequently not effective. Intrathecal methylprednisolone appears effective. Some reports of success. For topical pain relief (patch is FDAapproved). Some reports of success. Doses needed are much less than those used for depression treatment.

Valacyclovir, the orally administered prodrug of acyclovir, is effective in reducing the appearance of new zoster lesions, time to crusting, and time to 50% healing [126]. Compared to acyclovir, valacyclovir decreased the median duration of pain from 60 days to 40 days. Further, only 19% of patients taking valacyclovir had continued pain at 6 months compared to 26% of acyclovir recipients [127]. Valacyclovir has a side effect proﬁle similar to that of acyclovir but with no reports of nephropathy or neurotoxicity [128]. Famciclovir, the prodrug of penciclovir, has been shown to be at least equal to acyclovir in promoting cutaneous healing and decreasing the duration of acute pain [129]. Further, famciclovir has been found to decrease the duration of PHN among elderly patients compared to placebo [130]. Like all antiviral agents, treatment should be initiated as soon as possible after the zoster rash onset, preferably within 72 hr. It was reported recently that patients may also beneﬁt if antiviral therapy is started after 72 hr of rash onset, but the upper limit of time for initiation of antiviral therapy has not been determined [131]. Controlled studies have demonstrated famciclovir to be equally as safe, convenient, and effective as valacyclovir at reducing the duration of

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PHN in immunocompetent patients over 50 years of age [132]. Famciclovir has also been shown to be equally as effective and safe but more convenient than acyclovir for the therapy of herpes zoster in immunocompromised patients [133] as well as for the treatment of ophthalmic zoster in immunocompetent patients [134]. Many practitioners have tried corticosteroids to decrease inﬂammation and thus decrease the progression of the nerve damage that leads to PHN. Corticosteroids have been used both alone and in combination with antiviral agents. Some clinical trials have shown a decrease in persistent pain [135] or accelerated healing [136]. However, a more deﬁnitive study showed no long-term beneﬁt when corticosteroids were added to the acyclovir regimen [137]. The combination treatment led to more rapid rash resolution and a reduction in acute pain, but no effects on PHN were seen. In addition, adverse effects were more likely with the addition of corticosteroids. A second study showed no difference in pain at 6 months in a comparison of a combination of acyclovir and prednisone with acyclovir alone, prednisone alone, and double placebo [138]. In a small study of patients with Bell’s palsy, a combination of prednisone plus acyclovir was more effective than prednisone monotherapy for those who presented with zoster sine herpete and also had VZV DNA in their saliva [139].

12

TREATMENT OF PHN

No single antiviral agent has been consistently efﬁcacious in the treatment of PHN. However, the severity and duration of this complication can generally be reduced to some degree by early treatment of herpes zoster with the appropriate antiviral agents. The majority of clinical trials have studied and recommended treatment within 72 hr of the ﬁrst vesicle appearance. However, patients often present after the 72 hr window of treatment. It appears reasonable to initiate treatment after 72 hr in speciﬁc situations if the lesions are not completely crusted and the individual is immunocompromised, older than 50 years of age, and/or has trigeminal zoster [131]. Several other modalities have been employed in the treatment of PHN, such as analgesics, biofeedback, capsaicin, narcotics, tricyclic antidepressants, nerve blocks, and cutaneous stimulation. Systemic analgesics and narcotics are not typically effective against PHN, and long-term narcotic use may lead to drug dependency. However, these agents may be useful for the short-term therapy of acute pain with herpes zoster (Table 10).

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Tricyclic antidepressants have shown considerable effectiveness in the treatment of PHN although the mechanism of action appears to be independent of the antidepressant effects. Some experts recommend the administration of amitriptyline therapy as soon as possible for all zoster patients older than 60 years [140]. It is usually begun at low doses (10– 25 mg) and gradually increased to doses of 50–75 mg over 2–3 weeks. Desipramine and maprotiline are also effective, and desipramine may be more preferable with its lower anticholinergic and sedative effects [141,142]. Antipsychotics such as haloperidol, chlorprothixene, and ﬂuphenazine have also been tried for the treatment of PHN, often in combination with antidepressants. A placebo-controlled study of chlorprothixene, however, demonstrated only marginal efﬁcacy [143]. In a randomized, controlled, multicenter trial gabapentin was studied for the therapy of PHN in patients whose pain had been present for more than 3 months after healing of a herpes zoster rash [144]. This agent was found to be successful in the treatment of pain and sleep interference associated with PHN. In this study, patients received a 4-week titration period to a maximum dosage of 3,600 mg/day of gabapentin or placebo. Therapy was maintained for another 4 weeks at the maximum tolerated dose. Using an intent-to-treat analysis, persons receiving gabapentin had a statistically signiﬁcant decrease in average daily pain scores from 6.3 to 4.2 points compared to placebo recipients, whose change was from 6.5 to 6.0 points (p < .001). Changes in pain and sleep interference as well as secondary measures of pain were reduced with gabapentin (p < .001). In this study, the following adverse events were observed more frequently in gabapentin recipients than in those persons receiving placebo: ataxia, dizziness, infection, peripheral edema, and somnolence. However, study withdrawals were comparable in the two groups. These results led to the recent FDA approval of gabapentin for treatment of PHN. The literature reveals that whereas a decrease in PHN severity has been reported with a variety of therapies, there is no clear expectation of what that decrease might be if such treatments were used acutely in combination with an antiviral drug. Studies are currently being conducted, however, to determine the beneﬁts on PHN of initiating gabapentin therapy at the same time that antivirals are used for the treatment of acute herpes zoster. Topical therapies have also been beneﬁcial in the treatment of PHN. Capsaicin cream acts by enhancing the release or inhibiting the reaccumulation of substance P from nerve terminals and cell bodies. A clinical trial demonstrated that nearly 80% of patients treated with capsaicin experienced some pain relief [145]. However, some patients

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cannot tolerate the burning sensation related to capsaicin treatment. Some authors recommend pretreatment with topical anesthetics such as lidocaine to alleviate this problem [146]. Patients should be directed not to apply capsaicin to any unhealed skin lesions. Topical lidocaine is available in several forms, such as gel, cream, and a patch, and the topical lidocaine patch is FDA-approved therapy for PHN [147–149]. Other local treatments such as injection of bupivacaine, cryoanalgesia, EMLA cream, and sympathetic nerve blockade have led to pain relief in some patients [150–154]. Transcutaneous electrical stimulation has also been tried with limited success for the treatment of PHN [155]. Steroids have been used in various doses, preparations, and schedules for the therapy of PHN, but generally with limited success. However, Kotani et al. [156] demonstrated that intrathecal administration of methylprednisolone with lidocaine produced excellent or good pain relief at 4 weeks and at 1 and 2 years in over 90% of persons who had suffered intractable PHN for at least 1 year. In contrast, relief was reported in only 6% of those who received only lidocaine and 4% of those who received no therapy. Further, allodynia was reduced by more than 70% in the methylprednisolone–lidocaine group and less than 25% in the lidocaine-only group. Of note, no serious side effects were observed in any of the patients. 13

PREVENTION OF PHN BY PROPHYLAXIS OF HERPES ZOSTER

Because the development of herpes zoster has been linked to a decrease in cell-mediated immunity, studies are under way to evaluate the prophylactic effect of the live attenuated varicella-zoster vaccine. Studies thus far have shown an enhancement in immunity against the virus in elderly recipients of the vaccine [157,158], although results from one study demonstrated enhanced immunity that lasted only 1 year [159]. Further, 10–15% of those vaccinated failed to develop enhanced immunity regardless of dose [159]. More studies with improved vaccines may show more promising results. 14

CONCLUSION

Although primary varicella is generally considered a mild self-limited disease of children, the increased population of immunocompromised patients will likely lead to an overall increase in morbidity from this illness. The potential morbidity of herpes zoster, particularly in the elderly and immunocompromised, can also be quite devastating.

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7 Human Papillomaviruses Guy Hewlett Bayer HealthCare, Bayer AG, Wuppertal, Germany

Philip S. Shepherd and Jenny C. Luxton Guy’s, King’s and St. Thomas’ Medical and Dental Schools, London, England

1

INTRODUCTION

Human papillomaviruses (HPVs) are small, epitheliotropic, doublestranded DNA viruses that consist of an 8 kilobase (kb) genome contained within an icosahedral capsid. Approximately 100 different genotypes have been identiﬁed to date, and these can be divided into two groups according to the type of stratiﬁed epithelium infected—skin or mucosa (Table 1). A further classiﬁcation parameter relates to the potential of the virus to induce malignant changes in the infected tissue, a particular, although not exclusive, property of the papillomaviruses that infect mucosal surfaces. Thus the group of ‘‘high-risk’’ viruses, which includes HPV16 and HPV18, is causally related to the induction of malignancies, especially of the uterine cervix [1–3], whereas members of the ‘‘low-risk’’ group, which includes HPV6 and HPV11, induce benign lesions, particularly in the anogenital mucosa. Benign hyperplasias of the skin, or warts, are very common, especially among children and young adolescents, and infection with viruses such as HPV1 and HPV2 can result in unsightly lesions on the 227

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TABLE 1 Human Papillomavirus Types Associated with Cutaneous and Mucosal Lesions Site Skin

Lesion Common wart Plantar wart Myrmecial wart Mosaic wart Flat wart EV

Mucosa

Condyloma Intraepithelial neoplasia

HPV type 1, 2, 3, 4, 7, 10, 26, 27, 28, 29, 41, 57, 65 1, 2, 4, 63 1, 63 2 3, 10, 27, 28, 38, 41, 49 5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 47, 50 6, 11, 42, 43, 44, 54, 55, 74, 79 16, 18, 26, 27, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 64, 66, 67, 68, 69, 71, 72, 73, 74

back of the hands (common warts) and painful lesions on the foot (deep plantar warts). Patients suffering from the rare genetic disease epidermodysplasia verruciformis develop extensive ﬂat warts that can develop into squamous cell carcinomas, especially if infected with HPV types 5 or 8. Similar lesions are observed in renal transplant patients and, with the increase in numbers of immunosuppressed patients, infections with HPV are becoming a serious medical problem. Cervical HPV infections are more common, for example, in HIVpositive women than in HIV-negative women, and there seems to be a greater risk for high-grade lesions to develop. Genital HPV infection is the second most common sexually transmitted disease, with a prevalence of 20 million and an incidence of 5.5 million in the United States, for example. The annual number of cervical cancer cases worldwide is estimated to be between 400,000 and 500,000. Most cases occur in developing countries, but there are still approximately 14,000 new cases of cancer per year in the United States with an annual mortality of 5,000, despite a screening program that involves 50 million tests per year [4]. Annual costs arising from sexually transmitted diseases in the United States of America were recently estimated to total almost $17 billion, of which almost $4 billion was associated with HPV infection [5].

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Genomically, papillomaviruses are quite similar. The early (E) open reading frames (ORFs) are expressed during the preassembly phase of viral replication, and the late (L) ORFs are activated during the ﬁnal phase of replication and express the viral capsid proteins, L1 and L2. Some of the early proteins—E5, E6, and E7—possess oncogenic properties and are indirectly responsible for the transformation of infected cells. This transformation causes hyperproliferation of the cells, which results in the benign wart or, if the infection becomes persistent, can lead to invasive, cancerous growth of the tissue. The proteins E1 and E2 are directly responsible for the replication of the viral DNA in that they recruit the cellular DNA replication machinery to the origin of replication of the viral DNA. E4 is thought to facilitate release of mature virions from the keratinized cell. It is assumed that the virus initially infects the basal cells of the epithelium where the DNA is replicated at a minimal rate, in synchrony with the host DNA, in order to maintain copy number. The structure of the normal, stratiﬁed epithelium is the result of the basal cells dividing to produce daughter cells, one of which moves outward to eventually reach the surface of the skin or mucosa. During this outward movement, the cell shuts down its DNA replication machinery and undergoes a series of differentiation steps, probably brought about by sequential toggling of cellular genes. After going through this differentiation process, the heavily keratinized cells form the outermost, corniﬁed layer of the skin (Fig. 1). However, the replication of papillomavirus DNA requires the cellular DNA synthesis machinery to be activated throughout the process of migration from the immediate suprabasal layer to the stratum granulosum. It appears that once the infected daughter cell has left the basal layer, the cell is induced to reenter the S-phase by the controlled expression of the early ORFs E5, E6, and E7, which modulate the activity of the appropriate cell cycle proteins. This eventually leads to the synthesis of many thousands of copies of viral DNA per cell while, at the same time, the infected cell is permitted to toggle its differentiation signals, which the virus probably requires for switching its own genes on and off in the correct order. Virus progeny are assembled in the corniﬁed layer, and the majority of virus particles are released as the infected cell disintegrates on the surface of the skin or mucosa. Cutaneous warts and low-grade mucosal infections actively produce virus particles, whereas the high-grade lesions of the anogenital mucosa do not produce virions. Viral DNA is often integrated into the host DNA of high-grade lesions, although it is not uncommon to also detect episomal DNA [6]. As explained above, papillomaviruses can replicate only in differentiating epithelial cells, and because it has proved difﬁcult in the

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FIGURE 1 Schematic of a transverse section of corniﬁed epithelium with a description of papillomavirus gene expression and DNA replication during a productive infection. (Adapted from Ref. 123.)

past to produce differentiating skin cultures, this has restricted the study of these viruses. This is one of the reasons that, up until recently, the papillomaviruses had received little attention from the biomedical community and, as a result, the status of diagnostic methodology and therapeutic modalities had not progressed much beyond that achieved by the middle of the last century. Recommended diagnostic procedures are based on clinical examination, cytology, and histology, whereas almost all therapeutic options are directed toward the symptoms of the disease and not to the elimination of the causative agent. It must be recognized that widespread cervical cytological screening with the Papanicolaou test and treatment of premalignant lesions of the uterine cervix have resulted in an enormous reduction in the mortality of cervical cancer in the industrialized nations over the last 50 years, but there are nevertheless still large numbers of women who have equivocal cytological diagnoses, and there is a perceived medical need for more precise diagnostic methods. There is also an urgent need in developing countries for effective diagnostic and preventive measures.

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Developments in molecular biology and gene technology have resulted in a boom in basic research into papillomaviruses during the last 15 years, and there is an ever-increasing understanding of the viral life cycle and of HPV-induced transformation processes. New diagnostic and therapeutic strategies for HPV-associated disease have also been developed as pharmaceutical and biotechnological companies become more aware of the unmet medical need in these areas. In the following section of this chapter we review the clinical features of HPV infections and their diagnosis. Thereafter we discuss current approaches to therapy and then, in the ﬁnal sections, we consider the immune response to HPV infection and the concepts of prophylactic and therapeutic vaccination. 2

THE DISEASES AND THEIR DIAGNOSIS

2.1 2.1.1

Cutaneous Warts Pathology

A large number of HPV types are associated with skin lesions. Indeed, the ﬁrst HPV to be characterized (HPV1) was isolated from a plantar wart. ‘‘Cutaneous warts’’ is the collective name for a group that is made up of common warts, plantar warts, mosaic warts, ﬂat or plane warts, intermediate warts (a mixture of common and ﬂat warts), myrmecia, and the plaquelike lesions associated with epidermodysplasia verruciformis. Common warts are spread by nonsexual contact and occur singly or in groups, most commonly on the dorsal surfaces of the hands and ﬁngers. In children, common warts can also be observed on the knees. Particularly well known is the butcher’s wart, which, contrary to popular belief, is not caused by bovine papillomavirus but by HPV7. The relatively high frequency of warts on the hands of butchers and related professionals, such as ﬁsh handlers, is thought to be due to the exposure of the hands to extreme conditions such as low temperatures as well as cuts and abrasions caused by intensive scrubbing. Recently it was found that HPV7 is also associated with papillomas of the face and mouth in HIV-positive patients; these ﬁndings suggest that the relationship between the site of infection, the immune system, and HPV is much more complex than was previously thought. A high incidence of nonregressing, cutaneous warts has also been reported to occur in immunosuppressed transplant patients. Five years after grafting, more than 50% of patients have cutaneous warts, mostly of the common wart type, and during ongoing immunosuppression the frequency of patients with warts approaches 90%. It is likely that this huge increase in

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incidence is due to the loss of cell-mediated immunity and a resulting reactivation of latent HPV infections [7,8]. Plantar warts are found mainly on the soles of the feet and the palms. The painful warts that often occur on the soles of children’s feet are called deep plantar warts or myrmecial warts and are caused by HPV1. These warts are extremely contagious because the viral content is usually quite high. Mosaic warts are also found in palmar and plantar locations and are caused by HPV2. Flat warts are smaller than common warts and are mainly found on the hands and the face. They are common in children, young women, and immunodeﬁcient persons and present as partially pigmented, multiple ﬂat lesions. These warts are typically associated with HPV3, 10, 27, and 28. A further group of lesions belonging to the group of cutaneous warts is associated with the rare autosomal recessive disease epidermodysplasia verruciformis (EV), which is characterized by a persistent cutaneous HPV infection with disseminated lesions resembling ﬂat warts. Squamous cell carcinomas occur in about 50% of this patient group, and there is a strong association with HVP5 infection in these cases. EV lesions are associated with speciﬁc HPV types, most typically with HPV5, 8, 12, 36, and 47, although EV involving ﬂat wartlike lesions is often associated with HPV3 [8]. 2.1.2

Diagnosis

Clinical examination is the usual method of diagnosis of cutaneous warts, and the location of the lesions is probably the most important diagnostic tool. However, atypical lesions and atypical sites are not uncommon, so biopsy and histology are sometimes required to distinguish viral warts from other conditions. There are no data to support the use of type-speciﬁc HPV nucleic acid tests in routine diagnosis of cutaneous warts. 2.2 2.2.1

External Warts of the Anogenital Region Pathology

There are more than 40 different HPV types that infect the genital tract, and most of these infections are asymptomatic or remain unrecognized. Approximately 75% of all sexually active persons have been infected with genital papillomavirus, and 1% of this population have genital warts [9]. External genital warts, or condylomas, are benign, soft, ﬂeshcolored outgrowths that occur individually or can merge into large cauliﬂower-like lesions. The morphology of the external genital wart is

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probably a result of the type of skin actually infected: acuminate warts tend to appear on moist, thinly keratinized, non-hairy skin; the smoother, papular wart on normally keratinized skin; and ﬂat warts on either of these surfaces. Up to 95% of condylomas are caused by HPV6 or 11. These ‘‘low-risk’’ HPV types can also cause warts on the uterine cervix and in the vagina, urethra, and anus. A rare condition is the ‘‘giant condyloma’’ or Buschke Lo¨wenstein tumor, which is also associated with the ‘‘low-risk’’ HPV6 and 11 but is characterized by an aggressive semimalignant growth of the condyloma into the underlying dermis. The high-risk viruses HPV16, 18, 31, 33, and 35 are also sometimes found in association with visible genital warts and with squamous intraepithelial neoplasias of the external genitalia, and it is not uncommon to detect several different HPV types at one time. This is probably a result of repeated exposure to different types in sexually active patients. Vulvar intraepithelial neoplasia (VIN) is caused by HPV 16 and is often found in association with cervical intraepithelial neoplasia (CIN) (see Sec 2.3). The rate of progression of VIN to invasive cancer is lower than that of CIN, although there is a clear division between women older than 40 years (30% risk of progression) and younger than 40 (<1%). There has been a worldwide increase in VIN—a doubling within the last 4 years—and this has also been associated with an increase in the incidence of vulvar cancers in women under 50 years of age [10]. A recent review [11] notes that although classic VIN and CIN are caused by the same group of high-risk viruses, an important difference between the two diseases is the type of tissue infected: most CIN derives from infection of endocervical glandular mucosa and the metaplastic squamous epithelium of the transformation zone whereas VIN develops from infection of a mature, stratiﬁed, squamous epithelium of the vulvar epidermis or squamous mucosa. As noted above for cutaneous warts, HIV-infected individuals also show a prevalence for HPV infection and anogenital squamous intraepithelial lesions (SILs) that is inversely related to the CD4 count. However, there have not been any signiﬁcant increases in invasive anogenital cancer seen in HIV-positive patients. Because the conversion from SIL to invasive cancer usually takes a number of years, it is possible that this will be seen in HIV patients who have a prolonged survival time as a result of modern antiretroviral therapy [12]. Anal cancer is relatively uncommon but has shown dramatic increases in incidence over the past 20–30 years [4]. This increase is probably the result of changes in sexual behavior against the background of the HIV epidemic. Anal cancer and cervical cancer are very similar in

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their histology and in their tendency to originate in the transformation zone where columnar mucosa meets squamous epithelium. Recent data indicate that HPV infection is also one of the main risk factors for developing anal SIL [13]. Again, the impact of antiretroviral therapy on the progression of anal SIL is unclear in that it could lead to either an increased risk of progression due to greater longevity or to a higher probability of regression owing to an improvement in immune function. 2.2.2

Diagnosis

As with cutaneous warts, external genital warts are diagnosed without instrumentation according to location and, if necessary, by biopsy. Routine biopsy is not considered to be necessary for patients under 35 years of age with ﬁrst-time multiple acuminate lesions; however, it is recommended for atypical cases and differential diagnosis, especially in patients over 35 years of age. Papanicolaou smears are also being used for the screening of anal lesions [14], and the potential beneﬁt of an anal cancer prevention program, similar to the cervical cancer program, is currently under discussion. Nucleic acid tests are not regarded to be essential for the diagnosis and management of external genital warts. 2.3 2.3.1

HPV Infections of the Cervix Pathology

Polymerase chain reaction methodology has been used to demonstrate that most young, sexually active persons carry genital HPV of one type or another [15]. However, a large number of persons with evidence of genital HPV never develop any disease related to their infection [16]. For example, epidemiological data clearly demonstrate that approximately 70% of women found to be positive for high-risk genital HPV will remain free of dysplasia [17]. The high prevalence of HPV associated with all stages of cervical disease was the ﬁrst indication for the causal role of this group of viruses in cervical dysplasia. Slight dysplasia is characterized by the presence of koilocytes in the Pap smear, which are themselves signs of an active production of papillomavirus virions by any type of HPV. Progression from slight dysplasia to carcinoma in situ (CIS) is usually associated with loss of virion production and a concomitant reduction in the amount of episomal DNA. Moderate and severe dysplasia show a stronger correlation with high-risk types than slight dysplasia, and the high-risk genital HPV types, e.g., HPV16, 18, 31, 33, 35, 45, 51, 52, 58, and 59, are recognized as the primary cause of cervical cancer [18]. Members of this

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group have been detected in more than 99% of cervical biopsies collected in the course of a multicentric study. However, it is clear that the disease is multifactorial and, although high-risk HPV is necessary [19,20], other factors such as age at ﬁrst sexual intercourse, number of sexual partners, and hormonal exposure also appear to be associated with risk of progression from the initial infection to invasive cancer. One of the most important predictors for progression is the persistence of HPV infection. Moscicki et al. [21] recently demonstrated that the risk of developing high-grade SIL (HSIL) was very high for women with three consecutive, positive HPV typing tests at intervals of 4 months. Immunosuppression also increases the risk for developing high-grade cervical lesions, both in transplant patients and in HIV-infected patients [22]. 2.3.2

Diagnosis

A discussion of the diagnosis of cervical infections cannot begin without referring to a technique that is not regarded as a diagnostic test at all, the Pap smear. The Papanicolaou smear is a screening tool that, in principle, predicts the histology of the cervix epithelium. Since its introduction in the 1950s, Pap smear cytological testing has indirectly reduced the incidence of cervical cancer by more than 70% in the United States. However, a satisfactory diagnosis and a sound triage of the patient depend on the evaluation of abnormal cytology with colposcopy and biopsy where appropriate. The Pap screen is probably so effective in countries where cervical screening is carried out on a regular basis because it takes up to 15 years for a low-grade SIL (LSIL) to progress to invasive carcinoma, if at all, so that the screening program has time to identify potential cervical neoplasias during the preinvasive stage. Pap smears are highly efﬁcient at identifying high-grade lesions and invasive malignancies, but the rate of false negatives is between 15% and 40%. Nevertheless, it is a highly effective screening tool and continues to be the mainstay of all cervical cancer screening programs [23]. The diagnosis of LSIL is deﬁned by the presence of cells that correspond to ﬂat or exophytic condyloma of the cervix. Cytologically, the principal feature is an abnormal nucleus, mainly in the superﬁcial cells. Koilocytes are typical of LSIL, with their single or multiple enlarged nuclei with an irregular clear space around them, and are indicative of a productive HPV infection. Morphologically, HPV infection results in a lesion that has altered growth properties and that is characterized by a distinct cytopathic effect in the cells at the surface of the mucosa. Two factors distinguish LSIL and HSIL: one is an increase in less mature cells, and the other is an increase in nuclear abnormality in cells from all layers of the epithelium, not just the terminally differentiated cells of the

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surface. A description of the management of advanced disease is beyond the scope of this book, but, brieﬂy, the exclusion of invasive cancer, which will inﬂuence the decision as to whether to carry out surgery, requires that all the parameters of the triage agree, i.e., that cytology and histology agree, within limits, on the nature of the lesion. If there is any discrepancy, the appropriate tests should be repeated. Cervical intraepithelial neoplasia (CIN) is the term applied to all dysplastic, precursor lesions of invasive cervical cancer [24]. Thus all lesions of the cervix can be classiﬁed using the terminology condylomata, CIN1, CIN2, and CIN3 according to severity of disease (see Ref. 25, Figs 6.5–6.8, for illustrations of the different stages). Most CIN1 lesions will remain stable or disappear, but approximately 20% will progress to a higher grade disease. The likelihood of progression for CIN2 is 30% and for CIN3, 50%. These statistics represent the central problem facing cytologists, pathologists, and other clinicians confronted with cervical HPV infections and illustrates the urgent need for more predictive diagnostic technology and for improved therapeutic strategies. This problem of analysis and interpretation has recently been compounded by a suggestion that the generally accepted model of progression from CIN1 through CIN2 to CIN3 may well be incorrect [26]. The Bethesda system of lesion classiﬁcation was devised to simplify the cytological and histological characterization of cervical dysplasia by describing only two categories, low-grade and high-grade squamous intracellular lesions (LSIL and HSIL) [27]. The advantages of this system are that the number of terms used to describe the lesion have been reduced to just two, there is no need to use the term ‘‘neoplasia,’’ and much terminology has been eliminated that has little bearing on the treatment of the lesion. However, there are also disadvantages to the system, particularly when a lesion does not quite ﬁt the LSIL or HSIL categories. Such difﬁculties of classiﬁcation have led to the emergence of the ASCUS (atypical squamous cells of undetermined signiﬁcance) dilemma, especially in the screening of older women. The ASCUS or ‘‘borderline’’ group of women, which accounts for 3–6% of all Pap screening results, has caused concern among clinicians because of a lack of cytological reproducibility. This is a problem because although most ASCUS patients will have a normal histology, about 8% have HSIL and 0.1% have cervical cancer. Thus, for medical, ﬁnancial, and, particularly in the United States, legal purposes, ASCUS patients are usually subjected to cost-intensive follow-up examinations that can result in overdiagnosis and overtreatment. A method that has been heralded as a potential solution to this problem of descriptive screening involves testing for the presence of high-

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risk HPV DNA sequences by polymerase chain reactions or by a DNAhybridization method known as hybrid capture [28–30]. This proposed strategy for improving the accuracy of screening and diagnosis has unleashed an unprecedented amount of criticism, however [31]. There is no consensus on whether HPV typing could be used as a stand-alone triage method, possibly replacing cytological screening, or whether it should just be an adjunctive test to conventional screening. Current opinion seems to be that until clinical trials have been carried out in sufﬁcient depth, HPV testing should remain an investigational tool [32–35]. Human papillomavirus testing has also been used for evaluating the success of surgical removal of HSIL whereby the detection of HPV appears to correlate with an increase in the risk of recurrence. There is also evidence that a positive, high-risk viral DNA test 3 months after treatment of HSIL is more predictive than abnormal cytology for residual or recurrent disease [36]. 3

TREATMENT OF HPV INFECTIONS

On a global scale, carcinoma of the cervix may be the most preventable major form of cancer. Proposals for disease control have included prophylactic vaccination against HPV, HPV typing, improved cytological screening, and, above all, novel therapeutic strategies. The need for treatment of HPV infections is a much discussed topic. Obviously, advanced disease requires treatment, but because most HPV infections regress spontaneously, the treatment of low-grade mucosal infections and most cutaneous infections remains controversial. Even though most nongenital warts are not life-threatening, many patients will be distressed, for example, by cutaneous warts and will request appropriate treatment. The patient will expect the treatment to cure the afﬂiction or at least lead to a lengthy remission and freedom from warts. Current therapies (Table 2) do not necessarily eradicate warts, maintain clearance, or eliminate the virus or its DNA. Thus there is a variable degree of treatment success—recurrence rate can be high due to residual virus—and cost-effectiveness calculations will often determine whether or not a certain treatment is to be used. An additional drawback of many current therapies is that they are associated with skin reactions that range from itching through burning to pain. Some erosive therapies can also cause scarring and disﬁgurement [37,38]. Recently there has been a series of publications containing guidelines for the treatment of cutaneous warts, anogenital warts, and cervical dysplasia [39–43]. However, a close inspection of Table 2 reveals the lack of any speciﬁc treatment for the viral infection itself. Most treatments

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TABLE 2 Current Treatments for HPV Infectionsa Cutaneous warts Photodynamic therapy Salicylic acid Bleomycin Retinoids Formaldehyde Thermocautery Glutaraldehyde Chemical cautery CO2 laser Pulsed dye laser Topical sensitization Cimetidine Homeopathy Podophyllin Folk remedies Hypnosis Heat treatment Interferon Imiquimod

Cervical dysplasia Cold knife conization CO2 laser conization LEEP Electrocoagulation Cryosurgery CO2 laser vaporization Hysterectomy

Anogenital warts Podophyllotoxin Imiquimod Scissor excision Electrosurgery Cryotherapy Trichloroacetic acid Laser surgery Interferon 5-Fluorouracil Podophyllin

a

For details see Refs. 23, 42, and 43.

involve freezing, burning, etching, local chemotherapy, or surgical removal of the tissue. The recommended treatment for cutaneous warts is cryotherapy, although photodynamic therapy and salicylic acid are also accepted therapeutic choices. The currently recommended patient-conducted therapy of external anogenital warts is the application of podophyllotoxin or imiquimod, but there is insufﬁcient evidence to suggest that either of these compounds is suitable for treating cutaneous warts. It is likely that there is a poor level of penetration of active substance into the more heavily corniﬁed cutaneous wart tissue and the efﬁcacy of the treatment is reduced accordingly. Recommended physician’s ofﬁce therapies for anogenital warts are electrosurgery, laser, curettage, or scissor excision as well as cryotherapy or trichloroacetic acid. Once the bulk of the wart has been removed, an interferon-beta gel can be applied as an adjuvant. Treatment is not recommended for subclinical genital HPV infections that have been diagnosed by Pap smear, colposcopy, or HPV

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DNA detection. This is quite simply because there is no therapy currently available that will eradicate the infection; observation of the infected tissue by the physician at regular intervals is the preferred procedure in such cases. If lesions develop, an appropriate therapy can be initiated; if the infection remains dormant, the ‘‘watchful waiting’’ strategy should be continued. The treatment for high-grade premalignant lesions, on the other hand, involves the immediate ablation of the lesion by one of the methods mentioned above. Invasive cancer requires radical surgery, radiotherapy, and chemotherapy according to the status of disease [44]. None of the above treatments for warts has a particularly high rate of success; clearance rates range between 20% and 70%, and recurrence rates are high, and it is clear that new approaches to disease control, such as antiviral chemotherapy and vaccines, are urgently required. Antiviral chemotherapy is still very much in the preclinical, investigational stage, but other options that involve the exploration of the immune response to HPV and the related concepts of prophylactic and therapeutic vaccination are currently under intensive study. The main reasons for this are ﬁrst that the immune response to HPV, especially the mucosal types, has been the subject of detailed analysis in recent years and second that advances in biotechnology have made it possible to manufacture viral proteins and DNA on such a scale that vaccination schemes are now feasible.

4

IMMUNE RESPONSES TO HPV INFECTION

Evidence that the host immune response is important in the control of HPV infections comes from a number of observations. For example, the regression of genital warts is often accompanied by an inﬁltrate of predominantly CD4 positive cells [45]. This suggests that the cellmediated immune response is effective in most cases but fails to control HPV infection in lesions that progress. In order for progression to take place, HPV infection must persist for long periods of time, also implying that the host immune response remains ineffective during this time. In immunosuppressed individuals, such as transplant recipients [46] or patients with the genetically determined condition epidermodysplasia verruciformis [47], an increase in the incidence and severity of HPVrelated lesions is seen. Most studies on immune responses to HPV have involved HPV16, the viral type most commonly associated with the development of HSIL and cervical cancer.

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Cytotoxic T-Lymphocyte Responses

Cytotoxic T-lymphocytes (CTLs), characterized by the CD8 cell surface marker, are the immune effector cells classically associated with the killing of virally infected cells. This occurs via the presentation of viral peptides in the peptide-binding groove of the major histocompatibility complex (MHC) class I molecules on the surface of infected cells to the T-cell receptor of the CTL. Interaction between other accessory molecules on cells and the T-cell surface are required to fully activate the T-cell. Evidence that CTLs are likely to be important in elimination of HPV16 infections ﬁrst came from studies in mice where CTLs speciﬁc for the HPV16 E7 oncoprotein were shown to protect against tumor induction by HPV16 positive cells [48] and to eradicate existing HPV16 positive tumors [49]. Historically, HPV16 E6- and E7-speciﬁc CTLs have proven difﬁcult to detect, suggesting that they may be present at only low frequencies in the peripheral blood of women with cervical SIL and cancer [50–52]. The low frequency of these T-cells may be a result of their speciﬁc downregulation. Indeed, a study by Nakagawa et al. [53] showed that HPV16 E6- and E7-speciﬁc CTLs were more common in HPV 16-positive women without SIL than in those with SIL. Even though E7-speciﬁc CTLs may be detected in the lymph nodes and tumor tissues of cervical cancer patients [54], reduced levels of the cytolytic effector molecule granzyme B and of the cytokine IL2 suggest that these cells are poorly activated [55]. Furthermore, there is strong evidence for the altered expression of some of the molecules involved in MHC class I–mediated antigen processing and presentation in patients with cervical cancer, and, to a lesser extent, those with high-grade SIL. In this regard, multiple mechanisms for the dysregulation of MHC class I expression have been described [56]. Downregulation of MHC class I expression may also result from decreased levels of a transporter protein associated with antigen presentation known as TAP-1. This has been observed both in cervical cancer [57,58] and in recurrent respiratory papillomatosis [59]. In the latter case, the reduction in TAP-1 expression also correlated closely with a rapid recurrence of disease. Our ability to detect low-frequency T-cells has been greatly improved in recent years following the development of soluble, ﬂuorogenic MHC–peptide complexes or tetramers. These reagents have facilitated the direct detection of HPV-speciﬁc CTLs in short-term T-cell lines and even in peripheral blood mononuclear cells stained directly ex vivo [60]. It is hoped that the use of a more sensitive technique such as this might aid in the identiﬁcation of T-cell responses that are important in the control of cervical disease.

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T-Helper Cell Responses

T-helper cells characterized by the CD4 cell surface molecule probably do not have a direct antiviral or antitumor effector function but perform a critical role in the regulation of both CD8 positive CTL- and B-cellmediated immune responses. The antigen-speciﬁc activation of T-helper cells leads to the secretion of cytokines, including IL2, that provide help for the activation of CTLs, as well as IL4, IL5, IFN-g, and TGF-b, which are required for B-cell maturation and Ig class switching. Evidence that CD4 positive cells are required for an optimal antitumor response comes from vaccination studies in mice involving HPV16 E7-expressing DNA constructs [61,62]. Peripheral blood T-helper cell responses to HPV16 L1 [63] as well as to L2, E2, E4, E5, E6, and E7 antigens [64] have been described in women with SIL of the cervix. As for CD8 positive CTLs, evidence from crosssectional studies suggests that HPV16-speciﬁc T-helper cell responses to the HPV16 E5, E6, and E7 antigens are reduced in women with cervical SIL compared to controls [65–67]. The evidence linking such responses with protection from disease is far from clear: Some prospective studies have demonstrated a link between T-helper cell responses to certain E6 and E7 peptides [68], whereas others have shown that E7 responses and persistence and progression of cervical disease are linked [69,70]. Little information is available regarding the nature and speciﬁcity of cervical lesion–inﬁltrating T-cells, the vast majority of studies having been performed on PBMCs. In a study of wart-inﬁltrating lymphocytes, 75% of patients made responses to the HPV6 L1 antigen [71]. Varying numbers of inﬁltrating CD4 positive T-cells have been observed in cervical SIL and cancer, but their presence has not been positively linked with the regression of disease. 4.3

Humoral Immune Responses

HPV16-speciﬁc serum IgG levels are traditionally measured by using enzyme-linked immunosorbent assays (ELISAs). The replacement of fusion proteins and peptides of HPV16 capsids with conformationally correct virus-like particles (VLPs) as antigen in these assays led to an improvement in the assay sensitivity and speciﬁcity [72]. Even so, the antibodies detected in prospective studies of patients with cervical SIL and cancer were more strongly linked with persistent HPV16 infection than with protection from or clearance of viral infection [73]. The strongest evidence for the importance of neutralizing antibodies in prevention of papillomavirus infections comes from a number of animal studies involving vaccination with virus-like particles (VLPs)

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and subsequent experimental challenge with virus [74–76]. In each case, the protection induced following experimental challenge correlated with the presence of VLP-speciﬁc IgG antibodies. Although B-cells have been observed in the subepithelial stroma that lies beneath cervical lesions [77], both antigen-presenting cells and B-cells were reported to be markedly reduced in number in HSIL and in invasive disease [78]. Reports on HPV16 capsid-speciﬁc cervical IgA antibodies in patients with SIL have given differing results. Some suggest that local IgA antibodies and HPV16 infection are linked [79,80], others that local IgA antibody levels correlate more closely with recent disease [81] and decline rapidly after successful treatment [82]. No association was found between local IgA antibodies and viral clearance or regression of cervical disease [83]. Many studies have shown an association between HPV16 E7speciﬁc serum IgG and cervical cancer. In a few of these studies, patients with SIL were also investigated, but levels of seropositivity detected were low [84–87]. This may reﬂect the fact that in high-grade lesions and cervical cancer, HPV16 DNA integrates into the host genome such that HPV antigen expression is restricted largely to E6 and E7. The presence of these antibodies in patients with high-grade SIL and cancer indicates that they are unlikely to have any protective effect. 4.4

Antigen Presenting Cells

Antigen presenting cells (APCs) are critical for the effective development of virus-speciﬁc and tumor-speciﬁc cell-mediated immunity. For this reason, recent approaches in the development of therapeutic vaccines have turned to the manipulation of dendritic cells as a means of enhancing their effectiveness. Because HPV infections associated with cervical SIL are nonlytic and therefore remain within the epithelial keratinocyte until the cell dies, it is likely that antigen presentation by keratinocytes would be required for the HPV-speciﬁc immune response to be effective. Keratinocytes, however, do not normally express MHC class II molecules except during inﬂammation or following stimulation by IFN-g [88]; neither do they express the costimulatory molecules B7.1 and B7.2 normally found on professional APCs. It is therefore possible that presentation of HPV early antigens by keratinocytes to T-cells may result in their incomplete activation and thus lead to a state of T-cell tolerance. Some support for this hypothesis comes from experiments in the mouse, where expression of HPV16 E7 has induced T-cell unresponsiveness or tolerance [89,90]. Professional antigen presenting cells (Langerhans cells) are present in cervical epithelia and are likely to

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be involved in the processing and presentation of HPV virions to T-cells. It has been reported that the numbers of Langerhans cells are reduced in cervical SIL [91,92]. Also, patients who did not respond to IFN-g treatment of their genital warts were found to have decreased CD4positive and CD8-positive cells, Langerhans cells, and MHC class I and II expression, in addition to decreased expression of the cytokines IL1a and b, granulocyte macrophage colony stimulating factor (GMCSF), and tumor necrosis factor alpha (TNFa) [93]. Interestingly, nonresponders also had high levels of HPV6 E7 expression. 5

VACCINE DEVELOPMENT

The ﬁrst consideration in the design of an effective HPV vaccine should be to identify the immune responses that are important in prevention and/or control of HPV infection. As we have previously described, the strongest indicators as to the nature and speciﬁcity of these responses have come from animal studies. Although immune responses to many HPV16 antigens have been described in studies of peripheral blood from women with cervical SIL and cancer, the nature of the responses that protect from infection or control disease in humans is not known. Even less is known about the speciﬁcity of the lymphocytes that inﬁltrate lesions and play an active role in controlling disease. Nevertheless, HPV research has been directed toward the production of two major types of vaccine, prophylactic and therapeutic (Table 3). Prophylactic vaccines have been designed with the aim of preventing viral infection by stimulating a neutralizing antibody response directed toward the capsid proteins L1 and L2. Virus-like particles (VLPs) of HPV16 are the obvious choice of antigen for a prophylactic vaccine for a number of reasons. They are relatively easy to manufacture, have a 3-D structure indistinguishable from that of wild-type viral capsids, are free of the E6 and E7 oncogenes, and have been shown to be highly immunogenic in animal studies. In addition it is now known that VLPs can directly induce the acute activation of dendritic cells in the absence of adjuvant [94]. Therapeutic vaccines are intended to treat existing lesions or tumors by stimulating cytotoxic T-cells to target the E6 or E7 antigens that are expressed by the infected cells and thereby eliminate the lesion or tumor. A further consideration in the design of an effective prophylactic vaccine is the selection of a route of immunization that will stimulate a protective immune response in the genital mucosa. Intranasal immunization of mice with HPV16 VLPs has proven to be effective in stimulating VLP-speciﬁc mucosal IgG and IgA antibodies [95,96]. A recent report suggested that systemic immunization was more effective than either

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TABLE 3 Human Papillomavirus Vaccines in Clinical Trials Vaccine description Prophylactic Multivalent HPV6, 11, 16, 18 VLP HPV16, 18 VLP HPV16 VLP HPV6 VLP HPV16 bacteria Therapeutic HPV16 E7/hsp65 protein HPV16, 18 encapsulated DNA TA-HPV/TA-CIN protein HPV16 L1/E7 VLP HPV16 E7 peptide Naked DNA

Developing institution

Clinical phase

CSL/Merck Medlmmune/ GlaxoSmithKline National Cancer Institute CSL Ltd BTG

Phase III Phase II

Stressgen/Roche Zycos Xenova Medigene/Schering University of Leiden Merck

Phase Phase Phase Phase Phase Phase

Phase I/II Phase I/II Phase I II/III II II II I/II I

intrarectal or intravaginal VLP immunization in inducing mucosal immunity. Reports of phase I trials of HPV16 VLP-based vaccines in healthy adults show these vaccines to be well tolerated and to induce high neutralizing antibody titers and T-helper cell responses [97,98]. In another phase I trial involving patients with genital warts, vaccination with VLPs of HPV6b resulted in the regression of lesions in over 50% of patients [99]. Following the success of early trials, pharmaceutical companies such as Merck/CSL and GlaxoSmithKline and organizations such as the National Cancer Institute (NCI) are taking VLP vaccines into late-stage trials. It may be some years, however, before it is known whether such vaccines are capable of inducing long-lasting protection at genital mucosal surfaces. Many different approaches to the design of therapeutic HPV vaccines are currently under investigation. These can be broadly classiﬁed into fusion protein, chimeric VLP, peptide or DNA-based vaccines, and those that use a combination of antigen types. Additionally, some vaccines are designed around live recombinant viral or bacterial vectors, whereas others use the ex vivo manipulation of dendritic cells to enhance immune responses.

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Protein vaccines that have reached clinical trials include a heat shock protein (hsp)-65/HPV16 E7 fusion protein used to treat patients with high-grade anal SIL [100] and an HPV6 L2E7 fusion protein used to treat patients with genital warts [101]. A reduction in the severity of lesions was seen in all eight patients entered into the former study, and this vaccine is now in phase III trials [102]. Regression of genital warts was observed in ﬁve of 27 cases in the latter study. The results of two phase II trials involving the HPVL2/E7-based vaccine (TA-GW vaccine developed by the former Cantab Pharmaceuticals PLC), however, showed no signiﬁcant difference between wart recurrence rates of patients and controls at 6 months postvaccination. TA-HPV, Xenova’s vaccine for treatment of cervical cancer, has reached phase IIa trials in patients with high-grade anogenital intraepithelial neoplasia and is also being tested in conjunction with TA-CIN in a phase II ‘‘prime boost’’ type study [103]. Another variation of the protein-based vaccines are chimeric VLPs (cVLPs). These are VLPs that incorporate early antigens such as E6 and E7 (for a review, see Ref. 104). The potential advantages of such a vaccine are that the conformationally correct viral capsid could induce a protective Ig response while cytotoxic T-cells directed toward E6 or E7 would target existing lesions. Such a vaccine might be used to treat existing lesions and at the same time reduce the chance of new infections in a combined therapeutic and prophylactic approach. Data from studies of mice immunized with cVLPs showed that protection against challenge with E7-expressing tumor cells was dependent on E7-speciﬁc CD8 positive CTLs [105,106] and was not generated by the capsid alone [107]. Data on cVLP immunization in human volunteers are eagerly awaited. Studies involving peptide vaccines have focused on the HPV16 E7 peptide speciﬁc, HLA-A*0201 restricted CTL responses ﬁrst characterized by Ressing et al. [108]. A number of these vaccines have now reached clinical trials. Most trials have been carried out in patients with advanced cancer, principally to investigate issues of safety and toxicity, even though these patients are unlikely to experience any clinical improvement as a result of vaccination [109]. In one phase I trial conducted in women with high-grade cervical or vaginal lesions, three of 18 patients cleared their lesions and a further six patients showed partial regression following vaccination [110]. Because dendritic cells (DCs) are known to play a central role in mediating antitumor immune responses, recent approaches designed to enhance the efﬁcacy of peptide vaccination have involved pulsing DCs ex vivo with peptides of E6 and E7 or using a combined lipid-

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complexed E7 peptide/E7 peptide pulsed DC approach. Both of these approaches are currently the subject of NCI-sponsored clinical trials (see www.nci.nih.gov/clinical_trials). A further DC-based vaccine approach that has reached clinical trials involves pulsing DCs with fulllength E7 proteins and subsequent in vitro stimulation of HPV16 and HPV18 E7-speciﬁc CD4 and CD8 T-cells from the PBMCs of cervical cancer patients [111]. PBMCs were primed by this method and adoptively transferred to a patient with metastatic adenocarcinoma of the cervix [112]. The CTL population induced was CD8-positive, predominantly CD56positive, IFN-g, TNF-a, and IL2-secreting, and MHC class I restricted and accumulated in the lungs where metastatic disease was extensive. Vaccination of mice with GMCSF/HPV16 E7-expressing constructs resulted in protection from challenge with HPV16 E7-expressing tumor cells [113]. Effective antitumor immunity has been attributed to the effects of the cytokine on the growth and differentiation of DCs. Although CD8 positive cells are thought to be the main effector cell population in GM-CSF-mediated tumor rejection, induction of both Thelper-1 (Th1) and Th2 CD4 cell subsets has also been shown to be required for an optimal antitumor response [61]. DNA-based vaccines are considered to be better suited to tumor therapy, because they tend to induce strong CTL responses and weak humoral responses. A recent novel approach involves the use of recombinant adeno-associated virus (AAV) expressing a chimeric hsp/ HPV16 E7 DNA construct (AAV/hspE7 DNA). CTL-mediated tumor protection was induced in mice that was CD4 and CD8 T-cell-dependent [62]. Vaccination with an AAV/HPV16 E5 DNA construct led to a reduction in tumor growth that was CD8-dependent and CD4independent [114]. The latter report also provides some evidence that E5 is a tumor rejection antigen. Another approach taken has been to target HPV16 E7 DNA to the endosomal/lysosomal compartment of antigen presenting cells by linking it to lysosome-associated membrane protein-1 (LAMP-1) DNA. This vaccine produced effective antitumor immune responses in mice, where the numbers of E7-speciﬁc CD4 and CD8 positive T-cells and the level of E7-speciﬁc CTL activity were all increased [115]. Antitumor immunity could be further enhanced in this model by boosting with recombinant vaccinia virus (Vac-Sig/E7/LAMP-1) DNA [116]. The uptake of DNA by antigen presenting cells can also be improved by enclosing it in microparticles. A recent clinical trial of encapsulated DNA encoding multiple HPV16 E7 protein epitopes demonstrated that patients maintained an elevated speciﬁc immune response for the duration of the observation period [117].

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In this section we have described the immune response to papillomavirus infection with particular reference to HPV16 and its role in cervical disease. The nature and speciﬁcity of those responses thought to be important in protection from HPV infection and control of disease were described, although it is emphasized that the exact nature of such responses in humans has yet to be identiﬁed. Many different HPV vaccines have now reached clinical trials, and we have highlighted some of the most recent of these. Phase II and III trials of prophylactic VLP-based vaccines have given promising results, although the crossreactivity between different HPV types and the cross-protection of induced immune responses have yet to be convincingly demonstrated; polytope vaccines may provide a solution to these problems [118]. The design of an effective therapeutic vaccine for high-grade lesions is likely to prove a much greater challenge. This is because there is much evidence that immune responses to HPV in patients with high-grade SIL and cancer are dysregulated in a number of ways, and it is not clear how this immune dysregulation could be overcome. Therapeutic vaccines for low-grade lesions and external warts, i.e., productive HPV infections, may become a reality sooner, although the economics of their use is the subject of much discussion [37,38,119,120]. 6

SUMMARY

Human papillomaviruses establish largely innocent relationships with the host. Most infections are subclinical or give rise to wartlike manifestations in the skin and mucosa that usually regress over a relatively short period of time. In some cases, however, there is a distinct possibility that the lesion will develop into malignant cancer, particularly in the mucosa. Persistence of the high-risk viral infections, especially in the mucosal tissue, seems to correlate with progression of dysplasia and may be the trigger for development of invasive disease. Natural immunity is slow in developing, taking several months and in some cases years to become sufﬁciently effective to prevent horizontal transmission or reinfection by the same virus. The nature of HPV-associated disease, the perceived lack of precision of screening modalities, and the moderate efﬁcacy of current treatments indicate that there is a substantial need, not only for modern diagnostic procedures but also for novel and effective therapies, especially for precancerous lesions. However, the complexity of the host-virus interactions during chronic infection with HPV suggests that it will be some time before an efﬁcacious, low molecular weight antiviral medicine will be available for treatment of epithelial infections and

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tumors. Imiquimod represents an advance along the immunological path and has certainly shown some sucess in the treatment of external anogenital warts. Treatments of the near future will almost certainly involve vaccination schedules, involving either polyvalent peptides, fusion proteins, VLP, or nucleic acid coding for multiple viral epitopes. Recent results with a monovalent VLP-vaccine directed towards HPV16 clearly demonstrated its effectiveness in preventing infection with HPV16 [121]. To quote Harald zur Hausen, ‘‘The vaccination story has come a long way . . . [and we are] on the road to preventing a major human cancer’’ [122]. Ongoing vaccine trials will continue to provide important information regarding the nature of HPV-speciﬁc immune responses, but prospective clinical studies to further deﬁne those immune responses which control disease should not be overlooked.

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Ressing ME, Sette A, Brandt RMP, Ruppert J, Wentworth PA, Hartman M, Oseroff C, Grey HM, Melief CJM, Kast WM. Human CTL epitopes encoded by human papillomavirus type-16 E6 and E7 identiﬁed through in-vivo and in-vitro immunogenicity studies of HLA-A*0201-binding peptides. J Immunol 1995; 154:5934–5943. Van Driel WJ, Ressing ME, Kenter GG, Brandt RMP, Krul EJT, van Rossum AB, Schuuring E, Offringa R, Bauknecht T, Tamm-Hermelink A, van Dam PA, Fleuren GJ, Kast WM, Melief CJM, Trimbos JB. Vaccination with HPV16 peptides of patients with advanced cervical carcinoma: clinical evaluation of a phase I-II trial. Eur J Cancer 1999; 35:946–952. Muderspach L, Wilczynski S, Roman L, Bade L, Felix J, Small LA, Kast WM, Fascio G, Marty V, Weber J. A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulva intraepithelial neoplasia who are HPV 16 positive. Clin Cancer Res 2000; 6:3406–3416. Santin AD, Hermonat PL, Ravaggi A, Chiriva-Internati M, Zhan DJ, Pecorelli S, Parham GP, Cannon MJ. Induction of human papillomavirusspeciﬁc CD4(þ) and CD8(þ) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18positive cervical cancer. J Virol 1999; 73:5402–5410. Santin AD, Hermonat PL, Ravaggi A, Bellone S, Cowan C, Korourian S, Pecorelli S, Cannon MJ, Parham GP. Development, characterization and distribution of adoptively transferred peripheral blood lymphocytes primed by human papillomavirus 18 E7-pulsed autologous dendritic cells, in a patient with metastatic adenocarcinoma of the uterine cervix. Eur J Gyn Oncol 2000; 21:17–23. Chang EY, Chen CH, Ji HX, Wang TL, Hung K, Lee BP, Huang AYC, Kurman RJ, Pardoll DM, Wu TC. Antigen-speciﬁc cancer immunotherapy using a GM-CSF secreting allogeneic tumour cell-based vaccine. Int J Cancer 2000; 86:725–730. Liu DW, Tsao YP, Hsieh CH, Hsieh JT, Kung JT, Chiang CL, Huang, SJ, Chen SL. Induction of CD8 T cells by vaccination with recombinant adenovirus expressing human papillomavirus type 16 E5 gene reduces tumour growth. J Virol 2000; 74:9083–9089. Ji HX, Wang TL, Chen CH, Pai SI, Hung CF, Lin KY, Kurman RJ, Pardoll DM, Wu TC. Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumour immunity of DNA vaccines against murine human papillomavirus type 16 E7expressing tumours. Hum Gene Ther 1999; 10:2727–2740. Chen CH, Wang TL, Hung CF, Pardoll DM, Wu TC. Boosting with recombinant vaccinia increases HPV-16 E7-speciﬁc T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines. Vaccine 2000; 18:2015–2022. Klencke B, Matijevic M, Urban RG, Lathey JL, Hedley ML, Berry M, Thatcher J, Weinberg V, Wilson J, Darragh T, Jay N, Da Costa M, Palefsky

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119. 120. 121.

122. 123.

Hewlett et al. JM. Encapsulated plasmid DNA treatment for human papillomavirus 16associated anal dysplasia. Clin Cancer Res 2002; 8:1028–1037. Street MD, Tindle RW. Vaccines for human papillomavirus-associated anogenital and cervical cancer: practical and theoretical approaches. Exp Opin Invest Drugs 1999; 8:1–16. Landow K. Nongenital warts: when is treatment warranted? Postgrad Med 1996; 99:245–249. Gross G. Do we need antivirals for genital herpes simplex virus and human papillomavirus infection? Int J Antimicrob Agents 1999; 12:1–3. Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB, Chiaccherini LM, Jansen KU. A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 2002; 347:1645–1651. Zur Hausen H. Cervical carcinoma and human papillomavirus: on the road to preventing a major human cancer. J Natl Cancer Inst 2001; 93:252–253. Laimins LA. Human papillomaviruses target differentiating epithelia for virion production and malignant conversion. Seminars in Virology 1996; 7:305–313.

FURTHER READING Gross GE, Barrasso R, eds. Human Papillomavirus Infection. A Clinical Atlas. Berlin: Ullstein Mosby, 1997. Syrja¨nen KJ, Syrja¨nen SM. Papillomavirus Infections in Human Pathology. Chichester: Wiley, 2000.

8 Hepatitis A Virus Verena Gauss-Mu¨ller University of Lu¨beck, Lu¨beck, Germany

Reinhart Zachoval Grosshadern Medical Center, Munich, Germany

1

INTRODUCTION

Reports of epidemics of jaundice date as far back as the Greek and Chinese antique worlds [1]. In past centuries, epidemics of hepatitis were observed, particularly in association with military conﬂict. Only after World War II did it become clear that two etiologically and epidemiologically distinct forms of hepatitis exist. The short-incubation, fecalorally transmitted, epidemic (but also sporadic) form is caused by the hepatitis A virus (HAV), whereas the causative agent of the longincubation, parenterally transmitted disease was recognized as the hepatitis B virus (HBV) [2,3]. Very soon, the existence of additional hepatitis agents had to be assumed. Hepatitis A virus was ﬁrst identiﬁed in 1973 by electron microscopy [4]. The successful transmission of HAV to experimental animals such as marmosets and chimpanzees meant a giant leap forward in hepatitis A research, and it subsequently enabled the propagation of the virus in cell culture [5–7] as well as the development of safe and effective hepatitis A 259

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vaccines [8–10]. The availability of HAV vaccines and the self-limiting course of the disease without sequelae have curbed the development of an antiviral therapeutic concept so far. However, based on the increasing knowledge of the HAV biology, molecular targets for antiviral intervention have been deﬁned and investigated as will be described here.

2 2.1

VIROLOGY The Infectious Agent, Its Genome, and Proteins

Hepatitis A virus (HAV) is the only member of the genus Hepatovirus within the Picornaviridae family, which comprises diverse groups of human and animal pathogens. Within the genus, enterovirus, poliovirus, and coxsackievirus have been intensively studied. The Rhinovirus genus is characterized by its acid lability and was the ﬁrst target for antipicornaviral therapeutic tests. In many aspects HAV is substantially different from other picornaviruses, which complicates its molecular study. The HAV particle has an icosahedral symmetry with a diameter of 27–30 nm. The mature and infectious viral capsid is formed by 60 copies of the structural proteins (VP1, VP2, VP3, VP4). The viral genome encapsidated by the structural proteins is a linear RNA molecule of messenger-sense polarity and approximately 7500 bases in length. A small polypeptide (3B) is covalently linked to the 50 end of the RNA (see Fig. 1 for the location of the genetic elements and viral proteins on the viral genome). Like all picornaviral genomes, the HAV RNA has three parts: (1) a 50 nontranslated region (50 NTR) of about 740 bases, (2) a large open reading frame encoding a single polyprotein of approximately 250 kDa molecular mass, and (3) a short 30 NTR with a poly(A) tail. The 50 NTR can be functionally divided into two domains that both fold into stable secondary and tertiary RNA structures and that play essential roles in the viral genome replication and translation. Similar to the 50 NTR and possibly in synergy with it, the 30 NTR has functions in RNA and protein synthesis. The central region of the viral RNA encodes a single large polyprotein with domains P1-2A, P2, and P3. Whereas the P1-2A region contains the capsid proteins VP1 (or VP1-2A), VP2, VP3, and VP4, all polypeptides encoded in domains P2 and P3 appear to be components of the viral replication complex (RC) that provides the structure for and catalyzes viral genome replication. As part of the viral polyprotein itself

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FIGURE 1 Schematic presentation of the HAV genome and polyprotein. The names and known or proposed functions of the mature viral proteins are indicated. The hepatitis A virion is composed of the viral structural proteins and RNA genome. The viral nonstructural proteins encoded in domains P2 and P3 are parts of the viral replication complex (RC).

and a vital player in viral protein generation, the virus-encoded proteinase 3C catalyzes almost all cleavages within the precursor protein [11–14]. Timely regulated release of mature viral proteins from a large precursor polypeptide is the key step in the gene expression strategy of picornaviruses, and therefore great efforts have been directed toward understanding of the proteolytic cascade and the regulatory role of the viral proteinase system. Various recombinant expression systems and infected cell cultures have been used to explore the production of viral proteins and to study the effect of inhibitory substances that might be of therapeutic use (see below). The key enzyme in viral RNA synthesis is the HAV RNA polymerase 3D, whose function has not yet been demonstrated [15]. Protein 3B (also called VPg) is covalently linked to the 50 end of the viral RNA and functions as protein primer for RNA synthesis. Owing to its hydrophobic nature, protein 3AB might serve as anchor of the primer 3B to membranes of the endoplasmic reticulum where the HAV RC is supposedly located. HAV protection 2A fused to

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the structural protein VP1 is unique in its function as signal for capsid assembly [18]. The multiple functions of HAV proteins 2B and 2C are not unequivocally deﬁned. Genetic analyses suggest that both proteins are essential for viral replication, because replication efﬁciency is inﬂuenced by amino acid substitutions in 2B and 2C [16]. Both proteins seem to be involved in the rearrangement of cellular membranes resulting in vesicles that might function as a scaffold for the viral RC [17]. The capacity of some HAV nonstructural proteins, to bind to viral and host proteins and viral RNA has been studied in order to understand their role in the formation of the viral RC and in the regulation of genome expression. The RNA-binding capacity of 3C and its precursor proteins, in addition to their proteolytic activity, make these polypeptides particularly interesting as multiple targets for antiviral intervention [19] (see page 265). Details of the molecular biology of HAV are reviewed by Hollinger and Emerson [20]. 2.2

Viral Replication In Vitro

After its ﬁrst isolation in cell culture, it soon became obvious that HAV is quite unique in its replication mode and physical stability compared to the well-studied members of the picornavirus family, e.g., the entero- or cardioviruses. An HAV-speciﬁc feature most striking from the very beginning is its protracted replication without shutting off the host cell metabolism. Presumably, several of the individual steps in the HAV replicative cycle, schematically shown in Figure 2, contribute to the slow growth of HAV. HAV replicates in various established human and monkey cell lines and can be followed by detection of viral antigen or RNA. In contrast to other picornaviruses, destruction of the infected cell layer [cytopathic effect (CPE)] is not induced in cells infected with most strains of HAV. Virus binding to the cell surface receptor (Fig. 2, step 1) is the ﬁrst, highly speciﬁc step that initiates the infectious cycle [21]. During or after particle internalization, the viral RNA genome is uncoated [22] (step 2 in Fig. 2) and is used as a template for translation. The primary translation product, or polyprotein, is cleaved by the viral proteinase 3C in a regulated fashion and possibly with the assistance of viral and host cofactors (step 3), allowing the formation of the viral RC. By a switching mechanism that is not yet understood, the viral genome then serves as a template for RNA replication. Picornaviral genome replication occurs on membranes and is induced by some viral proteins. Negative-strand synthesis starts at the 30 end of the RNA and results in the replicative form consisting of a double-stranded RNA with complementary polarity

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FIGURE 2 Schematic presentation of the viral life cycle in the host cell. For details of the essential replication steps denoted by numbers, see the text. C ¼ capsid proteins; RC ¼ replication complex.

(step 4). Presumably facilitated by the action of a helicase, several new positive (þ) strands are subsequently transcribed from the doublestranded template. The newly formed positive strands are either translated to produce more viral proteins or packaged by the viral structural proteins (step 5). Before or after leaving the cell (step 6), the viral capsid has to mature to obtain its infectivity. Procapsid maturation involving proteolytic cleavage of VP0 seems to require viral RNA and is highly inefﬁcient in HAV [23]. An excellent overview of picornaviral replication is given by Racaniello [24]. 2.3

Molecular Targets of Therapy

In contrast to HAV, the individual steps in the rhinoviral and polioviral life cycles have been studied in great detail in order to determine their molecular mechanism and with the ultimate purpose of using them as targets for antiviral drug development. Almost all steps of the picornaviral replicative cycle described here (Fig. 2) have been

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considered as molecular targets for antiviral agents based on data obtained mostly for picornaviruses other than HAV. As an antipicornavirus therapeutic approach, only interference with viral receptor binding and polyprotein processing has so far reached clinical testing; other approaches discussed below have been considered solely on the basis of in vitro experiments. It can be assumed that future developments in anti-HAV therapeutic strategies will be guided by ﬁndings obtained from other picornaviruses.

2.4

Inhibition of Receptor Binding and Uncoating

Besides a few intracellular determinants, the host cell receptor seems to be the key player governing viral tissue tropism. The receptors of poliovirus and most rhinoviruses are membrane-spanning molecules of the immunoglobulin superfamily that speciﬁcally interact with a depression on the surface of the cognate virus [25]. By cocrystallization and genetic analysis, a detailed picture of the molecular partners on both the viral and receptor sides has emerged. Based on these data, peptidomimetic structures and soluble fragments of the receptor were tested for their ability to bind to rhinovirus and to compete for its interaction with the viral capsid and hence its infectivity [26,27]. These experiments suggested that soluble ligands competing for the receptor on the cell membrane can be used as antiviral substances. The efﬁcacy of tremacamra, a soluble form of the intercellular adhesion molecule 1 (ICAM1), was tested with some success in a randomized clinical trial [28,29]. In the past, the putative HAV receptor has been described, but antiviral approaches to speciﬁcally inhibit HAV entry into liver cells have not been reported [21]. A common structural feature of many picornaviruses is a hydrophobic pocket that is located at the ﬂoor of the canyon formed by the capsid proteins and is occupied by a small fatty acid-like moiety. This pocket factor seems to stabilize the viral capsid and to limit its ‘‘breathing,’’ the structural transitions required for uncoating [30,31]. After binding of the viral capsid to the cell receptor, the capsid structure undergoes a conformational change that releases both the pocket factor and the capsid protein VP4, and that is accompanied by RNA discharge into the host cell cytoplasm. Several capsid-binding compounds have been described that can replace the pocket factor and entropically lock the capsid structure, preventing viral genome release [32]. These compounds have inhibitory effects on the replication of rhinovirus and coxsackie-virus by interfering with viral spreading.

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Using a structure-based combinatorial library of synthetic compounds, a limited number of compounds were found as speciﬁc inhibitors of poliovirus and rhinovirus replication in a cell culture system [33]. Because the molecular structure of HAV has not yet been determined, a structure-based antiviral approach is currently unavailable. Studies with HAV using anti-poliovirus and anti-rhinovirus active drugs (e.g., chalcone, SCH 38057, WIN 52035, R61837, pleconaril) have not been reported. The inhibitory effect of various other substances that seem to interfere with early steps in the HAV life cycle was described earlier [34], but these compounds were not considered for clinical trials. 2.5

Inhibition of the Viral Proteinase

Once released from the virion, the picornaviral genome is initially translated into a single polyprotein (Fig. 2, step 3) before it is copied into the complementary negative strand. Mainly through the action of the viral proteinase 3C, which is a part of the polyprotein (see Fig. 1), this large polypeptide is speciﬁcally cleaved into structural and nonstructural proteins. The molecular characterization of HAV 3C by X-ray crystallography revealed a trypsin-like fold with a cysteine in the active site [12,14]. A similar pattern was found for proteinase 3C of rhinovirus and poliovirus [35]. Genetic and inhibition analyses of proteinase 3C of other picornaviruses had already shown that 3C is a unique cysteine proteinase, which renders it an ideal target for antiviral intervention [36]. For HAV 3C, in vitro and ex vivo inhibition by a tetrapeptidylic inactivator of the active site resulted in blocking proteinase activity as well as in reduced infectivity [37]. A large variety of other types of compounds such as peptide aldehydes, ﬂuoromethyl ketones, ß-lactams, isatins, homophthalimides, and sulfones tested positive in proteinase inhibition studies, yet only some of them showed antiviral activity [38]. Possibly due to both the low bioavailablity of peptidic inhibitors and the lack of a convenient small animal model for hepatitis A, none of these compounds was tested in vivo. Efforts to develop antiviral compounds targeted to the viral proteinase have been more advanced for rhinovirus 3C [29,39,40]. From a large array of compounds, AG7088, a potent tripeptidic inhibitor of rhinovirus 3C, was tested in a clinical trial [41]. 2.6

Antiviral Inhibitors Targeted to Viral RNA Replication

Among the many nucleoside analogs tested and in part used as antiviral agents, ribavirin was the only one indicated to be effective for positivestrand RNA viruses [42]. This inhibitor of inosinate dehydrogenase was found to act as a mutagen for poliovirus [43]. Its inhibitory activity on

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other picornaviruses, including HAV, has not been reported yet. In combination with interferon-alpha, ribavirin has been approved for treatment of hepatitis caused by hepatitis C virus, also a positive-strand RNA virus (see Chap. 10). 3

EPIDEMIOLOGY AND MODES OF TRANSMISSION

Hepatitis A virus is mainly transmitted by the fecal-oral route either through direct feces–hand–mouth contact or indirectly via contaminated food or water. Given the stability of HAV, it is clear that the prevalence of hepatitis A is closely linked to the overall level of hygiene. Because viremia during acute hepatitis A is short, parenterally transmitted infections by transfusion of blood or blood products are rarely seen [44,45]. Other body ﬂuids such as urine, nasopharyngeal secretions, or semen have been investigated, mainly under experimental conditions in animal models; their role as a vector for transmitting the infection under normal circumstances is not clear but might be minor. In the United States, the main sources of infection are (1) household or sexual contacts with HAV-infected individuals (12–26%), (2) children or employees in day care centers or their contacts (11–16%), (3) international travellers (4– 6%), (4) recognized food or waterborne disease outbreaks (2–3%), and (5) no source identiﬁed (>50%) [47–49]. In addition, cyclic outbreaks have been reported in illegal drug users and among male homosexuals [48]. 4

PATHOGENESIS

Hepatitis A virus is shed into the feces of acutely infected patients and transmitted by the fecal-oral route. The virus is highly stable, maintaining its infectivity even under unfavorable environmental conditions. Because of its acid resistance, HAV survives the gastric pH and passes through the stomach. The precise mechanism of viral uptake in the gastrointestinal tract and passage to the liver is still unknown, mostly due to the lack of a convenient animal model for the infection. Viral replication in intestinal epithelial cells or cells of the Peyer’s patches have been suggested but not formally shown [50]. In the liver, the virus replicates in hepatocytes that show degeneration (apoptosis), but no widespread necrosis of the liver parenchyma can be observed. Activation of cells in the reticuloendothelial system of the sinusoids and the portal tract marks the onset of immune mechanisms that have been proposed as the main pathogenetic principle of the disease. The detection of large amounts of virus prior to the onset of clinical symptoms supports the notion that T-cell-mediated immune response

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rather than the direct cytopathic effect of HAV gives rise to the destruction of liver cells. From the liver cells, the virus is released into the biliary system via bile canaliculi, appears in the small and large intestines, and is eventually excreted in feces. 5

SEROLOGICAL DIAGNOSIS

Laboratory ﬁndings in acute hepatitis A are not different from those observed in other forms of viral hepatitis. The diagnosis of acute infection is based on the detection of high IgM class antibody titers against the virion (anti-HAV IgM) in a single acute phase serum sample [51,52]. Anti-HAV IgG appears with the onset of hepatitis, rising to high titers in the ﬁrst year and persisting lifelong. Anti-HAV IgG is a sign of previous infection and indicates immunity [53]. Peak levels of alanine aminotransferase (ALT) as the most sensitive indicator of liver cell damage are usually below 2000 U/L and drop after the onset of jaundice. In most cases the bilirubin concentration in serum reaches maximal levels of 200 mmol/L after the maxima of transaminases, whereas other biochemical markers of cholestasis such as g-glutamyltranspeptidase (gGT) and alkaline phosphatase (aP) are only slightly to moderately elevated. Laboratory abnormalities usually resolve within 4– 6 weeks. Routine testing for HAV antigen or HAV RNA in the stool or in body ﬂuids is neither available nor indicated. Excretion of HAV in feces begins 1–2 weeks before onset of symptoms and peaks in the late incubation period, declining rapidly thereafter [54,55]. HAV viremia is short-lived, occurs in the late incubation/early hepatitis phase [56], and rarely leads to parenteral transmission of the virus. A synopsis of the clinical and virological ﬁndings in acute hepatitis A is depicted in Figure 3. 6

CLINICAL FEATURES

Acute infection with HAV may result in a wide spectrum of clinical outcomes ranging from mere seroconversion, through subclinical illness with abnormal liver function tests, to classical icteric hepatitis and fulminant hepatic failure. The most important factor responsible for the different disease patterns seems to be age: Less than 5% of children under 3 years of age but more than 80% of adults develop icteric clinical illness [46]. The average incubation period (infection until appearance of dark urine and rise of transaminases) is about 4 weeks (2–7 weeks) [56]. The prodromal phase is characterized by malaise, anorexia, fever, nausea,

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FIGURE 3 Immunological, virological, and clinical events associated with hepatitis A infection in humans. The rise in serum levels of transaminases parallels the IgM course (not shown).

and ﬂu-like complaints. In addition, tenderness of the right upper abdomen may be present due to enlargement of the liver. Appearance of dark urine is usually the ﬁrst objective sign of hepatitis, followed by acholic feces and icterus of the sclera and skin. Major symptoms of the acute disease comprise, in addition to dark urine, jaundice, fatigue and anorexia, fever, headaches, myalgias, and itching [57]. The symptoms usually disappear after 2–3 weeks. Liver enzymes are normal after 4 weeks in most cases. 7

GENERAL MANAGEMENT AND THERAPY

Patients with acute hepatitis A do not need to be hospitalized unless there is fecal incontinence or adequate care for the patient cannot be provided. There is no speciﬁc treatment, and dietary restrictions are not necessary apart from the recommendation to restrain from alcohol

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consumption. Symptomatic therapy of pruritus (e.g., by cholestyramine 4–8 g day) or nausea (e.g., by metoclopramide) is recommended as needed. The value of bed rest has not been proven by controlled trials. Corticosteroids have no place in the treatment of uncomplicated acute hepatitis. Because of the benign course of the disease, there is no need for antiviral therapy. Interferon-alpha has been given to individual patients with fulminant hepatitis A outside controlled trials without evidence of clinical beneﬁt [58]. Instead, in these rare cases when HAV runs into a fulminant course, the patient should be evaluated for liver transplantation [59].

8 8.1

PREVENTION Active Immunization—Preexposure Prophylaxis

The availability of a hepatitis A vaccine provides the opportunity to substantially lower disease incidence and potentially eliminate infection. To achieve this, widespread routine vaccination of children is needed. In many countries, current recommendations restrict active immunization to certain risk groups: 1. Persons from areas with low endemicity or working in countries that have high or intermediate endemicity of infection 2. Persons who are at occupational risk for infection 3. Illegal drug users 4. Men who have sex with men 5. Individuals who have clotting factor disorders In addition, susceptible patients with chronic liver diseases and nonimmune persons who either are awaiting or have received liver transplants should be vaccinated. Besides locally available hepatitis A vaccines, two formalininactivated vaccines prepared from cell-culture-adapted HAV have been licensed in many countries; HAVRIX1 (manufactured by Glaxo SmithKline) and Vaqta1 (produced by Merck and Co.). The antigen content of HAVRIX is expressed as enzyme-linked immunosorbent assay (ELISA) units (EL.U.); for Vaqta the antigen content is measured as units of HAV antigen. The vaccines should be given twice intramuscularly according to the vaccination schedule recommended by the manufacturer (Table 1). For children and young adults (ages 2–18 years), doses with a lower antigen content are available. Both vaccines are highly immunogenic in children, adolescents, and adults and produce protective antibody levels in 94–100% of those vaccinated 1 month after the ﬁrst

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TABLE 1

Vaccination Schedule and Dosage of Hepatitis A Vaccines

Vaccine

Age of patient

Dosea

Volume

Havrix1 Vaqta1 Havrix1 Vaqta1

2–18 yr 2–18 yr >18 yr >18 yr

720 EL.U 25 U 1440 EL.U 50U

0.5 mL 0.5 mL 1.0 mL 1.0 mL

a

Schedule (months) 0, 0, 0, 0,

6–12 6–18 6–12 6

EL.U ¼ ELISA unit.

injection [60–64]. The protective efﬁcacy of both vaccines was evaluated in double-blind, controlled, randomized clinical trials conducted in Thailand among 40,000 children and in a semiclosed New York community with a high incidence of HAV infections and found to be 94–100% [8–10]. Surveillance data were collected to monitor long-term protective efﬁcacy in these vaccinated individuals and to determine the possible need for a booster injection. In the longest follow-up study available, no hepatitis A cases were detected among children followed for 7 years after vaccination [65]. Protective levels of anti-HAV are estimated to last for at least 20 years [66–68]. Reduced immunogenicity is observed when the hepatitis A vaccine is administered simultaneously with serum immunoglobulin [69,70]. Reduced anti-HAV antibody levels after vaccination were also found in risk groups, such as HIV-infected men and patients with chronic liver disease of viral or nonviral origin [71,72]. Other factors, such as age, smoking, and obesity, that might affect the immunogenicity of hepatitis A vaccines have not been elucidated so far. Other vaccines (e.g., for hepatitis B, DTP, yellow fever, rabies, Japanese encephalitis, poliovirus) can be administered simultaneously with hepatitis A vaccine without affecting either vaccine’s immunogenicity or increasing the rate of adverse events [73,74].

8.2

Side Effects and Adverse Events

Pre- and postlicensure studies have demonstrated the safety of both vaccines. Among adults, the most frequently reported side effect is a local reaction at the injection site (soreness, 56%) and systemic reactions such as headache (14%) and malaise (7%). Serious adverse events are in the range of background incidence rates and cannot be attributed to the vaccine.

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271

Testing for Anti-HAV Before and After Vaccination

The decision to determine the immune status by serological testing prior to vaccination should be based on the expected prevalence of immunity and the cost of serological testing. In most industrialized countries screening of individuals >40 years might be cost-effective. Postvaccination testing is not indicated due to the high immunogenicity of the vaccine in adults and children. Anti-HAV concentrations after vaccination are usually 10–100-fold lower than those after natural infection. Highly sensitive assays [lower limit of detection 20 mlU/mL (milliinternational units per milliliter) minimal protective antibody level] can sometimes detect low antibody liters, whereas the routine anti-HAV (IgG and IgM) test (lower limit of detection 100 mlU/mL) is usually insufﬁcient. Anti-HAV tests are standardized by a WHO reference immunoglobulin, and the results are expressed in milli-international units per milliliter.

8.4

Postexposure Prophylaxis

Nonimmune persons who have recently been exposed to HAV should receive a single intramuscular dose of Ig (0.02 mL/kg) as soon as possible but not later than 10–14 days after exposure. Candidates for Ig administration are people who have had close personal contact with individuals acutely infected with HAV, unvaccinated staff or attendees of day care centers or homes where cases of hepatitis A have been recognized, classroom contacts of an index case, or those in comparable situations. The hepatitis A vaccine has been successfully used to control community-wide outbreaks because it prevents secondary infections in those in contact with infected people [75]. Active immunization can easily be combined with IG administration.

9

PERSPECTIVES

Hepatitis A virus infection of the liver causes signiﬁcant morbidity and even mortality. Great success has been achieved by taking preventive measures with the killed hepatitis A vaccine, which provides long-lasting protection from infection by all known viral variants. Therefore, the future goal in the control of HAV infections will be vaccination of people at risk. Therapeutic intervention will be restricted to patients refractory to vaccination.

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9 Hepatitis B Virus Guido Gerken and Christoph Jochum University of Essen, Essen, Germany

1

DIAGNOSIS OF INFECTION

The hepatitis B virus (HBV) is an enveloped partially double-stranded circular DNA virus with a diameter of 42 nm (Dane particle) (Fig. 1). It belongs to the Hepadna family of viruses. HBV can infect only humans and chimpanzees. Its genome consists of approximately 3200 base pairs. It codiﬁes various genes in an open reading frame (Fig. 2) [1,2]. The preC/C gene codes for polypeptides that make up the nucleocapsid containing the HB core antigen (HBcAg) and the HBe antigen (HBeAg), which is detectable in the blood after post-translational processing. The pre-S1/pre-S2/S gene codes for the lipoproteins that make up the viral envelope containing the HBs antigen (HBsAg). The P gene codes for the viral polymerase. This protein has several functions such as RNA pregenome encapsidation, priming of DNA synthesis, reverse transcription, and plus-strand polymerization [3]. The true function of the X gene is still unclear. It seems to be involved in regulatory functions and plays a role in hepatocarcinogenesis in chronic HBV infection. The HBV causes either acute or chronic inﬂammation of the liver. The incubation period from inoculation to disease ranges from 4 weeks to 6 months. About 50% of the cases do not develop jaundice. Of the 277

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FIGURE 1

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The structure of hepatitis B virus.

acute infections, 1–2% lead to acute liver failure. Between these two distinctively different courses of hepatitis B, a wide spectrum of symptoms exists. The prodrome usually consists of malaise, fatigue, nausea, elevated body temperature, and in some cases arthralgia and rash. Clinical hepatitis manifests as jaundice and elevated serum aminotransaminase values, pruritus, and nausea. Fulminant hepatitis shows symptoms of liver failure such as coagulopathy, encephalopathy, and rising bilirubin level. In most of the cases aminotransaminase levels decrease to normal within the following weeks. Eighty to ninety percent of the adults have normal aminotransaminase values and achieve clearance of the virus from the blood after 6 months. In 10–20% of the adults the virus persists in the blood. In these cases the hepatitis B advances to a chronic disease. In newborns and young children the rate of chronicity is remarkably higher. A perinatal infection becomes chronic in more than 90% of the cases. Chronic HBV infection is often completely asymptomatic and may not be detected until symptoms of cirrhosis appear. Chronic hepatitis B could appear with chronic fatigue and diffuse upper abdominal pain. In

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FIGURE 2 The structure of the HBV genome. For simpliﬁcation, the circular genome is shown as a straight line.

some cases of chronic hepatitis B, extrahepatic manifestations such as vasculitis [4], porphyria cutanea tarda [5], essential mixed cryoglobulinemia [6], and other autoimmune phenomena [7,8] may occur.

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The diagnosis of HBV infection is made by antibody and antigen tests and viral DNA detection. During HBV infection antibodies against HBcAg, HBeAg, and HBsAg are detectable: anti-HBc, anti-HBe, and antiHBs. Viral proteins such as HBeAg and HBsAg are detectable in the blood. Qualitative and quantitative DNA tests are available to prove viral load. The presence of anti-HBc of the IgM type and HBsAg conﬁrm the diagnosis of acute HBV infection. The HBeAg, if detectable, is a marker for active viral replication, but the lack of HBeAg does not exclude viral replication. Some viral strains have lost the HBeAg by a mutation in the precore region [9]. These precore mutant viruses are particularly common in the Mediterranean region and in Asia. Therefore the ‘‘gold standard’’ for conﬁrmation of viral replication is qualitative DNA detection. Virus DNA in blood or tissue may be detected by using the technique of molecular hybridization or polymerase chain reaction (PCR) assay. Molecular hybridization allows quantiﬁcation and is able to detect as little as 2 pg of HBV DNA/mL serum. With the PCR technique, as few as 200 copies per milliliter are detectable [10]. Seroconversion is indicated by the occurrence of anti-HBe. Loss of HBsAg is associated with the ‘‘healing’’ of the disease. The combination of the different markers is characteristic for different stages of the acute disease (Fig. 3). Chronic HBV infection is deﬁned by persistence of HBsAg for more than 6 months. The HBsAg-positive patient with normal aminotransaminase levels, low DNA levels, and positive anti-HBe detection is considered an asymptomatic carrier and does not need therapy in most cases. A therapeutic decision has to be made according to the individual constellation of HBV markers (Table 1). 2

VACCINATION

Today highly effective passive and active vaccines against HBV are available. HBV-speciﬁc immunoglobulin (HBIg) contains high anti-HBs titers. It has been shown to be effective for passive immunization against HBV infection if given prophylactically or within a few hours after infection. There is an indication for vaccination of persons who had sexual contact with an infected individual, neonates born to an HBsAgpositive mother, and persons who have had parenteral exposure (needle injury, etc.) to HBsAg-positive blood or body ﬂuids [11–15]. Passive immunization can be effective for up to 6 months. Persistent protection with a consequent reduction of the community acquired infection rate requires active immunization.

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FIGURE 3 Serum markers during an acute hepatitis B infection.

The ﬁrst active HBV vaccine was introduced in the early 1970s [16]; it was plasma-derived. Since the early 1990s the vaccine has been produced in the western countries by recombinant DNA technology. In both types of vaccine the essential immunogen is HBsAg. They confer a long-lasting protection against HBV infection [17]. The vaccines actually in use do not contain the highly immunogenic pre-S1 and pre-S2 determinants [18]. Incorporation of the pre-S1 and pre-S2 antigen determinants in a new vaccine can improve the seroconversion rate after vaccination [19]. Some data also suggest that the pre-S1 and pre-S2 vaccine is an effective immune therapy in chronic HBV infection [20], but these results are controversial. This vaccine has not yet been approved. In Germany and in over 80 other countries, immunization against HBV is generally recommended for children and adolescents [21]. Universal immunization of newborns against HBV in epidemic countries such as Taiwan has resulted in a remarkable decrease in the HBV carrier rate and in the incidence of hepatocellular carcinoma (HCC) in children [22]. In 1995 the World Health Organization (WHO) therefore recommended the general vaccination of all children worldwide [23]. In addition to this general recommendation, persons who are at special risk for HBV infection should be vaccinated in any case (Table 2).

HBeAg negative (HBV pre-core variants)

HBeAg-positive (wild-type HBV)

Treatment

IFN plus antivirals

Favor nucleotide/nucleoside analogs:

Favor nucleotide/nucleoside analogs with:

Favor IFN with:

Treatment in Chronic Hepatitis B

HBV-DNA-positive patients

TABLE 1

Long-term treatment usually required Favor therapies with low resistance risk (e.g., ADV) Inclusion into studies

High ALT Low HBV-DNA No contraindications Motivated patients Low ALT High HBV-DNA Patient’s preference

Options

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TABLE 2

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Individuals Who Are at Risk for HBV Infection

Illicit injection drug users Health care workers Chronically transfused persons Hemodialysis patients Homosexually active men Heterosexuals with multiple partners or other sexually transmitted disease Police ofﬁcers, emergency medical technicians, ﬁreﬁghters Household and sexual contacts of infected persons Clients and staff of institutions for the developmentally disabled Newborns of infected mothers

The currently recommended schedule for active vaccination consists of three doses given at 0, 6, and 24 weeks. A successful vaccination should result in an anti-HBs titer of 10 IU/mL or higher, which is achieved in 95% of young immunocompetent individuals. Adults younger than 40 years of age show a better response rate to vaccination than older individuals [24]. Persons with severe immunosuppression may not respond, even after a second course of vaccination. It remains unclear how long after vaccination the protection will continue. Anti-HBs may disappear within 10 years after successful immunization in about 40% of vaccinated adults. Immunity against clinical disease, however, may persist for years and even remain lifelong after the loss of measurable anti-HBs titer. Nevertheless, booster immunization is recommended in immunosuppressed individuals with anti-HBs levels below 10 IU/mL. 3

VIRAL REPLICATION STRATEGY AND TARGETS FOR THERAPY

The replication cycle of HBV is very complex and not completely understood. Viral replication takes place in the cytoplasm [1,25,26]. HBV is not cytopathic and does not kill the host cell, which is an outstanding feature of HBV infection. Only after long-term infection do secondary cytopathic effects occur. The initial steps of adhesion to the cell and cell invasion are not yet understood. The cellular receptor is still unknown. After invading the cytoplasm, the viral core particle is uncoated and is translocated to the nucleus of the host cell. Here the synthesis of the (þ)-strand HBV DNA is completed and DNA repair takes place. The HBV DNA is converted to a covalently closed circular DNA (cccDNA or

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supercoiled DNA). The cccDNA remains episomal and serves as a template for the cellular polymerase II, which produces pregenomic RNA and messenger RNAs [2]. The pregenomic RNA transcript serves both as a template for the reverse transcription of the ﬁrst ()-strand HBV DNA and as a messenger RNA for the viral polymerase and the nucleocapsid. The smaller pre-S and pre-C transcripts are translated to viral surface and core proteins. The synthesis of the ﬁrst ()-strand HBV DNA is followed by the production of a shorter (þ)-strand DNA to become a partially double-stranded DNA. This generated DNA can serve as viral nucleic acid in mature virions budding out from the host cells to infect new target cells. In addition, this new synthesized DNA can build new cccDNA, resulting in more than one cccDNA copy per nucleus [27]. This replication cycle offers a number of targets for speciﬁc antiviral treatment strategies. The transcription could be inﬂuenced by cytokines and antisense constructs. The viral polymerase with its reverse transcriptase activity is the main target of existing drugs, especially the nucleoside analogs. Another target is the protein synthesis and viral assembly, where cytokines and other non-nucleosidic inhibitors have been shown to be effective (27a). Figure 4 summarizes the replication cycle of the HBV and indicates the targets for therapy. The best target for antiviral therapy in HBV infection seems to be the inhibition of cccDNA formation in the nucleus. However, most of the antiviral agents that have been investigated so far show no or little effect against cccDNA. This accounts for the rapid reappearance of HBV after termination of antiviral therapy. In theory, complete viral clearance could be achieved if potent antiviral therapy, which completely inhibits viral synthesis, were administered as long as the duration of treatment outlasted the pool of existing cccDNA. However, the half-life of cccDNA depends on the loss of hepatocytes [28]. The half-life of infected hepatocytes is 10–100 days [29], so complete clearance requires an effective treatment of 1–10 years. However, costs, side effects, and resistant viral mutants preclude such a long therapy. Therefore the development of new therapeutic strategies should be focused on the blockade of cccDNA synthesis and/or cccDNA eradication.

4

TREATMENT AND MONITORING OF TREATMENT SUCCESS

Acute hepatitis B does not require speciﬁc therapy in most cases. In cases of progression toward liver failure, lamivudine, a nucleoside analog, could be used. Severe liver failure requires urgent liver transplantation.

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FIGURE 4 HBV replication cycle and possible targets of an antiviral therapy. The black arrows indicate actions that are of predominantly cellular origin. The dashed arrows indicate actions of viral proteins. Potential targets of antiviral therapy are shown in gray.

In the prevention of liver cirrhosis and hepatocarcinoma, chronic HBV infection is getting increasing attention. Currently there are two therapeutic strategies available for treating chronic HBV infection: (1) the use of immunomodulatory drugs such as interferon and (2) the use of nucleoside analogs. The second group serves to block the reverse transcriptase activity of the viral polymerase. Both therapeutic options are of limited success and/or have serious side effects. Therefore the indication for therapy and drug regimen should be carefully deﬁned. An asymptomatic carrier (see Table 1) does not need speciﬁc therapy, but periodic controls are required. In the case of superinfection with the hepatitis D virus no therapy is possible. All other cases of chronic hepatitis B should be treated. Immunomodulatory agents are mostly cytokines or derivatives, which are important in the regulation of natural defense against viral

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infection. The potential mechanism of action is the restitution of the host immune system, which is incompetent to eliminate the virus. Interferon-a was the ﬁrst drug approved for the treatment of chronic HBV infection. Although the exact mechanism of action of interferon-a is not yet understood, it has been shown to inhibit the replication of HBV and to establish disease remission under prolonged therapy. Interferon is administered subcutaneously (6–10 Mio IU [1 million international units ¼ 1 Mio unit] three times a week) for 4–6 months. In general, interferon therapy is effective in only 20–30% of the patients [30]. The best candidates for successful interferon therapy are patients with high aminotransaminase levels and low viral load. Interferon has a variety of side effects, which exclude some patients from therapy. In addition, the relatively low success rate makes interferon not an ideal antiviral agent. Table 3 gives a summary of the side effects of interferon-a and the contraindications to interferon treatment. A second hopeful immunomodulatory drug is thymosin. Thymosin is a thymic extract that mediates a variety of immunological effects including augmentation of suppressor T-cell activity and the stimulation of IgG production. Thymosin is able to promote disease remission and cessation of HBV replication in patients with HBeAgpositive chronic HBV without severe side effects [31–33]. Its synthetic derivative thymosin-a1 was effective and safe in therapy of chronic HBV infection in several multicenter phase III studies in the People’s Republic

TABLE 3

Side Effects of and Contraindications to Interferon-a

Side effects of interferon-a

Contraindications to interferon-a

Flu-like illness Bone marrow suppression Myalgia Headache Depression Irritability Sleep disturbance Weight loss Alopecia Skin rash Fatigue Arthralgia Hyperthrosis

Thrombopenia, <70,000/mL Leukopenia, <3000/mL Psychiatric disorders Hyperthrosis Decompensated liver cirrhosis Severe cardiopulmonary diseases Active alcoholism Active illicit intravenous drug use Autoimmune disorders Chronic nephropathy

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of China. However in order for the drug to be approved for use in Europe and North America further studies are needed. Another approach is the combination of interferon-g with granulocyte-macrophage colony stimulating factor (GM-CSF), which has been shown to have a signiﬁcant effect in chronic HBV infection [34]. It is well established that many nucleoside analogs showing activity against the human immunodeﬁciency virus (HIV) are also effective in HBV infection. This is not surprising because of the similarity between HIV reverse transcriptase and the HBV polymerase. Only nucleoside analogs lamivudine (3TC) and adefovir dipivoxilare are approved for therapy of chronic hepatitis B. Lamivudine inhibits the HBV DNA polymerase, acting as an obligatory chain terminator during HBV DNA synthesis. Lamivudine is active in interferon-naive patients as well as in interferon nonresponders. It has a low toxicity and is much better tolerated than interferon [35,36]. It is administered orally, usually at a dose of 100–150 mg once a day. It can also be administered to patients with decompensated liver cirrhosis [37]. Lamivudine’s potent activity against HIV and HBV makes this drug the ﬁrst choice in HIV-HBV-coinfected patients. Despite the initial high effectiveness and high safety of the drug there are two general limitations. First, after cessation of the drug, relapses were observed in most patients. Second, mutations in the polymerase gene lead to resistance of the virus against the drug. The most important mutation is the M539V exchange in the YMDD motif. These mutant viruses have replicative capacities and dynamics in vivo similar to those of the wildtype virus. However, they seem to have less pathogenicity [38]. How long lamivudine should be administered remains unclear. Some data suggest that it should be administered for more than 1 year even if a YMDD mutation takes place. Lamivudine administration should be stopped if seroconversion to anti-HBe has occurred. The YMDD mutation confers cross-resistance between lamivudine, emtricitabine (FTC), clevadine (L-FMAU), and L-Fd4C [45]. However, adefovirdipivoxil (ADV) and entecavir (ETV) suppress replications of both YMDD mutants and wild types of HBV (Table 4). The most promising drug seems to be adefovir. Adefovir and its oral prodrug adefovirdipivoxil in combination with lamivudine are now the subject of clinical trials for resistant mutants. Adefovir is primarily accumulated in the kidneys, which lowers the effective concentration in the liver and may result in nephrotoxicity. In a placebo-controlled trial of adefovir therapy in HIV infection, 60% of the patients developed renal impairment. Although these abnormalities were usually reversible after cessation of therapy, concerns about long-term deleterious effects

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halted adefovir development for HIV infection [39]. The lower doses required for effective therapy of HBV infection and the shorter duration of therapy seem to overcome this problem. Another strategy is the use of substance carriers able to deliver lipophilic prodrugs to the liver [40]. Another encouraging drug is entecavir. A recent study in 42 patients showed negative HBV DNA after 28 days of entecavir treatment in 33% of the patients. Entecavir is also effective against virus carrying the YMDD mutation. Only a few mild side effects were observed. Phase II trials with entecavir are ongoing worldwide [41]. There are many nucleoside analogs that are effective against hepatitis B. Table 4 gives a short survey. A special problem in the therapy of HBV infection is presented by virus variants. At present HBV is divided into six genotypes (A–F). The genotypes have different geographical distributions. They contribute to the various outcomes of the disease and the dissimilarities in therapeutic success. Virus mutants of special clinical interest are mutations in the precore and S genes. The precore region consists of 87 nucleotides. The predominant mutation is the G to A change at nucleotide 1896, which produces a stop codon that prevents the production of HBeAg [9]. The basis for the selection of this common mutation is still unclear. Immune escape and more effective replication are considered as the possible causes. This mutation shows a poor response to interferon. Mutations in the S gene are detected in infants born to carrier mothers who developed HBV infection despite vaccination and in liver transplant recipients who developed HBV reinfection despite prophylaxis with hepatitis B immunoglobulin. Although these mutants show weaker binding to

TABLE 4

Nucleosid Analogs Under Evaluation for Therapy Against Hepatitis B Infection Substance

Adefovir dipivoxil (ADV) Entecavir (ETV) Emtricitabine (FTC) Clevudine (L-FMAU) L-dT (L-thymidine nucleoside; telbivudine) L-Fd4C (L-cytidine-nucleosid)

Comment Approved September 20, 2002 Clinical trials (phase 2, 3 studies) Clinical trials (phase 2, 3 studies) Clinical trials (phase 1 studies) Clinical trials (phase 1 studies) Clinical trials (phase 1 studies)

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anti-HBs, there is no evidence of increasing prevalence of such escape mutants through the widespread use of HBs vaccines and hepatitis B immunoglobulin. In chronic hepatitis B the decision for therapy should depend on the extent of active viral replication. Prior to this decision several examinations should be performed: viral parameters and serum transaminases and a liver biopsy to determine the grade of inﬂammation and stage of ﬁbrosis. Relevant accompanying diseases should be ruled out. Patients with no detectable or very low levels of HBV DNA (i.e., <2.5 pg DNA/mL serum) have a good prognosis and will not beneﬁt from antiviral therapy. Patients with high levels of viral DNA with or without high aminotransaminase levels should be treated. In general, interferon is still the ﬁrst-line therapy. Indicative factors for a response to interferon-a are high serum transaminases, relatively low serum HBV DNA levels, anti-HBC IgM positivity, active necroinﬂammation in the liver biopsy, relatively short duration of disease, and absence of complicating disorders or diseases [42]. In patients with a more tolerant status against the virus (i.e., those with low transaminases and low-grade inﬂammation), lamivudine alone or in combination with interferon will be more successful. Response to interferon is poorer in individuals who are infected with a precore mutant HBV. Here the ﬁrst-line treatment is lamivudine [43]. Lamivudine is also the treatment of choice for patients who have contraindications to interferon. The ﬁrst objective of antiviral therapy is the suppression of viral replication. This is monitored by determiniation of DNA levels. Usually the therapy is successful if there is seroconversion from HBeAg positivity to negativity and from anti-HBe negativity to positivity. The response should be controlled by determination of the HBV DNA, HbeAg, antiHBe, and aminotransaminase levels. In these responders a loss of HBsAg is seen in 25–40%, which may take up to 7 years after termination of therapy [44]. Lamivudine is associated with suppression of HBV DNA and a substantial improvement in the liver histology. The main problem is the occurrence of resistant mutants, which show an increasing incidence under prolonged therapy. However, even in patients with resistant mutants the probability of response rises with the duration of therapy. Therefore it is recommended that the therapy be continued even after a resistant mutant has been detected. In the future, the development of new combination strategies of different nucleoside analogs will become a standard therapy for chronic HBV infection. In addition to vaccination programs a combination therapy seems to be the most promising approach for worldwide eradication of HBV.

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10 Hepatitis C Virus Miriam Kerstin Huber Johann-Wolfgang Goethe University Clinic, Frankurt/Main, Germany

Ulrike Sarrazin and Stefan Zeuzem Saarland University Hospital, Homburg, Germany

1

INTRODUCTION

Infection with hepatitis C virus (HCV) is a major cause of liver disease worldwide. Acute HCV infection is generally mild, with less than 25% of patients developing jaundice. The sequelae of chronic hepatitis C (CHC) are the real burden of HCV infection. Regardless of the route of acquisition, as many as 50–80% of acute HCV infections persist as chronic infection. Within 20 years after infection, one-half of patients will have signiﬁcant hepatitis, some with ﬁbrosis and some progressing to cirrhosis. With increasing duration of disease, the likelihood of disease progression to cirrhosis, hepatic failure, and/or primary hepatocellular carcinoma also increases. Although the rate of newly acquired HCV infections is decreasing, tens of thousands of individuals who were infected decades ago have not been diagnosed and will likely experience complications of HCV leading to hepatitis-related death in the future. 295

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EPIDEMIOLOGY AND RISKS OF TRANSMISSION

Approximately 170 million people worldwide are infected with the hepatitis C virus (HCV), with the reported prevalence of this infection ranging from 1% to 5% in most countries. Most (85–90%) of these individuals are chronically infected. HCV accounts for the majority of cases of viral hepatitis in the United States, and it has been supposed that 2.7 million individuals (1.4% of the population) are chronically infected with HCV in the United States and 9 million in Europe. Chronic HCV infection can lead to cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Population-based studies indicate that 40% of chronic liver diseases in the United States are HCV-related, accounting for 8000– 10,000 deaths annually. Hepatitis C is the most frequent indication for liver transplantation among adults, accounting for 20 to 40% of the 3600 liver transplantations performed in the United States each year, 30–50% of transplantations in some larger centers [1,2], and an even higher proportion in Europe [3,4]. In the United States, most individuals with chronic hepatitis C are between the ages of 30 and 49 years and have yet to manifest the long-term sequelae of the disease [5]. Throughout the world, the factors most strongly associated with HCV infection are intravenous drug use and receipt of blood transfusions and blood products before 1990. Furthermore, poverty, promiscuity, HIV infection, and concurrent alcohol consumption are linked to an increased risk of infection. In some cases no risk factors can be identiﬁed. Nosocomial transmission has been documented, such as from patient to patient by colonoscopy, during hemodialysis, and during surgery. Even though the prevalence of HCV infection is no higher among employees in public health care than in the rest of the population, needlestick injuries in the health care setting continue to result in transmission of the virus [6]. Blood transfusions posed the major risk of HCV infection in developed countries. The introduction of improved blood-screening measures based on the detection of HCV antibodies and most recently by nucleic acid testing (NAT) has dramatically decreased the risk of transfusionassociated HCV infection. Currently, the residual transmission risk results mainly from intravenous drug abuse leading to an increased prevalence of HCV subtypes 1a and 3a and a decline of subtype 1b.

3

CLINICAL COURSE OF DISEASE

The clinical progression of HCV-associated liver disease is slow and without signs or symptoms in most patients during the ﬁrst two decades after initial infection. Coinfection with hepatitis B virus and/or human

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immunodeﬁciency virus may lead to more severe disease. Chronic hepatitis C is frequently a progressive ﬁbrotic disease rather than an inﬂammatory hepatitis. The progression between stages of liver ﬁbrosis from ﬁbrosis without septa (F1) to cirrhosis (F4) is initially almost linear over time, although most likely some acceleration occurs in more advanced disease (Fig. 1). Histological activity grades are not as linearly correlated as ﬁbrosis stage, suggesting that ﬁbrosis stage provides a better estimation of the progression of chronic hepatitis C. Risk factors associated with an increased rate of progression of ﬁbrosis include age >40 years at infection, daily alcohol consumption >50 g, and male gender. No association with progression to ﬁbrosis was observed for HCV genotype and viremia [6]. Once cirrhosis is established, however, the prognosis is dismal. There is an increased development of complications secondary to liver failure and/or portal hypertension, including jaundice, encephalopathy, ascites, and variceal hemorrhage, any of which mark the transition from compensated to decompensated cirrhosis. Individuals with cirrhosis have a higher risk of developing hepatocellular carcinoma (HCC) than individuals without cirrhosis. The 5 year cumulative risk of HCC was 7% in a study of European patients with HCV infection and compensated cirrhosis and increased over time (the probability was 4% and 7% at 3 and 5 years, respectively). Liver disease-related mortality was 9%, with hepatocellular cancer and liver failure being the main causes of death. In Japan, the 5 year probability of development of HCC was &25% in untreated patients with chronic hepatitis C and compensated cirrhosis [7,8]. Both chronic hepatitis B virus (HBV) coinfection and concomitant alcohol use increase the risk of HCC in patients with HCV [9]. In some studies [10–12], but not in others [13], HCV genotype 1b has been reported to be associated with higher HCV RNA levels in the infected host and more advanced disease. These results are confounded by epidemiological factors (i.e., genotype 1b patients are older) and the previous lack of speciﬁcity of common genotyping methods and genotype-dependent HCV RNA quantiﬁcation [11,14]. The risk of HCC in HCV-infected patients with cirrhosis is higher than in those without, suggesting that hepatic cell damage and the associated regeneration play a signiﬁcant role in hepatic carcinogenesis [15,16]. However, HCV confers an additional risk for HCC beyond just cirrhosis. The molecular mechanisms of hepatocarcinogenesis are largely unknown. The ability of the HCV core protein to modulate gene transcription, cell proliferation, and cell death may be involved in the pathogenesis of HCC. Transgenic mice carrying the HCV core protein ultimately develop neoplasias with histopathological characteristics that closely resemble those of the early

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FIGURE 1 Relationship between age at liver biopsy and (a) histological activity grade or (b) ﬁbrosis stage in patients with chronic hepatitis C. Histological activity was scored on a four-point scale from 0 (no activity) to 3 (severe activity). Fibrosis was scored on a ﬁve-point scale from 0 (no ﬁbrosis) to 4 (cirrhosis). Points represent mean, and vertical lines represent 95% Cl.

stage of HCC in patients with chronic hepatitis C [17], indicating that the HCV core protein has an important role in the pathogenesis.

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299

DIAGNOSIS OF HEPATITIS C INFECTION

The work-up of chronic liver damage or cirrhosis of unclear etiology with elevated liver enzymes (e.g., ALT, AST) or reduced liver function test (e.g., thrombin time) should include a differential diagnosis of hepatitis C infection, one of the major causes. Patients chronically infected with HCV frequently present without any speciﬁc signs or symptoms. They may complain of uncharacteristic discomfort, such as fatigue or intermittent right upper abdominal pain, which does not necessarily implicate speciﬁc HCV testing. The main diagnostic tools to characterize the virus and to assess the degree of liver damage are serological and molecular classiﬁcation, ultrasound imaging, and histology. 4.1 4.1.1

Serological Diagnosis Enzyme Immunoassay

The ﬁrst-generation enzyme immunoassays (EIA-1) detected antibodies to the c100 antigen, a nonstructural recombinant protein located in the NS4 region of the HCV genome. The use of these early assays offered a 70–80% sensitivity, leading to false-positive results in numerous patients presenting with high levels of immunoglobulin [18–20]. Second-generation assays (EIA-2) identify antibodies against both structural (core antigen, C22) and nonstructural proteins of the NS3 and NS4 regions (C33, C-100) (Fig. 2). Using this enhanced technology not available in the ﬁrst-generation assays, anti-HCV is detectable in an additional 10–20% of HCV-infected individuals. In the case of acute infection those tests even provide improved sensitivity and become positive several weeks earlier [21–23]. The ﬁrst appearance of the antibodies to the epitopes in EIA-2 is approximately 4–6 weeks after infection (Fig. 3). Therefore, the diagnostic window complicates the serological veriﬁcation. The median lag time for detection of seroconversion is only insigniﬁcantly reduced by HCV-speciﬁc IgM antibody testing. Although of great value, EIA tests still have limitations, especially in certain populations such as immunosuppressed patients, in which inaccurate results, e.g., the lack of antiHCV antibodies, are relatively more common. In patients with acute or chronic disease the evidence of IgM antibodies against HCV core antigen is overlapping. In mild cases of chronic hepatitis C, IgM antibodies are rarely present; however, IgM antibodies are detectable in more than 50% of patients with aggressive liver disease [24]. IgG antibodies usually persist in patients with chronic hepatitis [25]. After successful eradication of the virus or recovery from acute hepatitis C, disappearance of anti-

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FIGURE 2 Genomic and proteomic map of the hepatitis C virus. The 50 untranslated region (UTR), comprising the IRES, and the 30 UTR ﬂank the genomic regions coding for the viral polyprotein. The 10 known polyprotein cleavage products are shown in their approximate sizes (in kilodaltons) and putative functions.

HCV IgG antibodies may be seen [26]. In chronic HCV infection, existence of HCV antibodies correlates with persistent viremia, i.e., HCV RNA seropositivity. At any rate, only a negligible number of individuals presenting with low HCV viral load lack anti-HCV [27]. Recombinant proteins of the NS5 region complete third-generation antibody testing [28]. However, improved sensitivity of the thirdgeneration enzyme immunoassay is attributed to increased reactivity of the NS3 antigen [29–31]. The supplementary NS5 antigen may even generate additional false-positive results [31]. 4.1.2

Conﬁrmatory Assay

Veriﬁcation of positive EIA test results and exact evaluation of the patient’s antibody response are based on various immunoblotting techniques, detecting monospeciﬁc antibodies against multiple membrane ﬁxed recombinant and single HCV proteins (RIBA, Ortho; Matrix HCV, Abbott) [32]. These systems (e.g., Matrix HCV) are based on modiﬁcation of EIA. Anti-HCV antibodies bind to recombinant hepatitis

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FIGURE 3 Biochemical and serological course of hepatitis C infection. Serum HCV RNA is detectable soon after infection, followed by an elevation of aminotransferases and detection of anti-HCV antibodies. Epitope-speciﬁc antibodies are detectable at different time points. EIA of the second and third generations detect antibodies against structural (core antigen, C22) and nonstructural proteins (C33, C100).

C virus proteins by forming an antigen–antibody complex. Prevention of unspeciﬁc reactions with recombinant proteins is guaranteed by dilution buffer containing yeast and E. coli proteins. After a stringent washing step that removes unattached antibodies, the presence of human IgG is analyzed in two steps. First biotin-labeled anti-human IgG antibodies (goat) and ﬁnally an alkaline phosphatase conjugated antibiotin antibody are added. After application of chromogenic substrate, the photometric analysis is performed. However, in up to 20% of blood donors with positive HCV EIA and negative or ambiguous immunoblot test result, HCV RNA may still be detectable by means of reverse transcription polymerase chain reaction (RT-PCR) (see also Sec. 4.2). Conﬁrmatory assays are reasonable in populations with low HCV prevalence (e.g., blood donation unit) but remain dispensable for clinical diagnosis of liver diseases. 4.1.3

HCV Core Antigen Assay

Serological identiﬁcation of HCV core antigen can be performed after accumulation of virus particles and dissolution of the virus envelope. The use of monoclonal antibodies in this assay makes it possible

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to recognize a particularly conserved core region. Indeed, various secondary structures of the core protein may inﬂuence antibody detection and attachment to the epitope. HCV antigen detection systems have recently become commercially available in Europe and the United States. 4.2 4.2.1

Molecular Diagnostics Qualitative HCV RNA Assay

Polymerase Chain Reaction (Roche Molecular Systems). Primarily in cases of minimal virus concentrations, direct identiﬁcation of HCVspeciﬁc RNA in serum or tissue requires sensitive methods. The combined technology of reverse transcription (RT) of HCV RNA with subsequent HCV cDNA ampliﬁcation by polymerase chain reaction (PCR) is established in molecular diagnosis of hepatitis C (RT-PCR). Principles of PCR are demonstrated in Figure 4. In the case of an acute infection, evidence of HCV-speciﬁc RNA will be veriﬁed by means of RT-PCR, mostly within the ﬁrst week [26,33]. Serological and molecular diagnostics and their clinical implications are compared in Table 1. Due to the relatively high conservation of the 50 noncoding region of the HCV genome, only primers complementary to this genome sequence have been qualiﬁed for diagnostics [34,35] (Fig. 4). Several teams worked on improving sensitivity and speciﬁcity. By ﬁnishing the reverse transcription procedure, they were running PCR twice, using two different primer pairs (the so-called nested PCR) [36,37]. When primer and incubation conditions are optimized, a sensitivity approaching that of nested PCR can even be obtained with normal PCR with just one primer pair [38,39]. The need to open the incubation tubes between the two PCR reaction mixtures is the major disadvantage of nested PCR because it increases the risk of contamination and the generation of falsepositive results. In 1993 Eurohep organized the ﬁrst quality control study, determining that false-positive test results occurred in about 29% (9/ 31) of participating laboratories. Only 16% of the involved laboratories attained perfect results [40]. The ﬁrst standardized RT-PCR assay for detection of HCV RNA was introduced by the end of 1993 (Amplicor2 HCV, Roche Diagnostics). This system uses DNA polymerase of Thermus thermophilus, which under suitable buffer conditions provides reverse transcriptase as well as DNA polymerase activity [41]. RT-PCR can be carried out in only one incubation tube without the need to open the tubes during each of the following reaction steps: reverse transcription

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FIGURE 4 Reverse transcription polymerase chain reaction (RT-PCR). After extraction of viral RNA, reverse transcription (RT) is performed to translate RNA into complementary DNA (cDNA). During polymerase chain reaction (PCR) the two strands are separated by heating (denaturation). Short oligonucleotides complementary to the (þ)-strand (primer 1) and the ()strand (primer 2) at both sites of the sequence of interest hybridize. A thermostable DNA polymerase elongates the missing parts complementary to the target (in the 50 ? 30 direction). Two DNA double strands result, which in turn will be denatured into their single strands. For the following cycles of PCR, exponential replication of the deﬁned genome sequence (ﬂanked by the two primers) takes place.

(RT) and PCR. Uracil-N-glycosylase (UNG) and the replacement of dTTP by dUTP serve in this system as an additional means of preventing contamination. Furthermore, it is possible to degrade potentially contaminating amplicons containing dUTP selectively enzymatically without destroying the dTTP-containing native HCV RNA. Also, selective enzymatic digestion of potentially contaminating ampliﬁcates containing dUTP is possible without destruction of native HCV RNA,

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TABLE 1 Comparison of Serological (Anti-HCV) and Molecular (HCV RNA) Test Systems for the Diagnosis of Hepatitis C Anti-HCV Detection of an acute infection Differentiation between resolved, cured, and chronic infection Diagnostics in immunosuppressed patients Diagnostics in newborns to HCVinfected mothers Reinfection after organ transplantation Virus quantiﬁcation Organ-speciﬁc detection

HCV RNA

After *10–12 weeks

Within the ﬁrst week

þ/

þ

þ/

þ

þ

þ

þ þ

which in turn contains dTTP. The commercially available HCV RT-PCR assay of the ﬁrst version reached an analytical sensitivity limit of 1000 RNA copies/mL [39,42]. Sensitivity was genotype-dependent; e.g., HCV2 and HCV-3 isolates attained minor sensitivity compared to HCV genotype 1. Nevertheless this technique attained excellent sensitivity and speciﬁcity for clinical settings [39,43]. Systems of the second version offer equal sensitivity for all genotypes with an improved detection limit (100 copies/mL). HCV RNA will be extracted from 200 mL of serum (in ﬁrst version 100 mL was needed). Molecular analysis of HCV ()-strands represents a direct parameter for virus replication. Analyses of the ()-strand are complicated because positive HCV RNA strands serve as templates for the synthesis of negative strands, either due to false primer binding during cDNA synthesis or because of insufﬁcient inactivation of RT during PCR ampliﬁcation. Formation of thermostable hairpin structures and selfpriming possibly lead to false-positive HCV ()-strands [44]. Ligase Chain Reaction. In the ligase chain reaction two contiguous oligonucleotides are joined by a thermostable DNA ligase in the 50 ! 30 direction under adenosine triphosphate (ATP) consumption. The target sequence is denatured, and another set of oligonucleotides complementary to the ﬁrst one hybridizes to the DNA molecules. The product is then

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cleaved, and the process is repeated cyclically. Ampliﬁcation starts with the addition of complementary oligonucleotides to the reaction. This cycling procedure continues as soon as another ligase joins two more oligonucleotides for subsequent hybridization. Reaction products are easily detected by gel electrophoresis. Alternatively, ﬂuorescence-labeled ﬂanking regions of each oligonucleotide permit detection in an ELISAlike format. For example, a biotin-labeled oligonucleotide is able to bind to a streptavidin-coated microtiter plate. The ﬁrst oligonucleotide can produce a certain signal only if the second oligonucleotide, even one carrying a signal, is attached to it. Although the ligase chain reaction appears to be quite elegant, the main disadvantage is low speciﬁcity. To increase speciﬁcity, oligonucleotides would have to be further apart. The arising gap is closed by a ﬁlling reaction, which violates PCR patent law. The development of HCV RNA detection systems based on the ligase chain reaction is stagnating. Transcription-Mediated Ampliﬁcation (Bayer Diagnostics). Detection of HCV RNA by the technique of transcription-mediated ampliﬁcation (TMA) consists of three steps that are performed in a single tube: (1) target capture, (2) target ampliﬁcation, and (3) speciﬁc detection of target amplicons by hybridization protection assay. Five hundred microliters of serum is used for extraction. HCV RNA is released from viral particles. By hybridization of the viral RNA to capture oligonucleotides and binding of the virus probe complex to magnetic microparticles following several wash and aspiration steps, potential assay inhibitors or interfering factors are removed. Ampliﬁcation of target RNA is performed by autocatalytic, isothermal production of RNA transcripts using two enzymes (reverse transcriptase and T7 RNA polymerase) and two primers, one of which contains a T7 promoter. The promotercontaining primer hybridizes to target RNA and synthesizes cDNA by reverse transcriptase (Fig. 5). The RNA of the resulting RNA–DNA duplex is degraded by the RNase H activity of the reverse transcriptase. The second primer then binds to the cDNA that already contains the promoter sequence from the ﬁrst primer, and new DNA is synthesized by the reverse transcriptase. The RNA polymerase recognizes the T7promoter sequence in the double-stranded DNA molecule and synthesizes numerous RNA transcripts. Each of the newly synthesized RNAs reenters the TMA process and serves as template for a new round of replication, resulting in an exponential ampliﬁcation of target RNA. The RNA amplicons are detected by a hybridization protection assay with amplicon-speciﬁc acridinium ester–labeled DNA probes. The labeled probe hybridizes to the RNA amplicons. During the selection

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FIGURE 5 Principle of ‘‘transcription-mediated ampliﬁcation’’ (TMA). Detection of HCV RNA by this technique consists of three steps that are performed in a single tube: target capture, target ampliﬁcation, and speciﬁc detection of target amplicons by hybridization protection assay. Ampliﬁcation of target RNA is performed by autocatalytic, isothermal production of RNA transcripts using two enzymes (reverse transcriptase and T7 RNA polymerase) and two primers, one of which contains a T7 promotor.

step when alkaline reagent is added, the unhybridized probe is hydrolyzed due to the high pH, whereas the hybridized probe is protected from the hydrolysis and thereby retains the chemiluminescent label. After addition of detection reagents, chemiluminescence from the acridinium ester label retained within the hybrids is measured in a luminometer and signals are expressed numerically in relative light units. In-process control and amplicon characterization are achieved via a dual kinetic assay. This procedure involves the use of two different chemically modiﬁed acridinium esters that differ in their reaction kinetics and therefore in the time and duration of their light emission. Both probe sets (i.e., target- and internal control-speciﬁc) are added to the same tube and are hybridized and selected at the same time. The acridinium ester on the internal control probe will emit a short, intense

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ﬂash of light (‘‘ﬂasher’’), whereas the second acridinium ester will produce a longer lasting glow of light (‘‘glower’’) that can be measured shortly afterward. Therefore, the differences in chemiluminescent reaction kinetics allow the simultaneous detection of two acridinium ester derivatives in a single reaction tube, providing internal control of the assay reactants. The analytical sensitivity of TMA is 93% and 100% at 25 and 50 copies/mL, respectively. According to the World Health Organization, HCV RNA standard sensitivity is 96% (5 IU/mL) and 100% (10 IU/mL). In this assay HCV genotypes are detected in parallel with comparable efﬁciency (HCV-1a/b, 2b/c, 3a, 4c/d, and 6a). The clinical speciﬁcity is greater than 99.5% [45]. Nucleic Acid Sequence-Based Ampliﬁcation. The mechanism of nucleic acid sequence-based ampliﬁcation (NASBA) resembles the transcription-mediated ampliﬁcation method. Standard reaction of NASBA contains T7 RNA polymerase, RNase H, reverse transcriptase (RT), nucleotides, deoxynucleotides, and one sense primer [S-primer (1)]. This S-primer contains nucleotides complementary to the target RNA, as well as the T7 RNA polymerase promoter sequence on its 50 end and an antisense primer [AS-primer (2)]. During the ﬁrst stages of the NASBA process, S-primer (1) attaches to target RNA using the complementary binding region. T7 RNA polymerase promoter sequence on the 50 end is incapable of binding and extends past RNA. Reverse transcriptase serving as RNA-dependent DNA polymerase elongates the S-primer (1) on its 30 end by encoding deoxynucleotides beyond the binding region of the antisense primer. A complementary cDNA to the existing RNA has been formed. The RNase H degrades the RNA template, leaving behind single-stranded cDNA. AS-primer (2) then anneals to the single-stranded cDNA and is extended by RT on its 30 end in the opposite direction. RNA is replaced by a second DNA strand. Synthesis continues up to the 50 end of the ﬁrst cDNA strand with completion of the T7 polymerase promoter sequence attached to S-primer (1). Noncyclic prephase is terminated. The ampliﬁcation cycle starts as soon as T7 polymerase enters the system. This DNA-dependent RNA polymerase requires DNA as template, nucleotide triphosphates as substrates, and a speciﬁc promoter sequence for initiation. The main advantage of T7 polymerase is that it is possible to synthesize thousands of RNA molecules out of a single DNA molecule, using only one suitable promoter. During the cyclic phase of NASBA the RNA molecules (as compared to cDNA) are complementary to the original RNA, assuming that only AS-primer (2) is able to bind. RT transcribes these RNA molecules to cDNA. RNase H removes RNA followed by the binding of primer 1, and again RT will continue

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lengthening them on their 30 end (second-strand synthesis). Concurrent with the second-strand synthesis, a double-stranded DNA is being formed that carries a complete T7 promoter, because cDNA has been completed on its 30 end. Again T7 polymerase engages, and the cycle is closed. NASBA is dedicated to RNA ampliﬁcation. Using ampliﬁcation of DNA, the DNA molecules have to be transcribed to RNA during the noncyclic initial steps of the procedure, which require heating up to 1008C twice in order to break the double strand. The option of an ampliﬁcation keeping a constant temperature of 418C represents the great advantage of NASBA. Due to the fact that NASBA reaction is continuous, single-stranded RNA ampliﬁcation results; RNA–DNA hybrids and double-stranded DNA are equally involved in the system. However, single-stranded RNA is signiﬁcantly preponderant. Detection succeeds after hybridization of single-stranded RNA with a speciﬁcally labeled probe. Sensitivity and speciﬁcity as well as the risk of contaminated amplicons are comparable to those of PCR. Because of patent law, an HCV NASBA kit is not yet available. After introduction of internal RNA standards, NASBA is qualiﬁed for virus quantiﬁcation, and it is used for, e.g., HIV quantiﬁcation. 4.2.2

Quantitative HCV RNA Detection

Quantitative HCV RNA testing is important for diagnosis, therapy indication, and monitoring. Various studies showed a signiﬁcant predictive value of HCV viremia in terms of interferon-a-based therapy. More favorable response rates are achieved in patients with less than 26106 copies/mL serum [equivalent to 800,000 IU/mL (WHO standard)] than in those individuals who present with a higher pretreatment viral load. As soon as several antiviral substances are available for chronic hepatitis C, quantitative HCV RNA measurement will become essential for therapy monitoring. As in the treatment of HIV-infected individuals, HCV RNA quantiﬁcation checks antiviral efﬁcacy. Potential development of arising resistances may be detected at an early stage, and appropriate therapy can be initiated. PCR-Based Technology. The key to quantifying PCR products is correlating the deﬁnite ﬁnal amount of PCR reaction products with the number of molecules originally assayed [42]. The permanent doubling of reaction products from previous reaction cycles characterizes PCR. Indeed, efﬁcacy of the system is affected by various components (nucleotides, Taq polymerase, buffer conditions, primers, PCR

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inhibitors, and others). A high standardization of reaction conditions and avoidance of extraordinary reaction steps (e.g., nested PCR) are necessary to exclude high variability and to obtain reproducible quantiﬁcation. Although the different parameters mentioned may be easily controlled and optimized, some methodical variabilities remain, which can be tackled only by the introduction of internal standards. Quantitative RT-PCR combines transcription of the target sequence and an internal control in a single reaction mixture, followed by coampliﬁcation of the target sequence and standard cDNA. Numerous criteria have to be fulﬁlled by the internal control: (1) identical primers for target sequence and standard; (2) no differentiation between lengths of amplicons to reach a similar ampliﬁcation efﬁciency; (3) both reaction products, target sequence and internal control, should provide a comparable succession of nucleotides but remain distinguishable for a single detection system [42]. HCV-SUPERQUANT

2

(NGI). Quantitative RT-PCR systems are available in special laboratories [42,46]. HCV-SuperQuant2 of the National Genetics Institute (NGI) is based on quantitative RT-PCR. This assay achieves an equal dynamic range for each HCV genotype. The test is highly sensitive with an improved detection limit of <100 copies/mL providing a linear range between 100 and 56106 copies/mL. Ampliﬁcation is carried out by polymerase chain reaction, and cDNA is detected by hybridization to a digoxigenin-labeled DNA probe. This technique was used to perform multiple controlled phase III trials for medication approval, e.g., combination therapy of interferon-a and ribavirin. AMPLICOR MONITOR (ROCHE MOLECULAR SYSTEMS). A commercially available assay (Amplicor2 HCV Monitor) has been developed by Roche Molecular Systems. Compared to the qualitative assay, sensitivity of the quantitative test is tenfold decreased. Samples with values exceeding 106 copies/mL serum (400,000 IU/mL) are considered to be above the linear range of the quantitative assay, and HCV RNA levels may then be underestimated up to 2–3 log if they are not prediluted [47]. In contrast to Amplicor2 HCV Monitor, version 1.0, Amplicor2 HCV Monitor, version 2.0 attains an almost equivalent quantiﬁcation for each HCV genotype. Minor modiﬁcations now guarantee a similar sensitivity for the Amplicor2 HCV Monitor assay, version 2.0 conducted on the automated Cobas Amplicor analyzer. This is an automatic thermocycler that includes an integrated detection unit and 2 6 12 tubes circularly arranged for the ampliﬁcation procedure. After PCR termination, tube caps are penetrated and reaction products are automatically transferred to the detection device. Enzymatic analysis follows denaturation,

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annealing of a labeled probe, and stringent washing procedures. Virus RNA calculation is performed by means of a specialized software program following the internal standards. 50 -NUCLEASE (TAQMAN) ASSAY. Precise and reliable quantiﬁcation is offered by 50 -nuclease PCR assay or TaqMan technology, also patented by Roche and soon available as a commercial kit for HCV quantiﬁcation. This assay permits both real-time detection of the ampliﬁcation product during the ampliﬁcation procedure and ‘‘closed-tube’’ detection, excluding contamination of reaction products during post-PCR sample processing. The total duration of this procedure is roughly reduced by half compared to that of Cobas Amplicor (from more than 4 hr to less than 2 hr). This method uses an oligo probe with a reporter dye (50 end) and a quencher dye (30 end) attached. Both anneal throughout the reaction process to each newly formed molecule prior to primer attachment. As soon as polymerase hits the probe, it will be enzymatically degraded by Taq polymerase during primer elongation. Therefore the reporter is separated from the quencher and is now capable of emitting its characteristic ﬂuorescence upon speciﬁc excitation. The cleavage described excludes inhibition of the ﬂuorescent signal across the spine of oligonucleotides. The stimulating laser light is transmitted via ﬁberoptic cables ending in the thermocycler cap on the top of each tube. The energy is transferred back the same way, crosses a dichromatic mirror, and is guided toward a photomultiplier that measures the emitted signals of each cycle. Light emission is proportional to the amount of reporter molecules and the ampliﬁcation product, which is responsible for the quantitative quality of this system. The number of cycles leading to a certain threshold value of light emission, the so-called Ct value, permits proximate quantiﬁcation. In order to obtain precise quantiﬁcation, an internal standard runs in each reaction mixture. The internal control is hybridized, with a separate oligonucleotide carrying a different reporter dye. By switching to the characteristic ﬂuorescence of the internal standard, the relevant Ct value can be determined. If a dilution sequence of a quantiﬁed standard with unknown virus sequences is ampliﬁed, it is possible to generate a standard curve. The exact concentration of virus molecules can be deﬁned. LIGHT CYCLER. The Perkin-Elmer 7700 Prism detection system is used for the TaqMan technology and is based on conventional cyclers using a microtiter format. In contrast, the light cycler of Boehringer Mannheim (actually now also Roche Diagnostics) is a small compact unit of totally different design. PCR is carried out in glass capillaries that have

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to be ﬁlled by centrifugation to a maximum volume of 25 mL. These capillaries are aligned in a cycle of 32 positions that are air heated and cooled. The relatively large surface-to-volume ratio of the glass capillaries allows signiﬁcant shortening of the PCR cycles, with a complete ampliﬁcation time of 20–30 min. As with the ‘‘real-time’’ TaqMan technique, samples are detected by probe annealing and ﬂuorophore labeling as quencher and reporter dye. Contrary to 50 nuclease assay, two probes bind to the newly synthesized DNA molecules within each cycle such that the 30 end of probe 1 attaches directly in front of the 50 end of the second probe. While the ﬁrst probe is labeled on its 30 end and the second one on its 50 end, this close relationship allows energy transfer of the ﬂuorescent dye from the ﬁrst (30 end) to the second (50 end) probe. A photomultiplier will transform the emitted light of probe 2 into a signal. Analysis is performed analogous to TaqMan assay via Ct . Exact quantiﬁcation proceeds by means of an internal standard similar to TaqMan technology. Branched-DNA Technology (Bayer Diagnostics). Branched-DNA technology is a sandwich nucleic acid hybridization procedure for the direct quantiﬁcation of HCV RNA in human serum and plasma (signalampliﬁcation nucleic acid probe assay), which presents an alternative method to HCV-cDNA ampliﬁcation by means of the polymerase chain reaction (target sequence ampliﬁcation). HCV RNA is released from virions in the sample by the use of proteinase K and detergents and is hybridized to speciﬁc oligonucleotide capture probes, which are in turn hybridized to speciﬁc oligonucleotide probes (linkers) bound to a microtiter plate. Target probes hybridize to the bound viral RNA and preampliﬁer probes in the reaction mixture. The capture and target probes bind to multiple regions of the 50 untranslated and core regions of the HCV genome, which allows the assay to detect all HCV genotypes. Ampliﬁer probes bind to preampliﬁer probes to form a branched DNA (bDNA) complex. Multiple copies of an alkaline phosphatase (AP)labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the AP-bound complex with a chemiluminescent substrate. Light emission is directly related to the amount of HCV RNA present in each sample, and results are recorded as relative light units (RLUs) by luminometer (Fig. 6). The signal ampliﬁcation technique differs from target-sequence ampliﬁcation in that it lacks the risk of contamination by amplicons. Despite signal ampliﬁcation, Quantiplex2 HCV RNA, version 1.0 achieved a lower detection limit of only 350,000 copies/mL serum [48]. An improved sensitivity of 200,000 copies/mL serum was reached by

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FIGURE 6 Branched DNA (bDNA) signal ampliﬁcation (Bayer Diagnostics). After HCV RNA is released from virions, the RNA is captured to a microwell by a set of speciﬁc synthetic oligonucleotide capture probes. A set of target probes hybridizes both the viral RNA and the preampliﬁer probes. The capture probes and the target probes bind to the 50 untranslated and core regions of the HCV genome. The ampliﬁer probe subsequently hybridizes to the preampliﬁer, forming a branched DNA (bDNA) complex. Multiple copies of an alkaline phosphatase (AP)-labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the AP-bound complex with a chemiluminescent substrate. Light emission is directly related to the amount of HCV RNA present in each sample. Results are recorded as relative light units (RLUs) by the analyzer.

Quantiplex2 HCV RNA, version 2.0. Over 95% of chronically infected individuals (without antiviral therapy) were diagnosed by this system and 80–90% by using Quantiplex2 HCV RNA, version 1.0. In addition to that, version 1.0 underestimated genotype HCV-2 by a factor of 3 and HCV-3 by a factor of 2 [49,50]. Branched chain DNA assay version 2.0 and all subsequent versions of the kit quantify HCV RNA with equivalent efﬁciency across all genotypes. Compared with the ﬁrst- and second-generation assays, sensitivity in version 3.0 was improved by background reduction and simultaneous signal reinforcement. Application of artiﬁcial nucleotides isocytidine (iso-C) and isoguanosine (iso-G) offered an augmented signal-to-background ratio. Because of their unique molecular properties these

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synthetic nucleotides are linked with higher mutual afﬁnity than their four natural counterparts. Unspeciﬁc binding, which has been responsible for background noise in the past, could be minimized. Modiﬁcations of speciﬁc capture and target probes as well as application of preampliﬁer probes led to signal enhancement (Fig. 6). The HCV RNA bDNA assay version 3.0 (Versant2 HCV RNA) achieves a lower detection limit of 1000–2000 copies/mL (615 IU/mL) and provides a wide linear dynamic range (<2 6 103–40 6 106 copies/mL) without the necessity of sample dilutions. 4.2.3

HCV Typing

Although multiple HCV isolates have been sequenced completely, others have been only partially analyzed. Today sequencing offers the opportunity to differentiate six HCV genotypes (HCV-1 to HCV-6) worldwide, providing variations in nucleotides in over 30% of the envelope proteins [47,51] (Fig. 2). Supplementary subtypes can be deﬁned within those groups (subtypes a, b, c, and others). Homology of subtypes varies between 80% and 90% [49,52]. Minor sequence discrepancies are not given any further deﬁnition and are summarized under HCV quasi-species. Knowledge of the genotype is important because it has predictive value in terms of the response to antiviral therapy, with better responses associated with genotypes 2 and 3 than with genotype 1. The prevalences of HCV genotype and subtypes in Central Europe are HCV-1a 19%, HCV-1b 46%, HCV-2a 4%, HCV-2b 3%, HCV-2c 6%, HCV-3a 18%, and HCV-4 4% [53–56]. Variations of HCV genotypes within smaller geographical regions seem to exist. In intravenous drug abusers, genotypes HCV-1a and HCV-3a account for the majority of infections. HCV genotypes 5 and 6 have been described in South Africa and Hong Kong, respectively. Further identiﬁcation of genotypes will certainly be obtained by sequencing HCV isolates from other populations [57]. Coinfection of various HCV genotypes as well as superinfections with a heterologous HCV genotype have been demonstrated, especially in hemophilic patients and after polytransfusion. HCV genotypes are remarkably discrepant in Japan and the Western countries. Subtypes HCV-1b and HCV-2a are most common in Japan, whereas genotype HCV-1a is diagnosed in less than 1–2% and also genotype 3 is rare in Japan [58]. Serotyping. Epitope mapping of NS4 protein reveals that HCV antibodies interact with two main antigens. Several genotypes provide high variabilities of these genome regions. Genotype-speciﬁc peptides

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concordant with the speciﬁed regions permit serological genotyping [59]. Serological HCV genotyping is rapid and cost-effective, but it fails to differentiate between subtypes, which in turn is less clinically relevant (Table 2). ANTI-NS4-SEROTYPING

(MUREX HCV SEROTYPING 1–6, ABBOTT This technique is based on microtiter plates covered with puriﬁed HCV antigen of the NS4 region. Each reaction mixture contains immobilized NS4 antigens of the genotypes HCV-1 to HCV-6. Those are incubated with soluble NS4 antigens of ﬁve of the six existing genotypes together with patient or control serum. Only antibodies against the genotype-speciﬁc immobilized antigen, for which a soluble antigen is not available, will be detected. As previously described, genotyping requires six reactions supplementary to the controls. The antigen–antibody complex is detected by means of a second, peroxidaselinked antibody and ﬁnal addition of a chromogenic substrate. In over 90% of the samples this system offers precise identiﬁcation of genotypes HCV-1, -2, and -3 as well as HCV-4, which occasionally is found in Germany. According to the manufacturer, HCV-5 and HCV-6 genotype characterization is also possible (Table 2).

DIAGNOSTICS).

Genotyping. Sequencing of HCV isolates represents the gold standard for HCV genotyping. Sequence analysis of the 50 noncoding region is insufﬁcient for subgenotyping and has to be complemented by additional evaluation of coding regions. Nonoverlapping evolutionary distances referring to isolates, subtypes, and genotypes could be validated for the nonstructural (NS)-5B region. Commercial typing techniques are available. Those assays use nucleotide sequence information of either the 50 noncoding region or core gene. Hepatitis C virus genotyping in the TruGene2 HCV 50 NC and NS5B (Visible Genetics) systems is based on sequencing of the 50 noncoding (NC) region. After reverse transcription of extracted HCV RNA, a PCR product is generated out of the 50 noncoding region by use of a genotype-independent primer pair. Primers labeled with different dyes amplify sense and antisense strands in one tube (CLIP2 sequencing). With the help of special software, sequences of both complementary strands can be proofread and sequence artifacts can be corrected. This sequence is ﬁnally compared to 50 noncoding sequences registered in databases (containing 50 noncoding sequences of known isolates) and related to one HCV genotype or subtype. As with other genotyping methods (e.g., Inno-LiPA2, Innogenetics), which are based exclusively on the 50 noncoding region, SEQUENCING.

Sufﬁcient þ/þþ None Low

Technique

Sensitivity Genotyping Subtyping Contamination risk

Labor-intensiveness, costs

Recombinant immunoblot serotyping assay (RIBA HCV 2.0 SIA)

RT-PCR, reverse hybridization, detection on a test strip Very good þþþ þþ Low (‘‘nested’’ PCR not necessary) Acceptable

50 -NC

Acceptable

RT-PCR, reverse hybridization, EIA Good þþþ þþþ High (‘‘nested’’ PCR)

Core

DNA enzyme immunoassay (GEN-ETI-K DEIA)

Genotyping Reverse hybridization assay (Inno LiPA 2.0)

Comparison of Commercially Available Geno- and Serotyping Assays

Antibodies against core and NS4 Recombinant immunoblot

Detection

TABLE 2

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differentiation between HCV subtypes (e.g., HCV-1a/HCV-1b or HCV2/HCV-2c) also presents a problem for sequencing. The most reliable method of subtyping hepatitis C virus requires sequencing of a coding region such as the NS5B gene (TruGene2 HCV NS5B, Visible Genetics). REVERSE HYBRIDIZATION ASSAY. In the reverse hybridization assay Inno LiPA II of Innogenetics after HCV RNA extraction and transcription, ampliﬁcation of cDNA for a fragment of 50 noncoding region (50 NCR) is carried out using biotinylated primers. The PCR product then hybridizes to oligonucleotides attached to a nitrocellulose membrane (reverse hybridization). Labeled PCR products obtained from 50 NCR will hybridize only to a probe that perfectly matches the sequence of the isolate, allowing exact discrimination at the subtype level. Such a high speciﬁcity can be obtained by using very stringent hybridization conditions. After an isothermal wash, detection is performed by an alkaline phosphatase-labeled streptavidin conjugate. Each nitrocellulose strip provides internal controls for color reaction and PCR ampliﬁcation [60]. This test allows discrimination of genotypes 1–6, and handling is quite easy. Due to the sequence of the 50 noncoding region, the differentiation speciﬁcity between subtypes HCV-1a and HCV-1b reaches 90%. The assay cannot distinguish between subtypes HCV-2a and HCV-2c (Table 2) [54]. DNA ENZYME IMMUNOASSAY. The test system. GEN-ETI-K DEIA HCV of Sorin Biomedica is based on PCR and DNA enzyme immunoassay. First HCV RNA is extracted and reverse transcribed, then a sequence of core region is ampliﬁed by means of ‘‘nested’’ PCR. The PCR product is hybridized by type-speciﬁc oligonucleotides that are attached to a membrane. Monoclonal antibodies against double-stranded DNA ﬁnally detect those cDNA–DNA hybrids [61]. Genetic information of the core region allows very high speciﬁcity of HCV genotypes and subtypes, including genotypes HCV-4, -5, and -6. Satisfactory ampliﬁcation of Central European isolates is achieved by using those primers despite their greater nucleotide sequence variability. The high risk of contamination represents the main disadvantage of ‘‘nested’’ PCR.

4.2.4

Molecular Virus Characterization

Analysis of Quasi-Species. The viral population in chronic HCV infection consists of a heterogeneous mixture of closely related virions known as quasi-species. Diversity of the quasi-species population has been found to be related to ‘‘natural’’ progression to liver disease irrespective of treatment. High quasi-species variability is driven by

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limited transcription accuracy and low ﬁdelity of the HCV RNA– dependent RNA polymerase, which cannot proofread. The number of heterogeneous genome ‘‘quasi-species’’ in vivo is attributable to a rapid turnover of hepatitis C virions and the host immune surveillance. Mutations evolving mainly in the hypervariable region of the amino terminal end of the E2 protein help the virus to evade host immunity response. These ‘‘escape’’ mechanisms presumably participate in the high chronicity rate of the HCV infection [62]. Cloning and sequencing of the hypervariable HCV genome region (position 1470–1550) represents the preferred technology to determine quasi-species in an infected individual. Multiple trials reveal a better therapeutic response rate in patients with less of the HCV quasi-species compared to those individuals infected by a greater number of HCV isolates. When patients are on antiviral therapy, certain isolates remain interferon-sensitive whereas others develop resistance. Conventional cloning and sequencing of quasi-species (isolates) is too labor-intensive for routine clinical diagnosis. Single-strand conformation polymorphism (SSCP) and gel shift analysis [heteroduplex gel shift analysis (HDA)] of hypervariable region PCR products have been found to be sensitive enough to detect minor populations of the virus. These techniques also allow discrimination between equal-sized DNA fragments due to their sequence-speciﬁc conformation. Though tremendous clinical and scientiﬁc interest is focused on these technologies of quasi-species analysis, they are not yet standard in clinical settings. Possible Interferon-Sensitivity-Determining Regions of NS5A and E2 Genes. Individuals infected with non-genotype 1 show higher response rates to interferon-a therapy than those infected with HCV genotype 1 [63]. Characteristic differences in the amino acid sequences between codons 2209 and 2248 [the so-called interferon-sensitivity-determining region (ISDR)] within the carboxy terminal end of the nonstructural (NS)5A protein were found in Japanese interferon-sensitive and resistant HCV-1b isolates. Interferon sensitivity is revealed in HCV-1b isolates providing four or more mutations in the deﬁned region as opposed to the interferon-resistant HCV-1b wild-type isolates (prototype sequence). Response to interferon-a in isolates showing one to three changes in the amino acid sequence varies. Mutation in codon 2218 predominates; histidine (prototype) in this position characterizes interferon-resistant isolates, whereas histidine is frequently replaced by arginine in interferon-sensitive isolates [50,64]. Variability within the region of the gene for nonstructural protein (NS5A) appears to have particular clinical signiﬁcance in determining

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the sensitivity to interferon, as shown in isolates of Japanese subtype 1b. However, European and American isolates of HCV do not share this property to the same degree. Evidence of the described mutations in HCV isolates in Western countries is rare compared to the Japanese population. The correlation between the number of mutations in the ISDR and the therapeutic response in HCV-1b was also reported in recent European studies. However, only one to three mutations within the ISDR could be detected in the majority of the sustained responders [65]. Similar ﬁndings resulted for HCV subtypes HCV-1a or HCV-2 [66,67], whereas in HCV-3a-infected individuals such a correlation could not be identiﬁed [68,69]. In vitro interaction of NS5A protein and IFN-a-stimulated doublestranded RNA–dependent protein kinase (PRK) has been demonstrated. The PKR binding domain includes the ISDR. Mutations within the PKR binding region impairs NS5A–PKR interaction in vitro [70]. Inconsistent results have been obtained in terms of cellular inhibition of PKR by in vitro expression of HCV NS5A [71]. Laser confocal microscopy was not able to verify the expected colocalization of NS5A protein and PKR [72]. Consequently, NS5A–PKR interaction as a potential strategy of HCV resistance to interferon-a has not been deﬁnitively clariﬁed. Furthermore, an interaction between PKR and the envelope protein E2 of HCV-1a/1b isolates has recently been reported. A PKR/elF2a phosporylation homology domain (PePHD, codons 659–670) containing 12 amino acids was described. HCV genotypes 2 and 3 lost the PKR linkage, implying that PePHD of HCV-1a/1b isolates may cause relative interferon resistance compared to HCV-2 and -3 isolates [73]. Clinical correlation of mutations within PePHD could not be conﬁrmed for either HCV-1b or HCV-3a isolates [68]. 4.3

Histological Diagnosis

Chronic hepatitis C is associated with inﬂammatory cell inﬁltration of the portal tracts and parenchyma of the liver, which is usually accompanied by focal liver cell necrosis. As the disease progresses, ﬁbrosis develops as a result of inﬂammation and cell death. The levels of matrix metalloproteinases, which exert ﬁbrinolytic effects on the extracellular matrix, are decreased with the progression of chronic liver disease [74]. Plasma levels of transforming growth factor-b1 (TGF-beta 1), a cytokine that stimulates the production and inhibits the degradation of extracellular matrix proteins, are elevated in patients with mild and moderate chronic hepatitis [75]. Severe ﬁbrosis leads to cirrhosis, deﬁned as a state

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of diffuse ﬁbrosis in which ﬁbrous septae separate clusters of liver cells into nodules [76]. The progression to HCV-related cirrhosis is accelerated in patients with initial biopsies showing high-grade inﬂammation and in those with an advanced histological stage (presence of septal ﬁbrosis and parenchymal nodularity) [77]. The use of liver biopsy has broadened to serve multiple purposes: (1) conﬁrmation of clinical diagnosis, (2) assessment of severity of necroinﬂammation and ﬁbrosis, (3) evaluation of possible concomitant disease processes, and (4) assessment of therapeutic intervention. After diagnosis of chronic viral hepatitis has been veriﬁed, a reproducible clinical evaluation of the inﬂammatory activity and histological ﬁbrotic stage is required. The ﬁrst histological classiﬁcation codiﬁed the terminology chronic persistent hepatitis (CPH) and chronic active (aggressive) hepatitis (CAH) and the supplementary chronic lobular hepatitis (CLH). This system has been abandoned owing to heterogeneous parenchymal ﬁndings and its negligible signiﬁcance for histological diagnosis. A classiﬁcation that considers etiology and provides an exact description of inﬂammatory activity within the portal tracts, septa, and lobules as well as the extent of ﬁbrotic remodeling is reasonable. Systems for grading and staging the lesions of chronic viral hepatitis incorporate the view that necroinﬂammation is a measure not only of severity but also of ongoing disease activity and the parameter most potentially responsive to therapy. This is referred to as the ‘‘grade.’’ The lesions of ﬁbrosis and parenchymal or vascular remodeling are referred to as the ‘‘stage’’ and indicate long-term disease progression. The grade may ﬂuctuate with disease activity or therapeutic intervention; the stage is considered to be more constant. Various scoring systems have been proposed (Table 3) [78–81] to characterize the activity and stage of chronic hepatitis with the primary goal of objectivity, practicality, and reproducibility. This also allows statistical analyses of histological ﬁndings. 4.3.1

Knodell Score

The Knodell score represents a classical scoring system to rank the activity of chronic hepatitis [histology activity index (HAI)]. The Knodell score incorporates four categories: I. Piecemeal necrosis and bridging necrosis (within the portal tracts or between portal tracts and central veins); seven levels of increasing severity (A–G) II. Intralobular degeneration and focal necrosis; four levels of increasing severity (A–D)

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TABLE 3

Scoring Systems for the Assessment of Fibrosis

Knodell et al. staging A. No ﬁbrosis

B. Fibrosis portal expansion

C. Bridging ﬁbrosis (P–P or P–C linkage)

D. Cirrhosis

Ishak et al. staging

METAVIR staging

0. No ﬁbrosis 1. Fibrosis expansion of some portal areas, with or without short ﬁbrous septa 2. Fibrous expansion of most portal areas, with or without short ﬁbrous septa 3. Fibrous expansion of most portal areas, with occasional P–P bridging 4. Fibrous expansion of portal areas with marked bridging (P–P as well as P–C) 5. Marked bridging (P–P and/or P–C) with occasional nodules (incomplete cirrhosis) 6. Cirrhosis, probable or deﬁnite

F0. No ﬁbrosis F1. Portal ﬁbrosis without septa (incomplete septa ¼ transition) F2. Portal ﬁbrosis with rare septa

F3. Numerous septa without cirrhosis (bridging ﬁbrosis)

F4. Cirrhosis

P–C, portal-to-central; P–P, portal-to-portal.

III. Portal inﬂammation; four levels of increasing severity (A–D) IV. Extent of septal distortion (ﬁbrosis); four levels of increasing severity (A–D) Each category is divided into several levels, which measure histological activity when they are added together. These values have a discontinuous distribution (e.g., for piecemeal and bridging necrosis, 0, 1, 3, 4, 5, 6, or 10). The summation of values from each category results in a mixture of activity and stage, which, as the main disadvantage of the Knodell score demonstrated the need for an updated terminology separating grading and staging. Based on the Knodell score, new scores such as the Ishak score have been developed.

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Ishak Score

The Ishak score separates the grading and staging of visible histological changes in the liver parenchyma. Grading assesses the pattern and distribution of liver cell damage, including A. Periportal and periseptal interface hepatitis (piecemeal necrosis); ﬁve levels of increasing severity (0–4) B. Conﬂuent periportal necrosis with or without bridging necrosis, differentiating porto-portal and porto-central linkage; seven levels of increasing severity (0–6) C. Focal (spotty) lytic necrosis, apoptosis, focal inﬂammation; ﬁve levels of increasing severity (0–4) D. Portal inﬂammation; ﬁve levels of increasing severity (0–4) Fibrosis, architectural distortion, and cirrhosis are estimated by staging and are divided into seven categories (0–6). The degree of activity (grading) ranges between 0 and 18, and the stage (staging) measures, between 0 and 6. 4.3.3

METAVIR Score

The French METAVIR scheme, which is comprehensive but complex, has been established as a competitive system to evaluate histological activity of chronic hepatitis C infection. Activity and ﬁbrosis are equally differentiated and described. Activity is estimated as follows: Piecemeal necrosis; four levels of increasing severity (0–3) Intralobular necrosis; three levels of increasing severity (0–2) Inﬂammation within the portal tracts; four levels of increasing severity (0–3) Bridging necrosis; two options (yes/no) Results are ‘‘translated’’ by means of an algorithm into four degrees of activity: A0. A1. A2. A3.

None Mild Moderate Severe activity

Five stages exist to classify ﬁbrosis: F0. F1. F2. F3.

No ﬁbrosis Stellate enlargement of portal tract without septa formation Enlargement of portal tract with rare septa formation Numerous septa without cirrhosis

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F4.

Cirrhosis

The METAVIR system offers the great advantage of easy handling and thus a high degree of reproducibility [80]. 4.4

Current Recommendation for a Diagnostic Algorithm

The method of choice to conﬁrm suspicion of HCV infection is anti-HCV antibody testing. There is no need to verify positive antibodies by supplementary tests. Suspected acute hepatitis C should be examined immediately for HCV RNA, by means of RT-PCR or TMA, for example. Anti-HCV IgG generally arises 4–6 weeks after infection; anti-HCV IgM is not capable of narrowing this diagnostic window. Before starting interferon-a-based antiviral therapy, HCV RNA should be detected by a molecular test. Other viral infections (particularly HBV and HIV), hereditary liver diseases, autoimmune hepatitis, and toxic liver damage should be considered for the differential diagnosis. Ultrasound imaging of the liver may indicate existing liver cirrhosis and its complications (portal hypertension, ascites). Furthermore, ultrasound represents a sensitive method to exclude hepatocellular carcinoma. The extent of inﬂammation (‘‘grading’’) and architectural distortion (‘‘staging’’) in the liver can be revealed only by histology. Liver enzymes and biochemical parameters do not correlate well with histological ﬁndings (Fig. 7). Furthermore, genotyping together with HCV RNA quantiﬁcation has to be considered as a pretherapeutic diagnostic standard for initiation of treatment. Available HCV RNA quantiﬁcation assays (qRT-PCR, bDNA) were recently standardized. Following WHO standards, test results are given in international units per milliliter instead of copies or genome equivalents per milliliter. Using the HCV Amplicor monitor, version 2.0 (Roche Molecular Systems), 2 6 106 copies/mL is considered equivalent to 800,000 IU/ mL, whereas in the VersantTM HCV RNA assay, version 3.0 (Bayer Diagnostics), 2 6 106 genome equivalents is considered equivalent to 400,000 IU/mL. Immediate centrifugation of the sample after a blood draw and secure transport are essential for precise HCV quantiﬁcation. Primary objectives of treatment are normalization of liver enzymes and undetectable HCV RNA in serum (and liver tissue) 6 months after the end of treatment (sustained virological response). Sustained virological clearance is deﬁnitely related to histological improvement.

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Diagnostic work-up algorithm in hepatitis C.

TREATMENT OF HEPATITIS C Indication and Objectives of Antiviral Therapy in Chronic Hepatitis C

The primary goal of antiviral therapy in patients with chronic hepatitis C is clearance of HCV-speciﬁc RNA 6 months after the end of treatment by a sensitive molecular test [polymerase chain reaction (PCR) after reverse transcription of HCV RNA or transcription-mediated ampliﬁcation (TMA)]. Long-term follow-up conﬁrms that a negative HCV RNA test 6 months after the end of treatment correlates in over 95% of the patients with HCV RNA clearance for years. Undetectable HCV RNA is accompanied by a signiﬁcant reduction of inﬂammation and even a decrease in liver ﬁbrosis. Trial analysis supports the hypothesis that patients with apparent virus eradication have no progress to liver

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cirrhosis and have a signiﬁcantly reduced risk of HCC. Patients with normalized transaminases but persistent HCV RNA detection (biochemical response) after the end of treatment also show histological improvement. Liver enzymes, however, often elevate again during the long-term follow-up period. 5.1.1

Treatment of Acute Hepatitis C

Chronic infection often develops in patients who are acutely infected with the hepatitis C virus (HCV). To determine whether treatment during the acute phase could prevent the development of chronic infection, several trials have evaluated the efﬁcacy of interferon therapy for acute infection [82–93], and all but one reported a beneﬁcial effect of treatment. A large prospective trial in a representative group of patients with acute hepatitis C virus infection treated with standard interferon-a has recently been published. Forty-four patients suffering from acute hepatitis C received 5 MIU of interferon a-2b a day for 4 weeks and then three times per week for another 20 weeks. This monotherapy prevented the development of chronic hepatitis C infection in 43 of 44 individuals [94]. It is likely that about 30–50% of the patients would have had self-limited disease regardless of whether they received interferon. So far, there are no means to identify such patients. Because the current treatment eliminates the virus in only about half of cases of chronic hepatitis C [95], early treatment of acute hepatitis C infection with interferon-a (5 MIU per day for the ﬁrst 4 weeks, followed by a dose of 5 MIU three times a week for another 20 weeks) is recommended. Other treatment options, such as pegylated interferon-a, are currently being studied [96]. 5.1.2

Standard Interferon-a Monotherapy of Chronic Hepatitis C

Interferon-a monotherapy offers sustained virological response in less than 20% of patients. Standard treatment is subcutaneous application of 3 MIU interferon-a three times per week. In contrast to patients with chronic hepatitis B, patients with chronic hepatitis C rarely show an elevation of liver enzymes (‘‘ﬂare-up’’) during interferon-based therapy. Treatment for chronic hepatitis C should be continued for 48 weeks in patients with undetectable HCV RNA after 12–24 weeks of treatment. End-of-treatment virological response rates to interferon therapy range around 50%, and in more than 50% of these patients cessation of treatment leads to a virological relapse with recurrent elevation of liver enzymes. A shorter treatment period (less than 48 weeks) yields greater relapse rates after discontinuation of interferon-a. Response rates are

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dependent on HCV genotype, with only 5–10% for HCV genotype 1 infected patients achieving a sustained response rate, whereas sustained virological response rates are 30–40% in patients infected with HCV-2 or HCV-3. A less than 2 to 3 log drop of HCV RNA measured by a quantitative assay within 4 weeks after initiation of therapy predicts nonresponse in patients undergoing interferon-a monotherapy. Positive baseline predictors for virological response to therapy are short duration 6 of disease, low viremia (<2 6 106 copies/mL ¼ *800,000 IU/mL), infection with genotype HCV-2 or HCV-3, and absence of cirrhosis. Recent analyses demonstrated inhibition of ﬁbrosis progression and a decreased incidence of hepatocellular carcinomas in patients with chronic hepatitis C after treatment with interferon irrespective of HCV RNA clearance. Characteristic side effects of interferon-a are ﬂu-like symptoms, e.g., headache and myalgia, lumbalgia, and fever, which can be ameliorated by paracetamol (Table 4). Administration of aspirin is not recommended. Premature discontinuation of interferon therapy should be considered in patients with a platelet count below 50 6 109/L, development of a psychiatric disorder, newly diagnosed retinopathy, and severe bacterial infection. Sufﬁcient experiences concerning the teratogenic potential of interferon-a have not yet been accumulated. Safe contraception has to be ensured during the treatment period. If conception still occurs,

TABLE 4

Side Effects of Interferon-a and Ribavirin

Side effects of interferon-a Flu-like symptoms (headache, myalgia, arthralgia, fever, etc.) Gastrointestinal symptoms (nausea, loss of appetite, weight loss) Leukopenia, thrombocytopenia Psychiatric side effects (depression, fatigue, impaired concentration, enhanced irritability) Alopecia Tinnitus, hearing loss Loss of vision and retinopathy Previous drug abusers often experience interferon side effects as symptoms similar to ‘‘deprivation’’ from drugs (risk of relapse) Side effects of ribavirin Symptoms of the respiratory tract (dyspnea, cough, pharyngitis) Symptoms of the skin (exanthema and pruritus) Decline in hemoglobin with increase of indirect bilirubin and uric acid due to osmotic hemolysis

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interruption should be discussed. Contraindications of interferon monotherapy are as follows: neutropenia (<1.0 6 109/L), thrombopenia (<50 6 109/L), psychiatric disorder (depression, psychosis), severe extrahepatic (particularly cardiopulmonary) disease, decompensated liver cirrhosis, autoimmune-type disease, pregnancy, and nursing. 5.1.3

Therapy of Chronic Hepatitis C with Other Interferons

Consensus Interferon. Consensus interferon (interferon alfacon-1) is a non-naturally occurring, synthetic type 1 interferon-a (IFN-a) that contains in each amino acid position the most commonly observed amino acid from 13 IFN-a nonallelic subtypes. Interferon-alfacon-1 is 89% homologous to interferon a-2b. Dosage of interferon-alfcon-1 is measured in mass units (micrograms) instead of biological units (MIU). Previous data seem to indicate that interferon alfacon-1 is a more potent inducer of natural killer (NK) cell activity than the tested interferon-a subtypes and shows a stronger enhancement of antiviral and antiproliferative effects, presumably due to a higher afﬁnity of consensus interferons to type 1 interferon receptors. Results from a trial showed that interferon alfacon-1 9 mg three times a week in patients with chronic hepatitis C and high viral load achieved superior virological and biochemical response rates compared to a standard therapy regimen including 3 MIU interferon a-2b. Some studies have suggested that the therapeutic effect of consensus interferon is better than that of standard interferon, particularly among relapsers (recurrence of HCV RNA after the end of treatment) or primary nonresponders (patients who did not show substantial reduction in viremia during previous therapy) [97]. A non-blinded multicenter study investigated the response to interferon alfacon-1 retreatment in 337 patients with chronic hepatitis C who were nonresponders or relapsers to previous interferon therapy [98] (3 or 9 mg interferon alfacon-1 and 3 MIU IFN a-2b three times a week). The patients were randomized to receive interferon alfacon-1 15 mg three times a week for a period of 24 (n ¼ 167) or 48 (n ¼ 170) weeks, and their response to retreatment was recorded after an additional 24-week observation period [99]. Sustained virological response rates were signiﬁcantly higher in the 48-week group including all patients (27% vs. 12%) or only prior relapsers to IFN treatment (58% vs. 28%). Also, in the 48-week treatment group 13% and 58% of nonresponders and relapsers, respectively, showed a sustained virological response (week 72). The efﬁcacy of high-dose induction therapy with interferon alfacon1 was investigated in another trial for the re-treatment of previous relapsers and nonresponders to combined IFN plus ribavirin treatment

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(n ¼ 511). Patients received interferon alfacon-1 15 mg/day for 8 weeks and then were randomized to an interferon alfacon-1 9 mg/day or 15 mg/ day three times a week treatment group for a total of 48 weeks (nonresponders at week 24 were excluded from the study). An intentionto-treat analysis at week 24 seems to indicate that interferon alfacon-1 9 mg/day may be more effective than interferon alfacon-1 15 mg/day three times a week (23.5% vs. 15.6%, respectively) in the treatment of nonresponders to IFN-a plus ribavirin treatment [100,101]. Consensus interferon (9 mg three times a week) is approved in several European countries and the United States. Data from large studies indicate that interferon alfacon-1 has a side-effect proﬁle similar to that of interferon a-2a or interferon a-2b [97–99,102]. Natural Interferon. Natural interferon is extracted from virusstimulated human donor leukocytes and consists of multiple naturally occurring interferon-a subtypes. The constitution and ratio of interferon subtypes of natural interferon determine the main difference to lymphoblastoid interferon. Lymphoblastoid Interferon. Lymphoblastoid interferon (interferonan1) is produced from a human lymphoid cell line and consists of nine IFN-a subtypes of which at least two are glycosylated. This differs from the recombinant IFNs (interferon a-2a and interferon a-2b), which are single unglycosylated proteins formed of individual IFN-a genes (subtype 2) expressed in E. coli. Trials investigating the efﬁcacy of interferon-an1 monotherapy in patients with chronic hepatitis C compared with interferon a-2a or interferon a-2b showed no signiﬁcant improvement in terms of virological, biochemical, or histological response rates. One apparent difference, however, was a possibly lower relapse rate after treatment with interferon-an1. Lymphoblastoid interferon is available for clinical purposes in only a few European countries [103,104]. High-Dose, Induction Dose, and Dose Escalation Strategies for Hepatitis C Monotherapy. Current dose recommendations for standard interferon therapy are 3 MIU interferon a-2a/2b or, alternatively, 9 mg interferon alfacon-1 three times a week [105]. Each interferon treatment exceeding 3 MIU three times a week or a total dose of 9 MIU IFN/week has to be considered as high-dose strategy, which includes both those regimens that use higher doses at the beginning of treatment and those that use increasing interferon-a doses (escalation). High-dose treatment (induction) can be achieved either by shortening the dosage intervals (e.g., daily dose) or by single-dose incrementation. In general, higher

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dosages of IFN seem to increase virological and biochemical response rates compared to a standard therapy regimen (3 MIU three times per week). Because higher relapse rates occur in patients assigned to receive higher doses of interferon, signiﬁcant beneﬁcial effects (sustained virological clearance) could not be achieved. An increase in side effects led to the conclusion that high-dose strategy (including induction and escalation regiment) cannot be generally recommended. Pegylated Interferon (Peginterferon). Pegylated forms of interferon-a (PEG-IFN) have been developed in which an attachment with polyethylene glycol (PEG) delays protein degradation and clearance and reduces immunogenicity. The resulting prolonged plasma half-life increases exposure to the drug and therefore may enhance efﬁcacy and allow once-weekly dosing (as opposed to daily dosing or standard threetimes-weekly dosing of nonmodiﬁed standard interferons). Less frequent dosing may also improve patient compliance and quality of life. Covalent attachment of a 40 kDa branched-chain polyethylene glycol moiety to interferon a-2a produces peginterferon a-2a. Conjugation of a 12 kDa linear chain PEG polymer to interferon a-2b forms pegylated interferon a-2b. The following pivotal trials investigating pegylated interferon-a monotherapy have been conducted. One hundred ﬁfty-nine patients with chronic hepatitis C participated in a randomized ascending-dose (45 or 90, 180, 270 mg) study comparing PEG (40 kDa) IFN a-2a administered once weekly with 3 MIU IFN a-2a administered three times weekly for 48 weeks to determine the most appropriate PEG (40 kDa) IFN a-2a dose for subsequent clinical trials. Efﬁcacy was assessed by measuring HCV RNA following a 24week treatment-free period. Sustained virological responses for PEG (40 kDa) IFN a-2a once weekly were 10% (45 mg), 30% (90 mg), 36% (180 mg), and 29% (270 mg) compared with 3% for the three times weekly 3 MIU IFN a-2a regimen. In conclusion, the 180 mg PEG (40 kDa) IFN a-2a dose appeared to be the optimal dose based on sustained virological response and its associated side-effect proﬁle [106]. A phase III randomized multicenter trial assessed 531 untreated patients with chronic hepatitis C receiving either 180 mg PEG (40 kDa) IFN a-2a once per week for 48 weeks (267 patients) or 6 MIU of IFN a-2a three times per week for 12 weeks followed by 3 MIU three times per week for 36 weeks (total treatment duration 48 weeks). Thirty-nine percent of the patients in the peginterferon a-2a group, compared with 19% in the standard interferon group, achieved a sustained virological response. Of the HCV-1-infected individuals treated with pegylated or

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standard interferon, 28% and 7%, respectively, had a sustained virological response [107] (Fig. 8). An additional study was conducted to examine the efﬁcacy and safety of PEG (40 kDa) IFN a-2a (90 mg or 180 mg once weekly) compared with IFN a-2a (3 MIU three times weekly) in 271 patients with HCVrelated cirrhosis or bridging ﬁbrosis. The sustained virological response associated with the dose of 180 mg PEG (40 kDa) IFN a-2a was signiﬁcantly more effective than standard interferon a-2a administered three times weekly (30% vs. 8%) [108]. For the clinical development of pegylated interferon a-2b, an international multicenter phase II/III trial compared various doses of

FIGURE 8 Virological response rates in patients with chronic hepatitis C treated with pegylated interferon a-2a. Patients with chronic hepatitis C (n ¼ 531) were randomly assigned to receive either 6 million units of interferon a-2a subcutaneously three times a week for 12 weeks followed by 3 million units three times a week for 36 weeks (n ¼ 264) or 180 mg peginterferon a-2a subcutaneously once a week for 48 weeks (n ¼ 267). All patients were assessed at the end of treatment (week 48) and at the end of the follow-up period (week 72) for virological response, deﬁned as an undetectable level of hepatitis C virus RNA (<100 copies/mL). (From Ref. 107.)

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pegylated interferon a-2b to standard interferon a-2b for the initial treatment of compensated chronic hepatitis C. One thousand two hundred nineteen (1219) subjects were assigned to either the standard three-times-weekly interferon a-2b (3 MIU) scheme or once-weekly peginterferon a-2b (0.5, 1.0, or 1.5 mg/kg) for 48 weeks. The rate of sustained virological response was approximately twofold higher with 1.0 mg/kg peginterferon a-2b (25% vs. 12%) and 1.5 mg/kg (23% vs. 12%), compared with interferon a-2b. In conclusion, peginterferon a-2b achieved similar (0.5 mg/kg) or improved (1.0 or 1.5 mg/kg) clinical efﬁcacy compared with interferon a-2b while preserving its safety proﬁle (Fig. 9). Furthermore, all three peginterferon a-2b doses decreased liver

FIGURE 9 Virological response rates in patients with chronic hepatitis C treated with pegylated interferon a-2b. Patients with chronic hepatitis C (n ¼ 1219) were randomly assigned to receive either 3 million units of interferon a-2b (n ¼ 303) subcutaneously three times a week for 48 weeks or peginterferon a-2b subcutaneously [0.5 mg/kg (n ¼ 315), 1.0 mg/kg (n ¼ 297), or 1.5 mg/kg (n ¼ 304)] once a week. All patients were assessed at the end of treatment (week 48) and at the end of the follow-up period (week 72) for virological response, deﬁned as an undetectable level of hepatitis C virus RNA (<100 copies/mL serum). (From Ref. 109.)

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inﬂammation to a greater extent than did interferon a-2b, particularly in patients with sustained responses [109]. In general, both peginterferons are well tolerated. Laboratory abnormalities and adverse events associated with their use are typical of those associated with unmodiﬁed interferon. Compared with standard interferons, psychiatric side effects are no more common with pegylated interferons. Also, the rates of neutropenia, anemia, and thrombocytopenia are comparable with the two drugs. 5.1.4

Therapy of Chronic Hepatitis C with Ribavirin

Ribavirin (1-b-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) (Fig. 10) is rapidly absorbed following oral administration. Ribavirin has virostatic activity against several DNA and RNA viruses after intracellular phosphorylation [110]. Described pharmacological mechanisms of action are (1) inhibition of inosine monophosphate dehydrogenase with consecutive intracellular deﬁciency of guanosine nucleotides; (2) inhibition

FIGURE 10

Chemical structure of ribavirin.

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of viral RNA–dependent RNA polymerase; (3) inhibition of capping of mRNA, which is important for protein synthesis; and (4) immunomodulation with a Th2/Th1 shift [111–113]. Immunomodulatory properties and inhibition of the hepatitis C virus polymerase may be critical in the interferon-based treatment of patients with chronic hepatitis C infection. In three controlled trials the antiviral activity of ribavirin monotherapy with daily application of 1000–1200 mg was investigated in patients with chronic hepatitis C; however, no signiﬁcant antiviral activity was observed in vivo. Administration of ribavirin led to a temporary normalization of liver enzymes (for the duration of the treatment period) in approximately one-third of the patients, which rarely persisted after treatment discontinuation. 5.1.5

Combination Therapy of Interferon-a and Ribavirin in Patients with Chronic Hepatitis C

A beneﬁcial effect of the combination of interferon-a and ribavirin was ﬁrst shown in a pilot study in 1994 [114]. Since then numerous controlled trials have conﬁrmed the superiority of a combination therapy with interferon-a plus ribavirin compared with IFN monotherapy [115–119]. Combination Therapy of Previously Untreated Patients. The beneﬁt of interferon-a–ribavirin combination therapy compared with interferon-a monotherapy in previously untreated patients with chronic hepatitis C virus infection has been conﬁrmed in several controlled trials [115,116,118,120,121]. In two pivotal studies (n ¼ 1744 treated patients), only 6% of the patients receiving an interferon-a monotherapy for a 24week period compared with 16% of those receiving therapy for a 48week period obtained negative HCV RNA test results at 24 weeks of follow-up (sustained virological response) [118]. Corresponding virological response rates among patients treated for 24 weeks vs. 48 weeks with combination therapy (3 MIU interferon-a three times weekly plus ribavirin 1000–1200 mg per day) were 33% and 41%, respectively. Differences were highly signiﬁcant. Dropout rates in the 24-week treatment period did not exceed 10% for either treatment regimen. Interferon-a monotherapy for 48 weeks led to a discontinuation rate of 13%. During combination therapy up to 20% of the patients withdrew before the end of treatment (for safety proﬁle, see below). In patients completing a 48-week combination (treated per protocol population) treatment, apparent virus eradication could be obtained in more than 50% of patients. The sustained virological response rates are summarized in Figure 11 according to HCV genotype and baseline viremia. Differences in the virological outcome between subtype HCV-1a- and

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FIGURE 11 Virological response rates in patients with chronic hepatitis C treated with standard interferon-a with and without ribavirin. Patients with chronic hepatitis C were randomly assigned to receive either 3 million units of interferon a-2b alone (n ¼ 278) subcutaneously three times a week for 48 weeks or 3 million units of interferon a-2b subcutaneously three times a week plus ribavirin (1000–1200 mg/day) for 24 (n ¼ 277) or 48 weeks (n ¼ 277). All patients were assessed at the end of a 24-week follow-up period for sustained virological response, deﬁned as an undetectable level of hepatitis C virus RNA (<100 copies/mL serum). Logistic regression analysis showed that in addition to the treatment regimen (p < 0.001), the following baseline parameters were independent predictors of sustained virological response: genotype 2 or 3 (p < 0.001), low baseline viral load (p < 0.001), younger age (p < 0.005), low ﬁbrosis stage (p ¼ 0.01), and female gender (p ¼ 0.04). (From Ref. 118.)

HCV-1b-infected patients have not been described. More than 95% of the patients with undetectable HCV RNA at the end of follow-up also had normalization of transaminases. Vice versa, HCV RNA was still detectable in approximately 20% of the patients with normalized liver enzymes at the end of the 24-week follow-up period [116,118]. Combination Therapy of Pegylated Interferon-a Plus Ribavirin. Data from the following trials show that combination therapy of pegylated interferon-a plus ribavirin improves sustained virological response rates compared with standard interferon-a and ribavirin [95,107–109,122–125].

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A multicenter phase III clinical trial (n ¼ 1121) showed a signiﬁcantly higher sustained viral response rate in patients with chronic hepatitis C treated with peginterferon a-2a (180 mg once weekly) in combination with ribavirin (1000–1200 mg/day) (56%) than among those treated with interferon a-2b (3 6 3 MIU/week) plus ribavirin (1000– 1200 mg/day) (44%). Those patients who received peginterferon a-2a alone achieved a 29% sustained virological response rate [95] (Fig. 12). Among patients with HCV genotype 1 infection (65%, n ¼ 298), 46% of

FIGURE 12 Virological response rates in patients with chronic hepatitis C treated with peginterferon a-2a with or without ribavirin or standard interferon a-2b plus ribavirin. Patients with chronic hepatitis C (n ¼ 1121) were randomly assigned to receive either 3 million units of interferon a-2b subcutaneously three times a week plus ribavirin (1000–1200 mg/day) (n ¼ 444), peginterferon a-2a alone 180 mg once weekly subcutaneously for 48 weeks (n ¼ 224), or peginterferon a-2a 180 mg once weekly subcutaneously in combination with ribavirin (1000–1200 mg/day) (n ¼ 453). All patients were assessed at the end of a 24-week follow-up period for sustained virological response, deﬁned as an undetectable level of hepatitis C virus RNA (<50 IU/ mL serum). (From Ref. 95.)

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those treated with peginterferon a-2a (180 mg once weekly) in combination with ribavirin (1000–1200 mg/day) attained a sustained virological response, compared to 36% of those treated with the standard care (n ¼ 285). In a different study, 1530 patients with chronic hepatitis C were assigned to interferon a-2b (3 MIU subcutaneously three times per week) plus ribavirin 1000–1200 mg/day orally, pegylated interferon a-2b 1.5 mg/kg each week plus 800 mg/day ribavirin, or pegylated interferon a-2b 1.5 mg/kg per week for 4 weeks and then 0.5 mg/kg per week plus ribavirin 1000–1200 mg/day for 44 weeks. The sustained virological response rate was signiﬁcantly higher in the higher-dose peginterferon group (54%) than in the lower-dose peginterferon (47%) or standard interferon (47%) group (Fig. 13). HCV genotype 1–infected individuals with a baseline viremia of less than 26106 copies/mL (*800,000 IU/mL) who received the standard therapy regimen (363MIU interferon-a plus ribavirin) had a sustained virological response rate of 45%, compared with 73% of those who were treated with pegylated interferon a-2b (1.5 mg/kg PEG interferon a-2b) plus ribavirin (800 mg/day). In patients with a baseline viremia of more than 26106 copies/mL who were treated with standard or pegylated interferon a-2b plus ribavirin, sustained virological response rates were 30% and 29%, respectively. The rates for patients with genotype 2 and 3 infections were about 80% in all treatment groups [123]. Logistic regression analyses were used to characterize the relation between sustained virological response rates and ribavirin doses. Sustained virological response were higher when the dose of ribavirin was greater than 10.6 mg/kg body weight. However, body weight itself represents an independent predictor of virological response. Prospective studies are required to analyze the therapeutic outcome by using different doses of ribavirin. Currently, the following ribavirin dosing schedules are recommended: patients <65 kg should receive 800 mg; 65– 85 kg, 1000 mg; and >85 kg, 1200 mg ribavirin daily. Positive baseline predictors for virological response to combination therapy are similar to those for interferon monotherapy: short duration of disease, infection with genotype HCV-2 or HCV-3, low viremia 6 ð< 26106 copies= mL ¼ 800,000 IU= mLÞ, absence of cirrhosis, young age (440 years), and female gender. Detection of HCV RNA 24 weeks after initiation of treatment has a high negative predictive value, and discontinuation of treatment is recommended. Similarly, combination treatment can be discontinued in patients with a decline of HCV RNA of less than 2 log at week 12 of treatment (negative predictive value 97– 98%).

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FIGURE 13 Virological response rates in patients with chronic hepatitis C treated with peginterferon a-2b or standard interferon a-2b in combination with ribavirin. Patients with chronic hepatitis C (n ¼ 1530) were randomly assigned to receive either 3 million units of interferon a-2b subcutaneously three times a week plus ribavirin (1000–1200 mg/day) for 48 weeks (n ¼ 505); peginterferon a-2b 1.5 mg/kg once weekly subcutaneously for 4 weeks, then peginterferon a-2b 0.5 mg/kg in combination with ribavirin (1000–1200 mg/ day) (n ¼ 514); or peginterferon a-2b 1.5 mg/kg in combination with ribavirin (800 mg/day) once weekly subcutaneously (n ¼ 511). All patients were assessed at the end of a 24-week follow-up period for sustained virological response, deﬁned as an undetectable level of hepatitis C virus RNA (<100 copies/mL serum). (From Ref. 123.)

Combination Therapy in Previously Treated Patients Who Initially Responded to Interferon-a But Relapsed After Treatment Discontinuation (‘‘Relapse’’). Patients undergoing antiviral therapy may achieve an endof-treatment virological response but subsequently relapse. An international randomized, placebo-controlled multicenter study

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assessed the therapeutic efﬁcacy of patients with relapse following interferon-a monotherapy. For re-treatment, patients were assigned to receive either a combination of interferon-a (3 6 3 MIU/week) plus ribavirin (1000–1200 mg daily) for 24 weeks or interferon-a alone (3 6 3 MIU/week) for 24 weeks [126]. Groups were stratiﬁed according to HCV genotype, baseline viremia, and existing liver cirrhosis. HCV RNA at the end of treatment (after 24 weeks of therapy) was undetectable by RT-PCR in 141 out of 173 patients with combination therapy (82%) compared with 80 patients out of 172 who received monotherapy (47%). A sustained virological response (after 24 weeks of follow-up) was reported in 84/173 patients (49%) with combination therapy and in 8/172 patients (5%) who were treated with interferon-a alone. Data on the re-treatment of relapse patients with pegylated interferon and ribavirin are not yet available. Furthermore, there are no recommendations for re-treatment of patients who relapsed after primary treatment with (peg)interferon plus ribavirin. Combination Therapy in Patients Who Did Not Respond to Interferon-a (‘‘Nonresponders’’). A meta-analysis of randomized trials revealed that combination therapy is more effective than re-treatment with interferon alone in patients with chronic hepatitis C who initially failed IFN monotherapy [127]. The efﬁcacy of the combination of IFN with ribavirin in re-treating patients with chronic hepatitis C not responding to interferon monotherapy is variable. Adopting different treatment protocols, a sustained virological response of 15% of primary nonresponders has been reported. The analysis of individual patient data of six controlled trials showed that the re-treatment response rate was independent of the presence of cirrhosis but dependent on the HCV genotype; patients with genotype 1 responded less frequently than those with genotype 2 or 3 infection (6% vs. 21%). A randomized four-arm multicenter study was designed to determine whether a higher dosage of interferon a-2b associated with ribavirin and/or a prolonged time of administration may improve therapeutic efﬁcacy in the treatment of patients with chronic hepatitis C not responding to interferon-a alone. Group 1 received 3 MIU IFN a-2b three times a week plus ribavirin 1000 mg/day for 12 months; group 2 received 5 MIU IFN a-2b three times a week plus ribavirin for 12 months; group 3 was assigned to 3 MIU IFN a-2b three times a week plus ribavirin for 6 months; and patients in group 4 were given 5 MIU IFN a2b three times a week plus ribavirin for 6 months. HCV RNA clearance at the end of the 6-month follow-up was achieved in 15% of group 1, 23% of

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group 2, 11% of group 3, and 16% of group 4. Among patients with HCV1 or HCV-4 infection, sustained response was signiﬁcantly higher in group 2 than in group 3 (18% vs. 7%), whereas virological response rates were not different in patients infected with genotype 2 or 3 (*30% in all groups) [128]. In each individual nonresponder case, the indication to re-treat with the combination of IFN and ribavirin should be based on general health and the patient’s anticipated compliance and on the severity of the underlying chronic liver disease and its propensity to evolve toward liver cirrhosis in the expected lifetime of the patient. Combination therapy with pegylated interferons may provide more efﬁcacious therapy than conventional IFN in the treatment of nonresponders. In a prospective study, triple therapy of IFN, ribavirin, and amantadine induced a sustained virological remission in as many as 48% of IFN nonresponders. This approach, if conﬁrmed in large trials, would point to triple therapy as the best option to re-treat previous nonresponders [129,130]. Therapy with Amantadine, Interferon þ Amantadine, or Interferon þ Ribavirin þ Amantadine. Amantadine, a tricyclic amine, provides antiviral efﬁcacy against the inﬂuenza A virus [131]. The molecular mechanism of action is based on inhibition of the early stage of viral replication, presumably uncoating of the virus [132,133]. Inhibition of HCV replication has been proposed; however, the molecular mechanism is unknown. Numerous clinical trials have been initiated to determine the antiviral efﬁcacy of amantadine in patients with chronic hepatitis C. First, a prospective pilot study was conducted to test the safety and efﬁcacy of amantadine as monotherapy in patients with chronic hepatitis C infection who had previously failed interferon-a monotherapy (nonresponders). Twenty-two patients were enrolled in the study and treated with amantadine 100 mg orally twice daily for 6 months. Four of 22 patients (18%) treated with amantadine achieved a sustained virological response [134]. The effectiveness of amantadine was subsequently challenged by additional trials in previously untreated individuals and interferon nonresponders. The aim of a large randomized, placebo-controlled German study was to compare the efﬁcacy and safety of interferon-a alone or in combination with amantadine for initial treatment of chronic hepatitis C. Previously untreated patients (n ¼ 119) with chronic hepatitis C were randomly allocated to treatment with interferon a-2a at a dose of 6 MIU three times a week subcutaneously for 24 weeks, followed by 3 MIU thrice weekly for an additional 24 weeks plus amantadine sulfate

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administered orally 100 mg twice a day for 48 weeks or the same IFN regimen plus a matched placebo. A virological end-of-treatment response was observed in 34% of patients treated with the combination therapy and in 33% on monotherapy, whereas a sustained virological response occurred in 10% and 22%, respectively [135]. Results from two large studies in Italy and the United Kingdom showed no statistically signiﬁcant synergistic antiviral action of amantadine. One hundred eighty (180) previously untreated individuals from Italy who were treated with IFN a-2a (6 MIU three times weekly for 24 weeks, then 3 MIU three times weekly for another 24 weeks) either with or without amantadine hydrochloride 100 mg twice daily for 48 weeks achieved sustained virological response rates of 24% and 17%, respectively [136]. Patients from the United Kingdom who received IFN a-2a 4.5 MIU three times weekly for 48 weeks with or without amantadine hydrochloride 100 mg twice daily achieved sustained virological response rates of 23% and 17%, respectively [137]. Another Italian group who enrolled 200 previously untreated patients with chronic hepatitis C and randomized them to 48 weeks of treatment with interferon a-2a at 6 MIU three times weekly for 48 weeks with or without amantadine hydrochloride at 100 mg twice daily reported a signiﬁcant difference. The rate of end-of-treatment and sustained virological response rate in their study were 46% for combination and 29% for monotherapy and 29% and 17%, respectively [138]. In further trials the efﬁcacy of triple antiviral therapy with interferon, ribavirin, and amantadine was evaluated in comparison with interferon/ribavirin combination treatment. In an Italian study 60 nonresponder patients were randomized to receive interferon-a (5 MIU three times weekly), ribavirin (800–1000 mg daily), and amantadine (200 mg daily) for 12 months or to receive the same treatment without amantadine. A sustained virological response was achieved in 19 to 40 (48%) patients in the triple therapy arm and only in 1 of 20 (5%) patients in the interferon/ribavirin combination treatment arm (p < 0.001) [129]. A randomized, placebo-controlled German multicenter trial evaluated high-dose interferon a-2a induction/ribavirin combination therapy (9 MIU IFN a-2a daily for 2 weeks, followed by 6 MIU IFN a-2a daily for another 6 weeks, then 6 MIU IFN a-2a three times a week until week 24 and 3 MIU IFN a-2a three times a week until week 48) with or without amantadine sulfate in 400 previously untreated HCV-infected subjects. Triple therapy with amantadine sulfate plus interferon a-2a induction and ribavirin led to a signiﬁcantly higher treatment response at week 24 (70% vs. 59%, p ¼ 0.02) and also improved sustained virological

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response compared with combination therapy with interferon a-2a induction plus ribavirin alone (52% vs. 43%, p ¼ 0.055) [139]. In conclusion, results for amantadine remain controversial in patients with chronic hepatitis C and are apparently inﬂuenced by various parameters (virological, biochemical, histological improvement, pharmacology and dosage of the drug, size of the trial, and therapeutic regimen). Therapy with amantadine may reduce IFN-a-related adverse effects and improve health-related quality of life in patients with chronic hepatitis C undergoing IFN-a therapy [135]. The role of amantadine in the treatment of patients with chronic hepatitis in combination with (peg)interferon-a and ribavirin should be further evaluated in large controlled trials. Antiviral Therapy in Immunocompromised Patients. Infection of the donor organ occurs in more than 95% of patients following liver transplantation due to HCV-associated cirrhosis. The antiviral effect of interferon-a alone in immunosuppressed patients is low [140]. In a pilot study, patients with liver transplants suffering from recurrent hepatitis C were treated for 6 months with a combination of 3 6 3 MIU interferon-a weekly plus 1000–1200 mg ribavirin daily. Subsequently, ribavirin was continued as monotherapy for another 6 months. HCV RNA was undetectable after 6 months of combination therapy in 10 of 21 patients. Normalization of liver enzymes and histological improvement were reported for each patient. In general, aminotransferases remained within the normal ranges during subsequent ribavirin monotherapy, although ﬁve patients became positive again for HCV RNA. Ribavirin-induced hemolytic anemia led to discontinuation of therapy in three cases; graft rejections were not observed [141]. Large-scale experience for the treatment of chronic hepatitis C infection in patients with concomitant immune suppression is limited. Controlled data evaluating the effect of interferon-based therapy in individuals after renal transplantation or in patients with endstage renal insufﬁciency are not available. In western countries about one-third of the HIV-infected population is coinfected with HCV. Before the era of potent antiretroviral therapy, patients died of AIDS and its complications before the sequelae of their hepatitis became clinically relevant. Nowadays, long-term survival from HIV infection brings HCV coinfected patients to an increased risk for morbidity and mortality associated with chronic liver disease. Thus, identiﬁcation and management of chronic hepatitis C is more and more critical for HIV/HCV coinfected patients.

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The human immunodeﬁciency virus may impair clearance of HCV in those acutely infected, resulting in an increased risk of developing chronic infection. In general, chronic HCV infection does not affect the natural course of the HIV infection. However, chronic hepatitis C is more progressive in HIV-coinfected patients. Treatment of chronic hepatitis C should be considered for all coinfected patients with compensated liver disease unless contraindicated (e.g., during pregnancy, severe depression, etc.). In patients with high levels of CD4þ cells (>500 m L1), treatment of hepatitis C should be a primary course of action (primarily initiated). In those coinfected patients with lower CD4þ cell counts, immune reconstitution should be achieved by highly activated antiretroviral therapy (HAART), and treatment for chronic hepatitis C should subsequently be initiated. Sustained virological response rates are dismal in patients with CD4þ cell counts of less than 200 mL1. Combination therapy with interferon-a plus ribavirin must take interactions with antiretroviral drugs into account. Chronic hepatitis C increases the hepatotoxic risk of antiretroviral drugs, possibly resulting in discontinuation of antiretroviral medication. This may blunt immune reconstitution in response to HAART and may enhance progression to AIDS and HIV-related death among coinfected patients [142,143]. A retrospective cohort analysis showed that response to interferon þ ribavirin is likely to be similar in HIV-infected patients (mean CD4þ cell count of 430 mL1) and non-HIV-infected patients. In this study, 20 HIV/HCV-coinfected patients were treated for 6–14 months with interferon/ribavirin at standard doses. A sustained virological response was achieved in eight patients (40%), including two of 10 patients infected with genotype 1 and in six of nine patients infected with genotype 2 or 3 [144]. Data from randomized controlled trials (in particular for pegylated interferons in combination with ribavirin) are pending for HIV/HCV coinfected patients. Safety Proﬁle of Combination Therapy with Interferon-a and Ribavirin. Ribavirin is generally well tolerated. Mild but characteristic side effects (see Table 4) are dyspnea, cough, sleeping disturbances, eczema, and pruritus. Ribavirin-induced osmotic hemolysis causes a decline in hemoglobin with increasing levels of reticulocytes, bilirubin, and uric acid. Side effects and laboratory abnormalities are rapidly reversible after discontinuation of treatment. Due to its teratogenic potential, ribavirin is strictly contraindicated in pregnancy. Strict contraception is required during therapy and for up to 7 months after discontinuation of ribavirin. During combination therapy of interferon-a

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plus ribavirin the incidence of depression, sleeping disturbances, emotional lability, nausea, weight loss, dyspnea, eczema, dry skin, and pruritus is increased compared with interferon monotherapy. A maximum drop in hemoglobin (*3 g/dL) is observed after 4 weeks of combination therapy. In about 15% of the patients the decline of hemoglobin exceeds 4 g/dL. Dose reduction of ribavirin is recommended either when hemoglobin values decrease below 10 g/dL or when hemoglobin values drop below 11 g/dL in patients with (suspected) coronary heart disease. Compared with interferon-a alone, combination therapy of interferon-a plus ribavirin causes a more pronounced decline in lymphocyte counts [116,118,126]. Safety Proﬁle of Combination Therapy with Pegylated Interferon-a and Ribavirin. Frequency and severity of adverse events of pegylated interferon plus ribavirin were typical of those with unmodiﬁed interferon-a. In particular, rates of depression and psychiatric disorders were not raised. The frequency of dose reduction for neutropenia according to the protocol was higher in peginterferon groups, but discontinuation of treatment because of low neutrophil count is rarely necessary. 5.2 5.2.1

Current Guidelines for Treatment of Hepatitis C Treatment of Acute Hepatitis C

Early treatment of acute hepatitis C with interferon alone (5 MIU per day for the ﬁrst 4 weeks, followed by a dose of 5 MIU three times a week for another 20 weeks) prevents the development of chronic HCV infection in the majority of the patients and is recommended. Most likely, similar results can be achieved with pegylated interferons; however, this should be conﬁrmed in additional trials. 5.2.2

Treatment of Chronic Hepatitis C in Previously Untreated Individuals

Based on data of recent trials, combination therapy for chronic hepatitis C with pegylated interferon-a plus ribavirin will replace the current standard of treatment with unmodiﬁed interferon-a (3 MIU interferon a-2a/2b week or 9 mg interferon alfacon-1 three times a week) plus ribavirin. Previously untreated patients with chronic hepatitis C should be treated either with 180 mg pegylated interferon a-2a or with pegylated interferon a-2b 1.5 mg per kilogram of body weight once weekly plus daily administration of ribavirin (800 mg for body weight of <65 kg, 1000 mg for 65–85 kg, and 1200 mg for >85 kg). Numerous trials suggest

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adjusting the duration of treatment individually according to HCV genotype and viral load. Patients infected with HCV subtype 1 should be treated for 48 weeks. Treatment periods of 24 weeks appear to be sufﬁcient for patients infected with HCV genotype 2 or 3. Unless there are any contraindications, therapy should remain as a combination treatment for the entire treatment period. In patients who do not achieve at least a 2 log drop of baseline viremia within the initial 12 weeks of therapy, a sustained virological response is highly unlikely (negative predictive value 97–99%). This can be considered as an algorithm for early discontinuation of treatment. Discontinuation of treatment that initially was planned for 48 weeks is deﬁnitely appropriate if HCV RNA is still detectable in serum after 24 weeks of therapy. 5.2.3

Treatment of Chronic Hepatitis C in Patients with Normal Aminotransferases

Histological progression to liver cirrhosis in patients with chronic hepatitis C and persistently normal aminotransferases is slow. Therefore, the therapeutic beneﬁt of interferon-based antiviral therapy in patients infected with chronic hepatitis C and persistently normal aminotransferases remains less deﬁned [146,147]. A general recommendation cannot currently be given. Combination therapy with pegylated interferon plus ribavirin may be considered in patients at substantial risk for viral transmission, e.g., surgeons and other health care providers. 5.2.4

Treatment of Chronic Hepatitis C in Patients with Liver Cirrhosis

Patients with compensated liver cirrhosis stage Child-Pugh A and in certain cases early stage Child-Pugh B should be treated with (pegylated) interferon-a plus ribavirin. Recent data show that patients who fail to eradicate the virus may still beneﬁt from interferon-a with respect to histological progression and risk of development of hepatocellular carcinoma. Trials have been initiated to evaluate this approach prospectively. Patients with decompensated liver cirrhosis should be evaluated for possible liver transplantation. 5.2.5

Treatment of Chronic Hepatitis C in Patients Who Initially Responded to Interferon-a Alone and Experienced Virological Relapse After Cessation

Patients who are virological responders to interferon-a alone and experienced virological relapse after cessation should be re-treated with pegylated interferon-a in combination with ribavirin (1000– 1200 mg/day). HCV-2- and HCV-3-infected patients should be treated

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for 24 weeks. Although not an evidence-based recommendation supported by controlled trials, a 48-week therapy appears appropriate for HCV-1-infected patients with virological relapse. No standard recommendation can be given for patients who relapsed following combination therapy.

5.2.6

Treatment of Chronic Hepatitis C in Patients Who Did Not Respond to Previous Therapy

Therapeutic recommendations for virological nonresponders to interferon-a or combination treatment are not well deﬁned. Patients should primarily be enrolled in controlled clinical trials. Criteria to consider patients for repeated therapy are as follows: high risk of progression, temporary or partial biochemical or virological response during their ﬁrst therapy, initial treatment with low-dose interferon-a, or a short duration of therapy [148]. According to the most recent data, triple therapy comprising (pegylated) interferon-a, ribavirin, and amantadine may be considered. Alcohol abuse accelerates the progression to liver cirrhosis, and patients must therefore be reminded to abstain from alcohol. There is controversy regarding the question of whether an acute hepatitis A or B infection in addition to chronic hepatitis C increases the risk of a fulminant course of disease. Current immunization recommendations support protection of patients with chronic hepatitis C by vaccination against hepatitis A or B in the case of relevant transmission risks.

5.2.7

Treatment of Immunocompromised Patients

Treatment of HCV should be considered in HIV/HCV coinfected patients due to their increased morbidity and mortality risk for HCVassociated end-stage liver disease. Combination therapy is preferred; however, careful clinical surveillance is necessary. In patients with CD4þ cells >400–500/mL, combination therapy should be performed before initiation of HAART; otherwise, immune reconstitution by HAART is recommended before initiation of treatment. Sustained virological response rates are marginal in patients with CD4þ cells <200/mL. In immunosuppressed patients, e.g., following organ transplantation, combination therapy (pegylated) interferon plus ribavirin is also more effective than interferon-a monotherapy. Deﬁnite evaluations in terms of dose and duration of treatment are not completed.

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FUTURE PERSPECTIVES FOR THERAPY OF CHRONIC HEPATITIS C

Several inhibitors of the protease/helicase and RNA-dependent polymerase of hepatitis C virus are currently in preclinical and early clinical evaluation. Furthermore, immunotherapeutic approaches (therapeutic vaccines, interferons, interleukins, histamine dihydrochloride, etc.) and molecular approaches (antisense strategies, ribozymes) are under development and may offer additional therapeutic perspectives. 6.1

Speciﬁc Enzyme Inhibitors

Potent antiviral substances are those that disturb the viral replication cycle and inhibit virus-encoded enzymes. Preferable targets of anti-HCV therapy are the helicase, the protease, and the RNA-dependent RNA polymerase (Fig. 14). The three-dimensional structures of these targets have been investigated, and their in vitro function has been examined [149–156]. Based on known enzyme structures, substances are developed that directly block catalytical centers of these enzymes. The recent achievement of an in vitro HCV replication system has substantially contributed to the development program of speciﬁc enzyme inhibitors [157]. 6.1.1

Helicase Inhibitors

Uncoiling of HCV RNA during translation and the replication process is catalyzed by a helicase that is functionally incorporated in the HCV NS3 protein. Inhibitors of the HCV helicase have been developed, but clinical data are not yet available. 6.1.2

Protease Inhibitors

Similar to other RNA viruses, HCV expresses its genetic information in a single polyprotein, which is processed by cellular and viral proteases into structural and nonstructural proteins. HCV protease inhibitors have been developed, and clinical development programs have been initiated. 6.1.3

Polymerase Inhibitors

Hepatitis C virus replicates by means of an RNA-dependent RNA polymerase (NS5B). Polymerase inhibitors have already been investigated in phase II clinical trials, but data have not been published. 6.1.4

Inhibitors of the Internal Ribosome Entry Site

Studies have shown that interaction of cellular proteins with the internal ribosome entry site (IRES) of viral RNA is necessary for translation [158].

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FIGURE 14 The hepatitis C virus is an enveloped virus with a (þ)-strand RNA genome encoding a polyprotein. This polyprotein is cleaved co- and posttranslationally into mature viral proteins by host cell peptidases and two viral enzymes designated the NS2-3 proteinase and the NS3/4A proteinase complex. It is assumed that virus replication takes place in a membraneassociated complex containing at least two viral enzymatic activities: the NS3 nucleoside triphosphatase (NTPase)/helicase and the NS5B RNAdependent RNA polymerase (RdRp). Based on their important role for the viral replication cycle, the NS3/4A serine-type proteinase complex, the NS3 NTPase/helicase, and the NS5B RdRp are the most attractive targets for development of HCV-speciﬁc antiviral therapies.

Speciﬁc inhibition of HCV IRES represents a promising strategy for antiviral treatment. Short RNA [inhibitor RNA (iRNA)] interrupting IRES-dependent translation of several viruses has been isolated from Saccharomyces cereviseae [158]. Inhibition of translation occurs by RNA binding to certain cellular proteins and not by antisense mechanisms. Most likely iRNA forms a stable secondary structure, which at least partly corresponds to the 50 noncoding region of the hepatitis C virus.

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Translation of cellular proteins will not be inﬂuenced by iRNA. HCV IRES inhibitors have not yet been investigated in clinical studies.

6.2

Antisense Oligonucleotides

Antisense deoxyoligonucleotides are DNA strands complementary to viral RNA or DNA. Hybrid formation inhibits translation and induces activation of RNase H, which itself leads to degradation of the formed hybrids (Fig. 15) [159,160]. Nonmodiﬁed oligonucleotides are rapidly degraded by nucleases. Based on various chemical modiﬁcations, nuclease-resistant oligonucleotides (ODNs) have been developed (SODN, M-ODN, B-ODN). S-ODNs cause speciﬁc inhibition in cell culture and in vivo but also impair gene expression nonspeciﬁcally due to their polyanion properties. High concentrations may cause inhibition of RNase H [159,161]. B-ODNs are less polar and show fewer unspeciﬁc interactions [159]. Highly conserved RNA regions are optimal target sequences for antisense oligonucleotides. Antisense oligonucleotides (mostly S-ODNs) complementary to the 50 noncoding region and the core region of HCV are able to substantially inhibit translation and replication of hepatitis C virus in vitro [159,161–165]. An S-ODN consisting of 20 nucleotides is currently being evaluated in phase I/II trials for treatment

FIGURE 15 Mechanism of antisense deoxyoligonucleotides (antisenseODNs). The inhibitory effect of AS-ODN may be mediated through the formation of DNA–RNA duplexes, followed by RNase H activation and subsequent cleavage of the RNA or blocked translation of the mRNA at the point of ribosomal assembly. In vitro data suggest that antisense molecules designed to target the 50 noncoding region of HCV exert inhibitory effects on subsequent protein expression.

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of chronic hepatitis C. This substance is administered intravenously three times a week.

6.3

Ribozymes

Ribozymes (ribonucleic acid and enzyme) are RNA molecules that catalyze sequence-speciﬁc cleavage of target RNA [166]. ‘‘Hammerhead’’ ribozymes consist of three helices: The variable helices I and III hybridize to complementary parts of the target RNA, and the conserved helix II forms the catalytically active site (Fig. 16) [167]. Ribozymes can be designed to hybridize and cleave RNA at any position. Chemical modiﬁcation of ribosomes is necessary to avoid rapid degradation in vitro and in vivo (168). The target sequence of a ribozyme must be easily accessible from outside. In particular, the IRES in the 50 noncoding region or the core gene of HCV serves as a target sequence for ribozymes [167]. Efﬁcient catalytical activity of hammerhead ribozymes for the cleavage of HCV RNA has been shown in vitro [169]. In HCV-infected human hepatocytes, (adenoviral) expressed ribozymes were able to reduce or even eliminate HCV RNA [161]. A nuclease-resistant hammerhead ribozyme that is directed against the 50 noncoding region of HCV has been developed by Ribozyme Pharmaceuticals, and phase II trials in combination with interferon-a are currently in progress.

6.4 6.4.1

Immunotherapy Interferon-o

Interferon-o (IFN-a II1) is a naturally occurring leukocyte interferon. Its protein sequence (172 amino acids) shows a 60% homology to other known interferon-a subtypes. Interferon-o binds to the same receptors as interferon-a and interferon-b. The biological and physiological properties of interferon-o have not yet been clariﬁed in detail [170]. Antiviral activity of interferon-o has been shown in vitro [171,172], and clinical trials with patients with chronic hepatitis have been initiated. 6.4.2

Interleukins

There is increasing evidence to implicate immune-mediated mechanisms in the pathogenesis of hepatocellular injury in HCV infection. CD4þ and CD8þ T cells, as well as their inﬂammatory and regulatory cytokines, have been implicated in both the hepatocellular damage and the perpetuation of chronic HCV infection. CD4þ cell responses are

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FIGURE 16 Catalytic cleavage by ribozyme. Ribozymes (ribonucleic acid and enzyme) are synthetic nuclease-resistant RNA molecules that catalyze sequence-speciﬁc cleavage of target RNA (substrate) in a fashion similar to restriction enzymes. Ribozymes can be designed to hybridize and cleave any RNA of known sequence with the presence of magnesium. Chemical modiﬁcation of ribosomes is necessary to avoid rapid degradation in vitro and in vivo. The target sequence of a ribozyme must be easily accessible from outside. In particular, the internal ribosome entry site (IRES) in the 50 noncoding region or the core gene of HCV serves as the possible target sequence for ribozymes.

polarized into T helper 1 (Th1) and Th2 types. Th1 cells secrete interleukin (IL-2) and IFN-g to accelerate the activation and proliferation of CTLs and NK cells. Type 2 helper cells (Th2 cells) produce IL-4, IL-5, IL-6, and IL-10, which promote B-cell differentiation into antibodyproducing plasma cells and the proliferation of such cells. Antigenpresenting cells produce IL-12 when stimulated by activated T cells, and

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IL-12 acts on Th1 cells, CTLs, and NK cells, leading to viral elimination and suppression of viral replication. IL-10 produced by Th2 cells acts on antigen-presenting cells to suppress the activation of Th1 cells by decreasing IL-12 production, leading to termination of the cellular immune response to the virus [173]. Cytokine analysis in the acute phase of HCV infection demonstrated a Th1-dominated T-helper cell response [173], and a Th2 cytokine proﬁle was observed in patients who developed chronic hepatitis with elevated levels of serum IL-2, IL-4, IL-10, tumor necrosis factor a (TNF-a), and IFN-g. The interplay between Th1 and Th2 cytokines may be important in regulating hepatocellular damage and disease progression in chronic HCV infection. Those with severe chronic hepatitis or cirrhosis have enhanced expression of both IFN-g and IL-2 messenger RNA that correlates with ﬁbrosis and portal inﬂammation, whereas IL-10 levels are reduced [174,175]. Interleukin-2. The glycoprotein interleukin-2 (IL-2) supports proliferation and differentiation of T lymphocytes and activates macrophages via interferon-g. IL-2 shifts the T-cell response toward Th1. Treatment of patients with chronic hepatitis C shows normalization of aminotransferases in approximately one-third of the patients. HCV RNA, however, remained detectable in all patients at the end of treatment [176,177]. Interleukin-10. Data from a pilot trial show that the administration of interleukin-10 (IL-10) was associated with a marked reduction of T-cell proliferation, a nonspeciﬁc immune response. The cytokine secretion pattern changed from a Th1- (IFN-g) to an IL-10-predominant Th0/Th2 response. Although there was no change in serum HCV RNA levels, IL-10 was associated with changes in serological markers, suggesting a reduction of immune response and ﬁbrogenesis [170,174]. Interleukin-12. Interleukin-12 (IL-12) plays a central role in mounting an effective cellular immune response directed toward elimination of intracellular pathogens. Recombinant human interleukin-12 (rHuIL-12) is a heterodimeric cytokine that promotes cell-mediated immunity by facilitating type 1 helper T lymphocyte responses, including the secretion of IFN-g from both T cells and natural killer (NK) cells, enhancing the lytic activity of natural killer cells and augmenting speciﬁc cytolytic T-lymphocyte responses [178]. A doseescalating phase I/II study was designed to assess tolerability, pharmacodynamics, and efﬁcacy of subcutaneous administration of recombinant human interleukin-12 (rHuIL-12) in the treatment of chronic

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hepatitis C. However, antiviral activity of rHuIL-12 has not been advantageous in comparison with other currently available treatments [179]. 6.5 6.5.1

Drugs That Modify Immune Response Histamine

Histamine dihydrochloride (HDC) is a naturally occurring immunomodulator. Phagocytic cells such as monocytes, macrophages, and neutrophils, which are present at the site of infections, strongly inhibit the killing activity of NK cells and T cells and prevent their activation by cytokines or other immunomodulators. The suppression of NK cells and T cells has been shown to be caused by the release of reactive oxygen species (ROS) by phagocytes. Administration of histamine dihydrochloride blocks the production and release of free radicals, thereby protecting NK cells and T cells and facilitating their activation by cytokines or lymphokines or other immunostimulators (IL-2, IFN-a), leading to improved antiviral activity [180–184]. The safety and effectiveness of histamine dihydrochloride is currently assessed in advanced stages of clinical investigation for use in combination with cytokines, interleukin-2 (IL-2), and/or IFN-a for the treatment of chronic hepatitis C. 6.5.2

Thymosin-a1

Thymosin-a is a naturally occurring peptide (28 amino acids) with immunomodulatory and antiviral properties in vitro and in vivo. The immunomodulatory properties comprise T-cell maturation, induction of cytokines including interferon-a and interleukin-2, and activation of NK cells [185]. The in vitro activity is characterized by an increase in Th-1 and an impairment of Th-2 response [186]. Thymosin-a is approved in several countries (e.g., Italy) for treatment of chronic hepatitis B and C [187]. 6.5.3

Inhibitors of Inosine-50 -Monophosphate Dehydrogenase

The enzyme inosine-50 -monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, namely, the conversion of inosine-50 -monophosphate (IMP) to xanthosine-50 -monophosphate (XMP). Two isoforms (isoforms I and II) of human (and mouse) IMPDH have been identiﬁed that share 84% identity. The major event occurring in cells exposed to competitive IMPDH inhibitors such as ribavirin or uncompetitive inhibitors such as mycophenolic acid (MPA) is a depletion of the intracellular GTP and

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dGTP pools. Intracellular guanine nucleotides are required for adequate RNA and DNA synthesis. Therefore, IMPDH inhibitors have potential antiproliferative, antiviral, and antiparasitic effects [188]. Mycophenolate Mofetil. Mycophenolate mofetil (MMF), a semisynthetic derivative of mycophenolic acid (MPA) is an immunosuppressive substance that noncompetitively inhibits inosine monophosphate dehydrogenase (IMPDH). The combination of interferon-a and MMF has been evaluated in patients with chronic hepatitis C; however, a synergistic antiviral efﬁcacy was not observed in pilot trials. Final results of larger clinical trials in Europe and the United States have not yet been reported. VX-497. VX-497 (Vertex Pharmaceuticals) is a selective, reversible, noncompetitive inhibitor of both isoforms of IMPDH. In vitro studies showed that VX-497 provides greater antiviral efﬁcacy than ribavirin against numerous DNA and RNA viruses. In a phase II trial, 30 patients with chronic hepatitis C who were nonresponders to previous interferona therapy received VX-497 for 28 days in increasing doses. In patients treated with 200 mg VX-497 three times a day, a considerable decrease in aminotransferases was observed; however, viremia remained unaffected. This study did not reveal any renal or hematological side effects [189]. Currently, the combination of interferon-a and VX-497 is being evaluated in clinical trials. 7

VACCINATION AND IMMUNE PROPHYLAXIS

The genetic variability of hepatitis C virus complicates the development of a universal HCV vaccine. Anti-HCV immunoglobulins are currently being investigated in clinical trials and may be useful to avoid reinfection of the donor organ in HCV-infected patients undergoing liver transplantation. REFERENCES 1. 2. 3.

Davis GL, Lau JY, Lim HL. Therapy for chronic hepatitis C. Gastroenterol Clin North Am 1994; 23:603–613. Belle SH, Beringer KC, Detre KM. Recent ﬁndings concerning liver transplantation in the United States. Clin Transpl 1996; 1:15–29. Prieto M, Berenguer M, Rimola A, Loinaz C, Barrios C, Clemente G, Figueras J, Vargas V, Casafont F, Pons JA, Herrero JI. Liver transplantation in hepatitis C. A Spanish multi-centre experience. Eur J Gastroenterol Hepatol 1998; 10:771–776.

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11 HIV Infection: Epidemiology, Pathogenesis, and Principles of Antiretroviral Therapy Reinhold Welker Bayer HealthCare, Wuppertal, Germany

Helga Ru¨bsamen-Waigmann Bayer HealthCare, Wuppertal, and University of Frankfurt, Frankfurt, Germany

1

INTRODUCTION

The acquired immunodeﬁciency syndrome (AIDS) was ﬁrst noticed in the United States of America in 1981, when the U.S. Centers for Disease Control and Prevention (CDC) reported the unexplained occurrence of Pneumocystis carinii pneumonia and Kaposi’s sarcoma in previously healthy homosexual men [1–3]. Soon thereafter the disease became recognized in male and female intravenous drug users as well as in recipients of blood transfusions and in hemophiliacs. As the epidemiological pattern unfolded, it became clear that an infectious agent transmissible by sexual contact and blood and from mothers to infants Disclaimer: The authors are employees of Bayer AG. The statements made in this chapter reﬂect the personal opinions of the authors and may not be considered ofﬁcial statements of Bayer AG.

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was the cause of the epidemic. In 1983 a retrovirus, later designated human immunodeﬁciency virus (HIV), was isolated from a patient with lymphadenopathy [4], and by 1984 HIV was demonstrated to be the most likely causative agent of AIDS [2,4]. Identiﬁcation of the causative agent allowed the development of diagnostic assays and provided the basis for a scientiﬁc approach to speciﬁc therapy. Epidemiological studies revealed a global pandemic [5,6], and its staggering worldwide growth was matched by an unprecedented explosion of information on all aspects of HIV disease. Despite tremendous efforts and signiﬁcant progress in many areas of HIV research, two decades after its discovery the worldwide spread of the virus continues at an alarming pace. Human immunodeﬁciency virus causes a chronic progressive disease leading to a severe life-threatening immunodeﬁciency, AIDS, as the end stage. Mainly for epidemiological surveillance, a case deﬁnition for AIDS was issued that has undergone several revisions over the years and may vary in some points among countries [5,7,8]. The most recent revision by the Centers for Disease Control and Prevention was issued in 1993 [7]. This revised CDC classiﬁcation system for HIV-infected adolescents and adults categorizes persons on the basis of clinical conditions associated with HIV infection and CD4þ T-lymphocyte counts as a measure of immune function (Tables 1 and 2). The system is based on three ranges of CD4þ T-lymphocyte counts and three clinical categories, yielding a matrix of nine mutually exclusive categories (Table 1). Using this system, by deﬁnition, any HIV-infected individual with CD4þ TABLE 1 1993 Revised Classiﬁcation System of the U.S. Centers for Disease Control and Prevention for HIV Infection and Expanded AIDS Surveillance Case Deﬁnitions for Adolescents and Adults Clinical categorya

CD4 T-cell category (cells/mL) >500 200–499 <200 a

A Asymptomatic, acute (primary) HIV or PGL

B Symptomatic, not A or C conditions

C AIDS indicator conditions

A1 A2 A3b

B1 B2 B3b

C1b C2b C3b

See Table 2 for deﬁnition of clinical categories. Categories indicating expanded AIDS surveillance case deﬁnition. PGL, progressive generalized lymphadenopathy. Source: Refs. 7 and 9. b

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TABLE 2 Clinical Categories of HIV Infection as Deﬁned for Expanded AIDS Surveillance Case Deﬁnitions for Adolescents and Adults Category A: Consists of one or more of the conditions listed below in an adolescent (> 13 years) or adult with documented HIV infection. Conditions listed in categories B and C must not have occurred. Asymptomatic HIV infection Persistent generalized lymphadenopathy Acute (primary) HIV infection with accompanying illness or history of acute HIV infection Category B: Consists of symptomatic conditions in an HIV-infected adolescent or adult that are not included in conditions listed in clinical categories A or C and that meet at least one of the following criteria: (1) The conditions are attributed to HIV infection or are indicative of a defect in cell-mediated immunity, or (2) the conditions are considered by a physician to have a clinical course or to require management that is complicated by HIV infection. Examples include, but are not limited to Bacillary angiomatosis Candidiasis, oropharyngeal (thrush) Candidiasis, vulvovaginal: persistent, frequent, poorly responding to therapy Cervical dysplasia Constitutional symptoms such as fever (38.58C) or diarrhea lasting >1 month Hairy leukoplakia, oral Herpes zoster involving at least two distinct episodes or more than one dermatome Idiopathic thrombocytopenic purpura Listeriosis Pelvic inﬂammatory disease, particularly if complicated by tubo-ovarial abscess Peripheral neuropathy Category C: Conditions listed in the AIDS surveillance case deﬁnition. Candidiasis of bronchi, trachea, or lung Cervical cancer, invasive Coccidioidomycosis, disseminated or extrapulmonary Cryptococcosis, extrapulmonary Cryptosporidiasis, chronic intestinal (> 1 month) Cytomegalovirus disease (other than liver, spleen, or lymph nodes) Cytomegalovirus retinitis Encephalopathy, HIV-related Herpes simplex: chronic ulcers (> 1 month), bronchitis, pneumonia, or esophagitis Continued on next page.

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Welker and Ru¨bsamen-Waigmann Continued

Histoplasmosis, disseminated or extrapulmonary Isosporiasis, chronic intestinal (> 1 month) Kaposi’s sarcoma Lymphoma, Burkitt’s or equivalent Lymphoma, primary, of brain Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary Mycobacterium tuberculosis, any site Mycobacterium, other species or unidentiﬁed species, disseminated or extrapulmonary Pneumocystis carinii pneumonia Pneumonia, recurrent Progressive multifocal leukoencephalopathy Salmonella septicemia, recurrent Toxoplasmosis of brain Wasting syndrome due to HIV Source: Refs. 7 and 9.

T-lymphocyte counts below 200 cells/mL has AIDS regardless of the presence of symptoms or opportunistic diseases [9]. Once individuals have had a clinical condition in a lower category their disease cannot be reclassiﬁed into a higher category even if the condition resolves. Because this deﬁnition of AIDS was not established for the practical care of patients but for surveillance purposes, the clinician should not focus on diagnosing AIDS. Rather, HIV disease should be viewed as a spectrum of conditions. Primary infection may or may not be associated with an acute disease syndrome and represents the initial stage of a chronic lifelong disease. In most cases primary infection is followed by an asymptomatic interval that can last for several years. If untreated, HIV disease usually progresses to an advanced stage and death (see Sec. 3). Due to the chronic progressive and multifactorial nature of the infection, each stage and each condition requires speciﬁc considerations. Treatment decisions are based on clinical ﬁndings as well as on laboratory parameters of viral activity (viral load) and immune function (CD4þ T-cell count). Despite enormous progress, current treatment options have certain limitations, posing unexpected challenges for both physicians and patients. The purpose of this chapter is to review aspects of HIV infection that are important for understanding the disease and speciﬁc approaches

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to antiretroviral therapy. Over the past two decades of HIV research enormous progress has been made in understanding the molecular biology of HIV replication, and this is reviewed brieﬂy in Section 3 where references to more comprehensive reviews are given. Subsequent chapters present in detail methods used in HIV diagnostics (Chap. 12) and speciﬁc aspects of currently approved and investigational strategies of antiretroviral therapy (Chaps. 13–16). It should be stressed that antiretroviral treatment regimens in clinical use are under constant and rapid evolution, and for up-to date comprehensive treatment guidelines for HIV infection the reader is referred to frequently updated recommendations issued by national authorities and national as well as international societies. Some of these guidelines are also available on the World Wide Web (e.g., http://www.hivatis.org; http://www.iasusa.org).

2 2.1

EPIDEMIOLOGY Transmission

Human immunodeﬁciency virus infection is predominantly a sexually transmitted disease (STD) worldwide [5,10]. The virus is transmitted by both heterosexual and homosexual contacts. In addition, HIV is transmitted by blood and blood products and by infected mothers to infants. There is no evidence that HIV is transmitted through casual contacts or through insect vectors [10]. Although in industrialized countries a large proportion of new HIV infections are among men who have sex with men, heterosexual transmission is clearly the most common mode of infection worldwide, particularly in developing countries [8,11,12]. In industrialized countries, however, the pattern of transmission is changing, and the frequency of heterosexual transmission is increasing [8,12,13]. The overall number of new HIV infections transmitted through heterosexual intercourse rose to 48% in western Europe, and in Great Britain and Sweden heterosexual transmission has become the main mode of HIV infection [8]. HIV has been demonstrated in seminal ﬂuid both in association with mononuclear cells and in the cell-free state. The amount of virus is greater in situations with inﬂammation and in conditions closely associated with other STDs. The virus has also been demonstrated in vaginal ﬂuids and in cervical smears. There is a strong association of HIV infection with receptive anal intercourse, especially in situations with sexual practices that are likely to traumatize the mucosa. Although the vaginal mucosa is several layers thicker than the rectal mucosa and less likely to be traumatized during intercourse, it is clear that the virus can be transmitted to either partner

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through vaginal intercourse. A chief predictor for heterosexual transmission is plasma viremia [9]. In addition, a history of STDs is strongly associated with HIV transmission [10,11,14–16]. In this regard there is a close association between genital ulceration and transmission from the standpoints of both susceptibility to infection and infectivity. However, infections with pathogens causing nonulcerative STDs are also associated with an increased risk of HIV infection. Several studies suggested that treating other STDs may help prevent HIV transmission. In commercial sex workers, the frequent use of spermicides containing nanoxyl N-9 (which is a detergent also capable of inactivating HIV) was also associated with an increase in ulcerative lesions and a higher rate of HIV transmission [17,18]. Interestingly, the use of oral contraceptives was also associated with an increased risk of HIV infection [15,19]. Lack of circumcision has been associated with a higher rate of HIV transmission in certain cohorts. Finally, HIV transmission has also been shown to occur during oral sex practices, although perhaps less efﬁciently than in anal or vaginal intercourse. Human immunodeﬁciency virus infection can be transmitted from an infected mother to her fetus or to her infant, and without intervention this occurs at a frequency of 10–30% [10,20,21]. This is an extremely important form of transmission in developing countries [5]. Mother-tochild transmission occurs most commonly during the perinatal period. Several conditions during pregnancy and at birth, including high maternal plasma HIV RNA levels, have been associated with an increased risk of transmission. Transmission can also occur during breastfeeding, especially in the ﬁrst days after delivery [22]. Efﬁcient antiretroviral therapy can dramatically decrease the rate of HIV transmission both during pregnancy and in the perinatal period. In industrialized countries, delivery by cesarian section, concomitant antiretroviral therapy, and refraining from breastfeeding have lowered the transmission rate dramatically [20–23]. HIV can be transmitted to individuals who receive HIV-contaminated blood transfusions, blood products, or transplant tissues [9,10]. In addition, HIV can be transmitted to injection drug users who are exposed to HIV while sharing certain instruments and materials needed for the preparation and injection of drugs. Since the beginning of the HIV epidemic, there have been only a few reported instances in industrialized countries in which HIV was transmitted to patients during invasive procedures, most likely through contaminated instruments. Between late 1970 and 1985, when mandatory testing of donated blood for HIV-1 was initiated, it has been estimated that over 10,000 individuals in the United States were infected through transfusions of

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blood or blood products. Transfusions of whole blood, packed red blood cells, platelets, leukocytes, plasma, and concentrates of clotting factors are all capable of transmitting HIV infection. However, gamma-globulin preparations have not been associated with transmission of HIV infection (although transmission of hepatitis B and C infections occurred), because the procedures involved in processing these products inactivate or remove the virus. Currently, in all industrialized countries, and to various degrees also in developing countries, several measures have made the risk of HIV transmission by blood and blood products or by transplanted tissues very small. These include laboratory tests for HIV antibodies and for HIV as well as self-deferral of donors on the basis of risk behavior [24]. There is also a small but deﬁnite occupational risk of HIV transmission in health care workers and laboratory personnel and potentially others who work with HIV-infected specimens. This is particularly the case in situations in which sharp objects such as needles and sharp medical instruments are used. The risk for HIV infection following percutaneous exposure to HIV-infected devices is higher when the exposure involves a larger quantity of blood. The risk also increases for exposure to blood from patients with advanced-stage disease, probably owing to the higher titer of HIV in the blood. With the existence of antiretroviral therapy, postexposure ‘‘prophylaxis’’ is now available that when used immediately lowers the risk of infection considerably. The exact recommendations of national authorities should be followed [23]. Antiretroviral therapy, however, is likely to be beneﬁcial also in circumstances where, owing to delayed administration, postexposure prophylaxis is unlikely to prevent infection [25] (see Sec. 4.1). Although with sensitive diagnostic tests HIV can be detected in virtually any body ﬂuid of infected individuals, there is no convincing evidence that HIV can be transmitted through exposure to saliva, tears, sweat, or urine. However, caution is required because bodily secretions are frequently contaminated with blood. 2.2

HIV/AIDS, a Global Pandemic

Infection with HIV or AIDS is a pandemic with cases reported from virtually every country [5,6,26,27]. The number of infected individuals has risen constantly since the beginning of the pandemic (Fig. 1), and more than 50 million people have been infected since the epidemic began. At the end of 2002, 42 million adults and children were estimated to be living with HIV/AIDS [5] (Table 3). About half of the infected individuals are women (Tables 3 and 4). The global distribution of these

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FIGURE 1 Development of the HIV pandemic. Estimated number of people living with HIV/AIDS by region, 1990–1999. (Data From Ref. 11.)

TABLE 3

Global Summary of the HIV/AIDS Epidemic, as of December 2002

Number of people living with HIV/AIDS Total Adults Women Children under age 15 People newly infected with HIV in 2002 Total Adults Women Children under age 15 AIDS deaths in 2002 Total Adults Women Children under age 15 Source: Joint United Nations Program on HIV/AIDS (UNAIDS).

42 million 38.6 million 19.2 million 3.2 million 5 million 4.2 million 2 million 800,000 3.1 million 2.5 million 1.2 million 610,000

Epidemic started

500

15,000

5 million

45,000

980,000

42 million

30,000

250,000

570,000

1.2 million

1.2%

0.1%

0.6%

0.3%

0.6%

2.4%

0.6%

0.1%

0.6%

0.3%

8.8%

50%

7%

20%

25%

27%

50%

30%

24%

36%

55%

58%

MSM, IDU, hetero MSM

MSM, IDU

IDU, hetero

IDU, hetero, MSM MSM, IDU, hetero Hetero, MSM

Hetero, IDU

Hetero, IDU

Hetero

Main mode(s) of transmissionb

b

The proportion of adults (15–49 years of age) living with HIV/AIDS in 2002, using 2002 population numbers. Hetero, heterosexual transmission; IDU, transmission through injection drug use; MSM, sexual transmission among men who have sex with men. Source: UNAIDS.

a

150,000

1.5 million 60,000

270,000

1.2 million

440,000

700,000

83,000

3.5 million

6.0 million

550,000

29.4 million

Living with HIV/AIDS

Adults and children

% of HIVAdult positive Newly infected prevalence adults who with HIV ratea are women

Regional HIV/AIDS Statistics and Features, End of 2002

Sub-Saharan Africa Late 1970s–early 1980s North Africa and Late 1980s Middle East Late 1980s South and southeast Asia East Asia and Late 1980s Paciﬁc Latin America Late 1970s–early 1980s Caribbean Late 1970s–early 1980s Eastern Europe and Early 1990s central Asia Western Europe Late 1970s–early 1980s North America Late 1970s–early 1980s Australia and New Late 1970s–early Zealand 1980s Total

Region

TABLE 4

HIV Infection 377

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FIGURE 2 The HIV pandemic 2002. (A) Estimated number of people living with HIV/AIDS by the end of 2002, by region. (B) Estimated number of adults and children newly infected with HIV during 2002 by region. (C) Estimated adult and child deaths from HIV/AIDS during 2002 by region.

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cases is illustrated in Figure 2a. Two-thirds of infected individuals are living in sub-Saharan Africa (Table 4) (Fig. 2a), where also the fastest increase in numbers was observed over the past two decades (Fig. 1). According to the Joint United Nations Program on HIV/AIDS (UNAIDS), in 2002 alone there were an estimated 5 million new cases of HIV infection worldwide (Table 4) (Fig. 2b). Moreover, in the same year there were an estimated number of about 3.1 million deaths from AIDS (Table 3) (Fig. 2c), making it one of the ﬁve leading causes of mortality worldwide [28]. The HIV epidemic has occurred in different regions of the world in waves, each having somewhat different characteristics depending on the demographics, the culture, and the timing of the introduction of the disease into the population (Table 4) (Fig. 2) [11]. Although the epidemic was ﬁrst recognized in the United States and western Europe, it very likely began in sub-Saharan Africa [29–32], which meanwhile has been particularly devastated by the disease [6] (Table 4) (Figs. 1 and 2). In subSaharan African countries the infection continues to spread at rapid rates [33]; for example, in Botswana available data indicate that the adult prevalence rate is 38.8%. Among high-risk individuals such as commercial sex workers seroprevalence may exceed 50% in many countries of this region. This has led to a dramatic fall in average life expectancy in sub-Saharan countries from 62 years to currently 47 years. In Botswana life expectancy has dropped below 40 years, a level not seen since before 1950 [5]. The epidemic in Asian countries, particularly in India and Thailand, has lagged temporarily behind that in Africa. However, the number of new cases in this region is rising rapidly. Although introduced later into the area (Table 4), a steep increase in the number of cases of HIV and AIDS is also reported in countries from eastern Europe, central Asia, and China [5]. In industrialized countries, the overall number of new infections stayed more or less constant over the past decade, with western Europe and North America recording approximately 30,000 and 45,000 new infections per year, respectively [5,8,12]. Owing to progress in therapy (see below), the number of individuals living with HIV/AIDS is steadily increasing. The major mode of HIV transmission worldwide is heterosexual sex, especially in developing countries, where the numbers of infected men and women are approximately equal (Table 4). In industrialized countries and in some countries in Asia, the epidemic was ﬁrst introduced by and spread among homosexual men and, to a greater or lesser degree (depending on individual countries or regions), among intravenous drug users. In this regard, the total numbers of AIDS cases

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in those countries still reﬂect a high proportion of cases among these high-risk groups. However, in most developed countries, there was a gradual shift such that the number of new cases with HIV/AIDS is steadily increasing among heterosexuals, whereas it is declining (or remains constant) among homosexual men and injection drug users [8,12].

2.3

Molecular Heterogeneity of HIV

Viruses closely related to HIV are found in several primate species (with HIV, collectively termed primate immunodeﬁciency viruses). Molecular analysis of these viruses suggest that the human pathogens most likely originated from monkey viruses [29–32]. The degree of sequence similarity among isolates is often depicted in phylogenetic trees [34]. Interestingly, some of these viruses do not cause disease in their natural host but lead to an AIDS-like syndrome when inoculated into different monkey species [32,35,36]. Molecular analysis of various HIV isolates derived from humans reveal sequence variants over many parts of the genome. Based on sequence similarity and serological tests, at present two types of HIV can be distinguished: type 1 (HIV-1) and type 2 (HIV-2) [37] (Fig. 3). HIV-2 is very closely related to simian immunodeﬁciency viruses (SIV) from sooty mangabeys and is found mainly in western Africa [5,38] and to a lesser extend in India, Europe, and other regions [32,39,40]. The majority of the pandemic, however, is caused by HIV-1. Based on molecular analysis, there are currently three groups of HIV-1: group M (major), group O (outlier), and group N [41]. Group O and N viruses are found mainly in central Africa, but group M viruses are responsible for most infections in the world. Sequence heterogeneity is also found within each group, and presently group M comprises 10 subtypes, or clades, designated A, B, C, D, E, F, G, H, J, and K (Fig. 3). In addition, several circulating recombinant forms (CRFs) between subtypes of this group are found (major CRFs representing the AE virus, often referred to as E, an AG recombinant, an AGI virus, and an AB recombinant). Again, within each subtype signiﬁcant sequence heterogeneity is observed between isolates. For example, subtype B viruses differ by up to almost 20% in their env coding sequences. With constant evolution of the virus and intense ongoing investigations in the ﬁeld, new recombinants, new subtypes, and perhaps also new clades are likely to be identiﬁed in the near future. Sequence variability is also found among HIV-2 and SIV isolates [32,37,42].

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FIGURE 3 HIV diversity. Primate lentiviruses are presently found in humans (HIV) and in non-human primates (SIV). The taxonomic tree depicted schematically in this ﬁgure must not be confused with a precise phylogenetic tree and does not necessarily reﬂect the degree of precise molecular relationships between these viruses. Although molecular heterogeneity exists within each type of primate immunodeﬁciency virus, for simplicity the degree of variability is shown only for HIV-1. Details of subtype diversity are given only for HIV-1 group M viruses. Again, within each subtype there is signiﬁcant heterogeneity as exempliﬁed for subtype B. (Data source: Los Alamos National Laboratory, New Mexico; HIV sequence database.)

The geographic distribution of different subtypes is not equal, and the global patterns of variation most likely result from the history of virus trafﬁcking [38,43]. For example, subtype C viruses ﬁrst arrived in India with their closest analogs in South Africa [40,44,45], and subtype E caused the heterosexual epidemic in Thailand [46,47]. However, with

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increasing individual mobility in our society, virus variants are traveling with their hosts, and new subtypes are being introduced into regions where they were previously not found. Subtype C viruses are the most predominant worldwide. The highest variability is observed in Africa, where >75% of isolates are of strains A, C, and D, with subtype C being the most common. Subtype B viruses are the predominant subtypes in North and South America, western Europe, and Australia. However, other subtypes are also present in these countries to varying degrees. To date, in Asia, subtypes E, C, and B predominate, whereas C is most prevalent in India and E accounts for most infections in southeast Asia. In China, subtypes A, B, C, and E as well as various recombinants thereof are found in different regions of the country. In eastern Europe and central Asia, an AB recombinant predominates. In geographical areas where clades overlap, recombination among viruses from different clades is observed, most likely as a result of individuals being infected with viruses of more than one clade. Variation in HIV-1 sequence is not just interesting from an epidemiological point of view but is also of utmost practical importance both for diagnosis and for therapy (see below) [48]. Diagnostic tests and antiretroviral agents were initially developed against subtype B isolates, which happened to be the most common in North America and western Europe. Given the variability of HIV-1, however, it is not surprising that viruses have been identiﬁed that show a natural resistance against existing antiretroviral drugs, in particular against protease inhibitors and non-nucleoside RT inhibitors but also against nucleoside reverse transcriptase inhibitors (see below) [49–52]. 3

PATHOGENESIS, CLINICAL MANIFESTATION, AND DIAGNOSIS OF HIV INFECTION

The hallmark of HIV disease is a profound immunodeﬁciency resulting primarily from a progressive quantitative and qualitative deﬁciency of CD4þ T lymphocytes [9,53,54]. When the number of CD4þ T cells declines below a certain level, the patients are at high risk of developing a variety of opportunistic diseases, particularly the infections and neoplasms that are considered AIDS-deﬁning illnesses (Table 2). The combination of viral pathogenic and immunopathogenic events that occur during the course of HIV disease from the moment of infection through the development of advanced-stage disease is complex and highly variable. It is important to appreciate that the pathogenic mechanisms of HIV disease are multifactorial and multiphasic and are different at different stages of the disease.

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The exact mechanism by which HIV causes disease is incompletely understood, but it is closely linked to the strategy employed by the virus to multiply. Therefore, understanding the biology of HIV is of utmost importance for understanding the pathogenesis of HIV disease and the approaches to treatment of HIV infection. A detailed review on the molecular biology of HIV goes beyond the scope of this book; however, this issue has been the subject of several extensive reviews [55–58]. Therefore, only a brief overview on the replication of HIV-1 is given here. Electron microscopy shows that the HIV virion contains a prototypical conical core that is surrounded by a lipid envelope [59,60] (Fig. 4). HIV particles have a diameter of approximately 120 nm [61], and the envelope contains numerous external spikes formed by the envelope glycoproteins that are anchored within the lipid bilayer. Beneath the membrane and in tight association with the inner leaﬂet of the lipid bilayer there is an electron-dense layer of protein, called the matrix (Table 5). The conical core (capsid) harbors the viral genome and the enzymes required for its replication [35,62]. HIV and related viruses contain an RNA genome, and the hallmark in the life cycle of these viruses is the generation of a DNA copy of their genome by a virally encoded enzyme in a process called reverse transcription. Therefore these viruses are termed retroviruses [35,55,63]. For infection of a new target cell, HIV-1 binds through its envelope glycoproteins to the CD4 receptor on the surface of the cell (Fig. 5) [64–66]. Subsequent interaction with a coreceptor (CCR5 or CXCR4) [67] triggers fusion of the viral and cellular membranes [68,69] and allows penetration of the core into the cytoplasm. In the cytoplasm of the new host the viral reverse transcriptase makes a DNA copy of the RNA genome that is transported into the nucleus [70–72]. Within the nucleus, viral integrase inserts the HIV genome into a chromosome of the target host cell [73,74]. An integrated viral genome is designated a provirus and behaves like a cellular gene. The host cell machinery is used for synthesis [57,75,76], processing [77–80], and translation of viral mRNAs and genomes (Fig. 5). Following synthesis, HIV virion components (Table 5) are transported to the plasma membrane, where they are assembled into virus particles that are released from the host cell by budding [59,81–84]. Finally, processing of protein precursors by the viral protease leads to particle maturation and yields infectious virions (Fig. 4a) [35,57,83,85]. Within the family Retroviridae, HIV-1 belongs to the primate lentivirus subgroup [35,55]. In addition to the prototypical retroviral

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FIGURE 4 Structure and genetic organization of HIV-1. (A) Schematic representation of the HIV-1 virion. Individual components (depicted by different symbols, line weights, and shading) and their localization within the virion are indicated. (B) Schematic representation of the DNA form of the HIV-1 genome. Long terminal repeats (LTRs), open reading frames (ORFs), and the encoded proteins are indicated to scale. Reading frames of the prototypical retroviral gag, pol, and env genes are indicated by gray boxes, and those of HIV-1 accessory genes are depicted as white boxes. For abbreviations and nomenclature of viral proteins, see Table 5. CypA, cyclophillin A. (Modiﬁed from Ref. 62 and from a ﬁgure originally provided by Hans-Georg Kra¨usslich, Heidelberg, Germany.)

HIV Infection

TABLE 5

Summary of HIV Proteins

HIV protein Gag

Gag-Pol Matrix

Capsid Nucleocapsid P6 Protease Reverse transcriptase Integrase

Vif Vpr Vpu Tat Rev gPr160 gp120 gp41

Nef a

385

Function/descriptiona Polyprotein that assembles into immature virions and yields MA, CA, NC, and P6 in mature particles. Polyprotein encoding MA, CA, NC, PR, RT, and IN. Forms lining beneath the membrane of the mature virion. Forms conical capsid shell in mature virion. Viral nucleic acid binding protein. Peptide derived from Cterminus of Gag. Cleaves Gag and Gag-Pol into their ﬁnal products. Viral polymerase.

Gag; Pr 55

Mediates integration of viral genome into host chromosome. Accessory protein, viral infectivity factor. Accessory protein, viral protein R. Accessory protein, viral protein U. Accessory protein regulating viral gene expression. Accessory protein regulating RNA metabolism. Viral glycoprotein precursor. Surface glycoprotein; mediates receptor binding. Transmembrane glycoprotein; mediates fusion to target cell. Accessory protein.

See text for details. Relative molecular weight in kilodaltons. Source: Data from Ref. 35. b

Abbreviation(s) and synonyms

Gag-Pol; Pr 160

Size (kDa)b 55

160

MA; p17

17

CA; p24

26

NC; p7

7

P6

6

PR

10

RT

51; 66

IN

33

Vif

23

Vpr

15

Vpu

16

Tat

14

Rev

19

Env; gp160 SU; gp120

160 120

TM; gp41

41

Nef

27

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gag, pol, and env genes, HIV-1 and related viruses encode for several so-called accessory proteins [35]. These proteins regulate various aspects of the viral life cycle, including gene expression, RNA export, virus release, and viral infectivity, and are important determinants of viral pathogenesis [57,86–89]. Therefore HIV-1 and related family members are also referred to as complex retroviruses [90]. 3.1

The ‘‘Natural History’’ of HIV Infection

Although there is substantial variation among individual patients, in the absence of antiretroviral therapy the course of HIV infection

FIGURE 5 Overview of HIV replication. Speciﬁc steps and events within the replication cycle are indicated. By convention, the viral life cycle is thought to begin with binding of HIV to its receptor, CD4. The ‘‘early phase’’ ends with integration of the DNA form of the viral genome into a host cell chromosome, establishing a provirus. In the ‘‘late phase,’’ HIV parasitizes the cell to synthesize progeny virus from the parental provirus. (Adapted from a ﬁgure originally provided by Hans-Georg Kra¨usslich, Heidelberg.)

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shows a common pattern (Fig. 6) [9,53,54,91]. Primary infection is followed by an interval of clinical latency, during which the patient is usually asymptomatic. Subsequently, constitutive symptoms and/or clinically apparent disease are observed, and death ensues within 2–3 years. 3.1.1

Primary Infection and Acute HIV Syndrome

The events associated with primary HIV infection are critical determinants for subsequent disease progression [9,25,53,91–95]. In primary HIV infection, viral replication intensiﬁes prior to the initiation of an HIVspeciﬁc immune response, leading to a burst of viremia [92,96] (Fig. 6). During this phase the virus disseminates to lymphoid organs, which is a major factor for the establishment of a chronic and persistent infection [53,91,92,97]. Once infection has been established the virus is virtually never cleared from the body [95]. In the phase of virus dissemination, about 50–70% of patients experience the ‘‘acute HIV syndrome’’ (Table 6) [9,23,98]. The symptoms resemble a ﬂu-like or mononucleosis-like illness

FIGURE 6 Typical ‘‘natural’’ course of disease in an individual with HIV-1 infection. (Adapted from Refs. 53 and 91.)

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and are usually noted approximately 3–6 weeks after infection. Upon clinical examination, in almost all patients lymphadenopathy at one or more sites can be observed (see below), although it is frequently not noted by the individual. An immune response to HIV can be detected within 1 week to 3 months following infection (see below), and HIVspeciﬁc immune response leads to a decline in plasma viremia (Fig. 6) [25,53,91,94,99]. Even without treatment, in most individuals, viremia declines weeks to months after the acute syndrome subsides. The level to which the plasma HIV RNA level declines within 6–12 months following primary infection strongly correlates with the subsequent course of disease [9,92,100–104]. Lower plasma HIV RNA levels were associated with a slower rate of disease progression. Therefore this level is referred to as the viral ‘‘set-point’’ [100,101,105,106] (Fig. 6). Interestingly, treatment of patients during the acute HIV infection was shown in some studies to lower the viral set-point and to improve laboratory markers of immune function [107–111]. Therefore, treatment of patients during primary HIV infection may positively affect the long-term disease progression [25].

TABLE 6 Symptoms Observed During the Acute Retroviral Syndrome Fever (96%) Lymphadenopathy (74%) Pharyngitis (70%) Rash (70%): Erythematous maculopapular with lesions on face and trunk and sometimes extremities, including palms and soles. Mucocutaneous ulceration involving mouth, esophagus, or genitals. Myalgia or arthralgia (54%) Diarrhea (32%) Headache (32%) Nausea and vomiting (27%) Hepatosplenomegaly (14%) Weight loss (13%) Thrush (12%) Neurological symptoms (12%): Meningoencephalitis or aseptic meningitis; peripheral neuropathy or radiculopathy; facial palsy; Guillain-Barre´ syndrome; brachial neuritis; cognitive impairment or psychosis. Source: Adapted from Refs. 9 and 23.

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3.1.2

389

Chronic Infection: The Asymptomatic Stage

After the acute infection and apparent curtailment of viral replication most patients experience a symptom-free period of ‘‘clinical latency’’ that usually lasts for several years [9,91] (Fig. 6). Originally it was thought that very little viral replication occurred during clinical latency. However, throughout the often protracted course of chronic infection, viral replication can almost invariably be detected in untreated patients, both by highly sensitive assays for plasma viremia and by demonstration of viral replication in lymphoid tissues [112–114]. Clinical latency is not a period of microbial latency, and a hallmark of HIV disease is the establishment of a chronic persistent infection, with varying degrees of active viral replication [95,97,115]. In patients who do not receive efﬁcient antiretroviral therapy, plasma levels of HIV RNA may vary from fewer than 1000 copies per milliliter of plasma to more than 10 million copies/ mL. Laboratory studies and mathematical modeling of viral dynamics revealed that in infected individuals 10 billion to 100 billion (1010–1011) virions are produced daily [110,115–123]. The virus is rapidly cleared from the circulation, with a half-life of less than 6 hrs. Over 90% of the circulating virus is produced by recently infected activated CD4þ Tlymphocytes. The time HIV needs for replication in these cells was estimated to be approximately 1.5 days. As a consequence of infection these cells undergo a rapid turnover, with a productively infected cell having a half-life of about 1–2 days. At any given time approximately 0.01–0.1% of the body’s total CD4þ T lymphocytes are productively infected with HIV, and perhaps 100 times greater that number contain HIV nucleic acid. The levels of HIV in the blood are determined by the rate of virus production. The rates of production are a function of the number of infected lymphocytes in the lymphoid tissue, so the steadystate levels of HIV RNA are directly related to the rate of decline of CD4þ T-Iymphocytes. The higher the RNA levels, the faster the loss of CD4þ cells and the shorter the duration of HIV infection before death [102]. Because the CD4þ cell count determines the risk of disease progression and the level of HIV RNA determines the rate of CD4þ cell decline, these values are used routinely to assess clinical status and the time scale for initiation of chemotherapy [23,124,125] (see also Chap. 12). The pathogenesis of HIV and its treatment are complicated by the existence of cellular reservoirs and tissue compartments [95,115,126]. In addition to activated CD4 lymphocytes, macrophages and dendritic cells have long been recognized as important host cells for HIV replication [127,128,235,236]. Macrophages and dendritic cells represent longer-lived sources for HIV production [95,97], because they have a much longer

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half-life in tissues than activated CD4þ T-lymphocytes and, at least in cell culture, macrophages are not readily killed by HIV infection. CD4þT cells may also become latently infected by HIV-1, but in a resting state these cells contribute little to the bulk of virus produced. However, they represent a long-lived reservoir that provides a low but continuous supply of infectious virions [57,95,115]. HIV also enters speciﬁc tissue compartments such as the central nervous system, the testes, and perhaps other tissues [95,129]. Although these reservoirs do not contribute signiﬁcantly to the viral load in the blood, they represent sanctuaries that may be difﬁcult for the immune system and antiretroviral therapy to access [94,95,130,131]. 3.1.3

Clinically Apparent Disease

Viral replication leads to gradual deterioration of the immune system, manifested particularly by the depletion of CD4þ T cells (Fig. 6) [9,53,54,93,97,132]. The progressive deterioration of the immune system that occurs in most patients with HIV infection leads to clinically apparent disease or an AIDS-deﬁning illness (Table 2). The profound immunosuppression that occurs during the late phase of HIV infection is the end stage of immunopathogenic events that began at the time of infection and continued for years. Clinically apparent disease is more likely to occur when CD4þ T cells fall below 200 per microliter of blood. During the ﬁnal phase, plasma HIV RNA levels tend to rise and CD4þ T-cell counts may fall to undetectable numbers (Fig. 6). Exceptions to the direct correlation between immune function and clinically apparent disease are progressive generalized lymphadenopathy (PGL), neurological disease, and Kaposi’s sarcoma. PGL and neurological disease may occur as a direct result of HIV infection, and Kaposi’s sarcoma is the result of coinfection with human herpesvirus 8 [9,129,133–135]. Symptoms of HIV disease can occur at any time during the course of infection, and virtually any organ system can be affected [9,54,136]. However, the spectrum of illnesses observed changes as the CD4þ T-cell count declines. The more severe and life-threatening complications of HIV infection occur in patients with CD4þ T-cell counts below 200 mL1 (Table 7). Characteristically, the causative agents of secondary infections are opportunistic organisms such as P. carinii, atypical mycobacteria, CMV, and other organisms that do not ordinarily cause disease in the absence of a compromised immune system (Table 2) [7,9,137,138]. However, common bacterial and mycobacterial agents also frequently cause disease. Patients may also be affected by malignancies such as primary central nervous system lymphoma that are not frequently found in the general population. Approximately 80% of

TABLE 7

RT-PCR 32.6% 9.5% 3.2% 2.0% —b

>750 32.6% 16.1% 8.1% 2.0% 3.7%

501–750 47.9% 16.1% 8.1% 2.0% —b

351–500

64.4% 40.1% 8.1% —b —b

201–350

CD4þ T-cell count (cells/mL)

85.5% 50% 14% —b —b

<200

a With the ﬁrst generation bDNA assay there was a 2–2.3-fold difference compared to the RT-PCR values. Values obtained with the newer generation bDNA assay version and RT-PCR are similar, except for the lower end of the linear range (see Chap. 12). b Too few subjects were in this category to provide a reliable estimate. Source: Data from the MACS cohort study; adapted from Refs. 101 and 102.

>55,000 20,000–55,000 7,000–20,000 1,500–7,000 <1,500

Viral loada

Likelihood of Developing an AIDS-Deﬁning Condition Within 3 Years as a Function of T-Cell Count and

>30,000 10,000–30,000 3,000–10,000 501–3,000 <500

bDNA

Viremia

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deaths among AIDS patients are a direct result of infection other than HIV, with bacterial pathogens heading the list. Tuberculosis also represents a frequent illness in individuals with HIV infection, particularly in developing countries where prevalence is very high. Following the widespread use of antiretroviral therapy and implementation of guidelines for prevention of opportunistic infection in industrialized countries, the incidence of secondary infections has decreased signiﬁcantly (see below) [2,8,12,137–141]. 3.2

Immunopathogenesis of HIV Infection

In HIV disease a progressive quantitative and qualitative deterioration mainly of the cell-mediated immune functions results in a profound immunodeﬁciency. The most pronounced defects are observed for CD4þ T lymphocytes [132]; however, the other limbs of the immune system are also affected. Lymphoid tissues are the major anatomic sites for the establishment and propagation of HIV infection [97,112,113,121,142–146]. For practical reasons, most diagnostic procedures and studies on the pathogenesis of HIV infection have been performed on peripheral blood mononuclear cells and on virus released into the circulation. However, lymphocytes in the peripheral blood represent only approximately 2% of the total body lymphocyte pool and so may not always accurately reﬂect the status of the entire immune system. Furthermore, viral replication occurs mainly in lymphoid organs such as the lymph nodes, spleen, and mucosa-associated lymphoid tissues, and not in the blood [97]. Despite these limitations, the level of plasma viremia reﬂects virus production in lymphoid tissues and represents an important diagnostic marker of disease progression [100–102,104]. Lymphoid tissue involvement may result in lymphadenopathy, which is experienced by most patients to varying degrees during the course of HIV infection [9,97,98]. During early stages of HIV disease the architecture of the lymph nodes is generally preserved. Germinal centers may even be hyperplastic owing to in situ proliferation and recruitment of cells. Folicular dendritic cells (FDCs) appear to be relatively healthy. As the disease progresses, lymph node architecture gradually deteriorates, especially the germinal centers. Concomitantly, an increase is usually observed in the level of plasma viremia and the relative number of infected cells in the blood as well as in the proportion of cells expressing virus. With further progression of the disease to the advanced stage, destruction of the immune system continues. In the end stage of disease, high levels of plasma viremia are observed, which represent a true increase in viral replication due at least in part to diminution of the

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immune control of viral replication and most likely also to a loss of clearance in the lymphatic organs [9,97]. Events similar to those in the lymph nodes (although less well characterized) most likely also occur in other lymphatic organs such as the gut-associated lymphoid tissue [97,144]. Upon exposure to the virus, the initial infection of susceptible cells may vary somewhat with the route of infection (e.g., inoculation of virus directly into the bloodstream as opposed to exposure on a mucosal surface). Studies in animal models suggest that following mucosal exposure to SIV, Langerhans cells (a dendritic lineage cell type) are the ﬁrst immune cells to encounter the virus [53,92,97,147]. Langerhans cells may become infected by HIV, but in addition they have the ability to bind and sequester HIV through the cell surface molecule DC-SIGN [128,148,149]. Subsequently these cells migrate to the draining lymph node, where they form foci surrounded by CD4þ T cells that are activated by antigen processed and presented by dendritic cells on their HLA class II molecules [93,128,148,150,151]. In the proximity of HIVinfected or ‘‘HIV-presenting’’ dendritic cells, activated CD4þ T cells are especially vulnerable to infection and destruction by the virus. CD4þ T lymphocytes and cells of the monocyte/macrophage lineage are the major ultimate targets of HIV infection [97,127,132]. Once a productive infection has been established, HIV is virtually never cleared from the body and disseminates rapidly within the organs of the lymphoid system, to the brain, and to other tissues [91,95,97,129,135,152]. Soon after the initial HIV infection, viral replication intensiﬁes, leading to a burst of viremia [91,92,96]. The HIV-speciﬁc immune response (see below) leads to a decline in plasma viremia weeks to months after the acute infection (Fig. 6). In most untreated patients, early in the course of chronic infection when the viral set-point has been reached and prior to signiﬁcant immunodeﬁciency, the levels of plasma viremia are variable but generally low. In lymph nodes, in situ hybridization revealed expression of virus in the paracortical area (where T cells mainly reside) and to a lesser extent in the germinal centers [97,113,121,142,143,145,146]. The number of cells expressing virus increases as disease progresses. Remarkably, substantial amounts of extracellular virions are trapped in the germinal centers of lymph nodes, most likely on the processes of folicular dendritic cells (FDCs). Trapping of antigen by FDCs is a physiologically normal function of these cells. However, in the case of HIV, the trapped virus appears to serve as a source of cellular activation, resulting in the secretion of proinﬂammatory cytokines (see below).

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Moreover, trapped virus appears to remain infectious for CD4þ T cells and macrophages [9]. HIV infection results in progressive loss of CD4þ T cells from the circulation as well as depletion of CD4þ T cells from total body stores [91,93,97,132]. Total body CD4þ T cells may be depleted in their absolute numbers because they are destroyed or because their production is impaired. In addition, the fraction of circulating cells may decrease as a result of redistribution out of the intravascular space into the conﬁnes of lymphoid organs. In the late stage of disease, depletion of CD4þ T cells explains most phenomena of immune dysfunction observed in HIV infection. However, T-cell dysfunction is also observed early in the course of infection. The balance of production and destruction of CD4þ T cells can be tipped by multiple mechanisms. HIV may kill CD4þ T cells either by direct productive infection or mediated through contact with the virus or its gene products [57,93,97,153]. Alternatively, physiological responses to HIV infection might initiate events that result in destruction of uninfected cells [154]. In either case loss of mature cells should be compensated for by increased production of new cells, and mature CD4þ T-cell depletion should occur only if cells lost cannot be replaced. Several mechanisms operating in concert have been proposed to explain how HIV infection can affect the production, function, redistribution, and destruction of CD4þ T cells: 1. Clearly, HIV infection of humans and SIV infection of rhesus macaques result in an increased turnover of CD4þ T cells [93,97,110,119,123,155]. Numerous experiments have demonstrated that HIV infection can result in direct destruction of CD4þ T lymphocytes. Interestingly, HIV-infected macrophages are not readily killed by the virus [235–238]. Analysis of viral dynamics in vivo indicated that accelerated destruction of CD4þ T cells by direct infection accounts for only a fraction of the observed T-cell deaths [110,118,123,156]. Moreover, turnover of other cell populations, especially CD8þ T cells, is increased to a similar extent [93,110,122]. 2. Concomitant with the increased turnover of CD4þ T cells in HIV infection, a redistribution of CD4þ T cells is observed from the circulation into lymphoid organs. This is a physiological function but may contribute to the reduced T-cell counts observed in the peripheral blood [93]. 3. Normally the immune system is in a state of homeostasis awaiting stimulation by foreign antigens [151]. In HIV infection, however, this balance appears to be perturbed and a generalized activation of the immune system is observed, driven in part by the

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antigenic stimulus of HIV and in part by antigen-independent mechanisms [93]. This activated state is reﬂected by phenomena such as hyperactivation of B cells, activation of monocytes, expression of activation markers on various cell types including CD4þ T cells, secretion of proinﬂammatory cytokines [237,238], lymph node hyperplasia, and autoimmune phenomena [9,97]. From the standpoint of the virus, activation of its host cell is necessary for progeny production [57]. Chronic activation may, however, adversely affect CD4þ T-cell production and may lead to defects in maturation, to increased rates of cell death, and to dysfunction of CD4þ T lymphocytes [93]. Although incompletely understood, the relevance of activation to CD4þ T-cell depletion is underscored by the observation that disease progression is associated with immune activation, and vice versa [9,93]. 4. In addition to increased rates of destruction, in HIV infection CD4þ T-lymphocyte production is also impaired. CD4þ cell production and maturation was observed to be affected both in the bone marrow and in the thymus [9,157]. Mature CD4þ T cells are often derived from early progenitors that may also express CD4. Such progenitors, including multilineage and lineage-restricted hematopoetic progenitor cells of the bone marrow, may be infected by HIV and destroyed or rendered nonfunctional. Moreover, the function of bone marrow stromal cells may also be impaired in HIV infection. The picture of bone marrow dysfunction in HIV disease is complicated further by opportunistic infections, malignancies, myelotoxic drugs, and nutritional deﬁciencies [9,93]. The thymus, housing many CD4þ T lymphocytes in varying stages of maturation, is another critical target organ in HIV infection, and several abnormalities are observed, including a decrease in thymic output [93,155,158–160]. 3.3

Immune Response to HIV

Following the initial burst of viremia, HIV-infected individuals mount an immune response to HIV that usually curtails the level of plasma viremia and in the absence of antiretroviral treatment likely contributes to delaying the ultimate development of clinically apparent disease for a median of 10 years. This immune response contains elements of both humoral and cellular immunity and is directed against multiple antigenic determinants of the HIV virion [9,25,94,161]. Antibodies to HIV usually appear within 6 weeks of primary infection and almost invariably within 3 months. Initially, HIV-speciﬁc antibodies are not neutralizing but can be detected by biochemical assays such as ELISA or Western blot (see Chap. 12). Neutralizing antibodies

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generally appear following decreases in plasma viremia and thus correlate with the appearance of HIV-speciﬁc CD8þ T lymphocytes. The ﬁrst antibodies are those against the Gag proteins, especially against p24 (the capsid protein), and the appearance of these antibodies is associated with a decrease in serum levels of free p24 antigen. Subsequently, antibodies against envelope proteins and products of the pol gene appear. In addition, antibodies against the accessory proteins may be detected. Although antibodies to multiple HIV antigens are produced, their functional signiﬁcance in controlling viral replication is not clear [9,25,94,154,161]. Yet their appearance is of clinical importance for diagnosis of HIV infection (Chap. 12). Naturally, antibodies that can control viral replication are directed against the envelope proteins. However, the HIV envelope proteins are heavily glycosylated and dynamic in their structure and are thus not readily accessible for the immune system [65,66,126,162]. Most of the anti-envelope antibodies are directed against an epitope in gp41 or against the V3 loop of gp120. However, the V3 region is a major site for development of mutations leading to immune escape [65,66,126]. Therefore, neutralizing antibodies directed to V3 are usually type-speciﬁc and can thus neutralize only virus of a given strain. Group-speciﬁc neutralizing antibodies are capable of neutralizing a variety of HIV isolates and are directed to a different region in gp120 or to a region in gp41. Given the fact that T-cell-mediated immunity is known to play a major role in host defense against viral infections [151,154,163], it has also been shown to be an important component of the host immune response to HIV and SIV [25,94,109,111,164–167]. T-cell-mediated immunity can be divided into two major categories, mediated respectively by the helper/ inducer CD4þ T lymphocytes and the cytotoxic/immunoregulatory CD8þ T cells [151]. The number of HIV-speciﬁc CD4þ T cells has been found to be markedly reduced in HIV-infected individuals, particularly in advanced disease [93,94,108,165,168]. This difﬁculty may be related to the fact that CD4 cells represent the main target cells of HIV-1 [132]. Those CD4þ cells reacting by proliferation may be among the ﬁrst to be destroyed or rendered nonfunctional during HIV infection [93,97,153]. MHC class I restricted, HIV-speciﬁc CD8þ T cells have been identiﬁed in the peripheral blood of HIV-infected patients [164]. From studies both in infected patients [108,109,166,169–171] and in animal models [111,167,172], a picture is emerging that HIV-speciﬁc cytotoxic T lymphocytes (CTLs) are most effective in controlling viral replication [25,94,154]. These virus-speciﬁc CTLs possess a range of antiviral activities, including the ability to kill infected cells and to produce certain cytokines and chemokines. Several of these functions may be

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involved in controlling viral replication. In addition, a strong HIVspeciﬁc T helper cell response is needed to maintain a strong CTL function [94,165]. Studies in SIV-infected monkeys provide convincing evidence that depletion of CD8þ T cells (and therefore also CTLs) during chronic infection resulted in rapid increase in the steady-state level of viremia [167,172]. In patients an inverse relationship between CTL and viral load has been demonstrated [94,169]. Moreover, patients have been identiﬁed who have been infected for 20 years and yet maintain low viral loads without drug therapy, indicating that immune control of viral replication may indeed be possible. These rare patients typically have strong HIV-speciﬁc CTL and helper cell responses [25,166]. Interestingly, preservation of helper T-cell response and thus perhaps better control of HIV replication may be obtained by treatment of patients with antiretroviral therapy during primary infection [25,107–109,111,168,173]. 3.4

Escape of HIV from the Immune Response

Despite a potent humoral and cell-mediated immune response to HIV, in most infected individuals the immune system is apparently unable to eliminate the virus or to suppress viral replication completely [9,91,95]. How HIV evades elimination by the immune system is not completely clear; however, several mechanisms are likely to play a role [57,93,94,126,161,174,175]. Because of the properties of the viral glycoprotein, it appears to be difﬁcult to mount an efﬁcient humoral immune response that is capable of efﬁciently neutralizing the virus [66,154,161,162]. In addition, HIV has an extraordinary ability to mutate (see Sec. 4.5), quickly allowing the virus to evolve new epitopes [35,176]. HIV-speciﬁc CTLs are most effective in controlling viral replication (see above). Interestingly, during primary infection HIV-speciﬁc CTLs expand greatly, but during chronicity these clones decline, and their function may no longer be detectable in the late stage of disease [9,25,94,108,153,168]. The reason for this is unclear, and several mechanisms have been proposed. Escaping elimination by mutation of CTL epitopes appears to be one mechanism that is used intensively by the virus [37]. In addition, and similar to other chronic infections, CTL clones may simply become exhausted in the course of disease [154]. HIVspeciﬁc CTLs are detectable, but they seem to be largely unable to kill virus-infected cells or to suppress viral replication by other means [94,169,177]. This defect may simply reﬂect the lack of support by HIVspeciﬁc T helper cells, which are being destroyed or also impaired in their function by the virus [153,165]. Other means by which HIV can escape CTL attack include infection of cells of the central nervous system

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and sequestration of infected cells in the central nervous system where T cells normally have no access [95,126,129,134,135,178]. Yet another mechanism to account for the immune evasion may simply be related to the intrinsic biological property of HIV as a retrovirus to integrate into the genome of its target cell. Once the provirus is established, several mechanisms have been proposed as being potentially involved in preventing the elimination of infected cells by effector cells of the immune system, including viral latency [57,95,115,126,175,179,180]. Viral latency is deﬁned as a period during which a provirus remains dormant and no active gene expression or virion release is detectable. Therefore latently infected cells are not recognized by the immune system as being infected and thus are not eliminated by CTLs. In virtually all HIV-infected individuals a pool of resting CD4þ T cells and cells of the monocyte/macrophage lineage can be detected that most likely serve as components of the persistent reservoirs of the virus [95,115,156,179–182]. Once these cells become activated, viral gene expression can occur [57]. Although viruses produced from these pools of latently infected cells represent only a small fraction of those released into the circulation, they have a very long half-life [95,115,118,183]. Moreover, these pools of latently infected cells are not affected by present regimens of antiretroviral therapy. The virus also appears to possess active mechanisms to escape immune surveillance [57,86,94,126,175] (see Sec. 3.3). 3.5

Diagnosis

The diagnosis of HIV infection depends upon the demonstration of antibodies to HIV and/or direct detection of HIV or one of its components (see Chap. 12). The standard screening test for HIV is the enzyme-linked immunosorbent assay (ELISA), and the most commonly used conﬁrmatory assay is the Western blot. HIV-speciﬁc ELISA tests are very sensitive and allow a high throughput of samples. However, these tests are not optimal in their speciﬁcity. The Western blot takes advantage of the fact that HIV infection elicits antibodies against multiple well-characterized viral antigens of different molecular weights. Most tests detect antibodies to HIV-1 and to HIV-2, but HIV variability (Sec. 2.3) has posed a challenge for test design and interpretation. In addition to the detection of antibodies, techniques were developed for the detection of HIV RNA in plasma or HIV DNA in blood mononuclear cells or tissues. Tests for the detection of HIV RNA are very sensitive (present detection limit, 50 copies of HIV RNA/mL) and use either the principle of reverse transcriptase polymerase chain reaction (RT-PCR) or

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branched chain DNA assay (bDNA). Assays for analysis of viral DNA by DNA-PCR can detect one proviral copy per 10,000–100,000 cells. Detection of plasma HIV RNA (viral load) was shown to be an important prognostic determinant [100–102,104,119,120] and has become crucial in monitoring the efﬁciency of antiretroviral therapy [23,124,125,184]. The CD4þ T-lymphocyte count is the laboratory test generally accepted as the best indicator of the immediate state of immunological competence of a patient with HIV infection [9,132]. CD4þ T cells are measured by ﬂow cytometry and, as described above, CD4þ T-cell counts below certain threshold levels indicate an increased risk of disease progression. According to most guidelines, CD4þ T-cell counts are also used for decision making for the initiation of antiretroviral therapy (see. Sec. 4). For monitoring of antiretroviral therapy, viral resistance testing and analysis of plasma levels of antiretroviral drugs are recommended (Chap. 12) [23,125,185–187]. 4

PRINCIPLES OF ANTIRETROVIRAL THERAPY

Measures for control of an infectious disease can be divided into two categories: prevention and therapy. In the case of HIV, strategies for prevention include all measures to reduce the rate of sexual transmission (e.g., education and use of condoms), parenteral transmission (e.g., testing of blood products), perinatal transmission, and the development of a vaccine. Due to intrinsic biological properties of HIV, no preventive vaccination strategy is yet available, and it is not clear whether this goal will ever be achieved [95,126,154,161,188,189,239]. The only therapeutic modality that has been shown to be speciﬁcally active against the pathogen is chemotherapy [139]. Therapeutic regimens and efforts to improve therapy have evolved over time [2]. At the beginning of the HIV epidemic, no speciﬁc antiretroviral compounds were available (Fig. 7), and treatment options for HIV-infected individuals were limited to supportive measures and treatment of AIDS-deﬁning conditions. Beginning with AZT in 1987, several potent antiretroviral agents became available in the 1990s (Table 8) (Fig. 7) that inhibited the viral reverse transcriptase and the viral protease (Chaps. 13–15). In the same time frame several important advances in knowledge regarding HIV biology and monitoring were also made. Plasma viremia was recognized as a marker for viral replication in the body, representing a key predictive factor for disease progression and serving as a tool to monitor the efﬁciency of therapeutic regimens

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FIGURE 7 Availability of antiretroviral drugs since the outbreak of the HIV/ AIDS pandemic.

[100,101,104,106,190]. Clinical and laboratory studies demonstrated that single agents are not effective in long-term control of viral replication, and resistant viruses emerged rapidly [191–194]. However, combination therapy involving two or more antiretroviral drugs was clearly shown to be beneﬁcial, and the concept of highly active antiretroviral therapy (HAART) emerged [23,124,125,194–196]. Based on theoretical considerations, the possibility for HIV eradication using potent antiretroviral therapy was proposed [116,117,119,120,176], leading to recommendations for early and aggressive treatment [105]. The concept of eradication was based on the assumption that complete suppression of viral replication was achievable and that the pool of infected cells was rather short-lived, suggesting the possibility of a ‘‘cure’’ within 2–3 years. However, the use of potent regimens has proven difﬁcult (see below), and subsequent studies indicated that low-level virus replication may occur despite suppression of plasma viral RNA levels below detection limits [178,179,181,197,198]. In addition, infected cells with much longer life spans have been discovered [91,95,97,115,178,180,182,199], and treatment interruptions were found to lead to a rapid rebound of plasma viremia [200–203]. Models underlying predictions on virus elimination were reﬁned [115,118,183,204], and, against the background of existing treatment regimens, HIV eradication is not considered a realistic goal [9,23,125].

Ziagen Agenerase Rescriptor Videx, Videx-EC Sustiva Fuzeon Crixivan Epivir Kaletra Viracept Viramune Norvir Fortovase,b Inviraseb Zerit Viread Hivid Retrovir Combivir Trizivir

NRTI PRI NNRTI NRTI NNRTI Fusion PRI NRTI PRI PRI NNRTI PRI PRI NRTI NRTIc NRTI NRTI NRTI NRTI

Class ABC AMP DLV ddI EFZ T20 IDV 3TC LPV/r NFV NVP RTV SQV d4T TDF ddC ZDV, AZT

Abbreviation GSK GSK Agouron/Pﬁzer BMS BMS Roche/Trimeris Merck GSK Abbott Agouron/Pﬁzer Boehringer Abbott Roche BMS Gilead Roche GSK GSK GSK

Manufacturer

1999 1999 1997 1991 1998 2003 1996 1995 2001 1997 1996 1996 1995 1994 2001 1992 1987 1997 2000

Year of approvala

Abbreviations: NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PRI, protease inhibitor; fusion, fusion inhibitor; GSK, Glaxo-Wellcome, or Glaxo-SmithKline; BMS, Bristol-Myers Squibb; Boehringer, Boehringer Ingelheim. a Dates of approval may vary between countries. b Saquinavir-SGC, soft gel capsule (Fortovase); Saquinavir-HGC, hard gel capsule (Invirase). c Tenofovir disoproxil fumarate is a nucleotide reverse transcriptase inhibitor (see Chap. 13).

Abacavir Amprenavir Delavirdine Didanosine Efavirenz Enfuvirtide Indinavir Lamivudine Lopinavir/ritonavir Nelﬁnavir Nevirapine Ritonavir Saquinavir Stavudine Tenofovir disoproxil fumarate Zalcitabine Zidovudine (azidothymidine) Zidovudine/lamivudine Zidovudine/lamivudine/abacavir

Trade name

Availability of Antiretroviral Agents

Compound

TABLE 8

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Patients clearly beneﬁt from therapy [139] (see below), but the foundation of HIV therapeutics is now long-term management of a chronic infection. The therapeutic goal that is presently considered realistic is maximal and sustained suppression of viral load to restore and/or preserve immunological function, thus reducing HIV-related morbidity and mortality [23,124,125,139]. 4.1

When Should Therapy Be Initiated?

Standards of care for HIV infection change rapidly, and it is advisable to consult the up-to-date recommendations that are available on the Internet (e.g., http://www.hivatis.org; http://www.iasusa.org). Decisions regarding initiation or changes in antiretroviral therapy should be guided by monitoring laboratory parameters of plasma HIV RNA and CD4þ T-cell count (Table 9) (see also Chap. 12) as well as the clinical condition of the patient [23,125]. Special considerations may be necessary for speciﬁc patient groups such as children or pregnant women [20,21,205–207]. Different considerations are needed for treatment of chronic infection and primary infection [23,25,124,200]. Many clinical trials have clearly shown that plasma viremia (viral load) is the most signiﬁcant predictor for disease progression [9,23,100,101,104,125,190], and a statistically signiﬁcant dose–response type of association was demonstrated between decreases in plasma viremia and improved clinical outcome (Table 7). The CD4þ T-cell count represents an additional important predictor of clinical outcome, especially in patients having low CD4þ T-cell counts [125,208], and present treatment guidelines rely on both parameters [23,125]. Physicians and patients must weigh the risks and beneﬁts of starting antiretroviral therapy and make individualized informed decisions. Early initiation of therapy allows better control of viral replication, which is likely to prevent or delay immune system destruction [23,25,107–109,125,208,209]. However, apart from adherence problems and impact on quality of life, the emergence of resistance and the potential for metabolic abnormalities raise important long-term concerns. When to initiate therapy and what regimen to choose are important decisions, because they also have an impact on future treatment options. Most antiretroviral drug–naive patients achieve maximal viral load suppression within 6–12 months after initiation of therapy [125]. Predictors of virologic success include low baseline viremia and high baseline CD4þ T-cell count [100–102,209–211], rapid decline in viremia to <50 copies/mL [212], adequate serum levels of antiretroviral drugs, and

Any value Any value > 55,000

<55,000

<200 >200 but <350 >350

>350

Asymptomatic

Treatment should generally be offered.b Some experts would recommend initiating therapy, because 3-year risk of developing AIDS is >30% in untreated patients. Other experts would defer therapy and monitor CD4þ T-cell counts more frequently. 3-year risk of developing AIDS is <16% in untreated patients; therefore many experts would defer therapy and observe.

Treat.

Treat.

Recommendation

With the ﬁrst-generation bDNA assay there was a 2–2.3-fold difference compared to the RT-PCR values. Values obtained with the newer generation bDNA assay version and RT-PCR are similar, except for the lower end of the linear range. b Clinical beneﬁt has been demonstrated in controlled trials only for patients with CD4þ T cell counts of <200 cells/mL3. However, most experts would offer therapy at a CD4þ T-cell threshold of <350 cells/mL3. Data from the MACS cohort study [101,102] suggest an increased 3-year risk of developing AIDS for individuals with CD4þ T-cell counts of >200 and <350 cells/mL3 and plasma HIV RNA >10,000 copies/mL (see Table 7). Source: Adapted from Ref. 23.

a

Any value

Any value

Plasma HIV RNAa (copies/mL)

Symptomatic (AIDS, severe symptoms) Asymptomatic, AIDS Asymptomatic Asymptomatic

Clinical category

CD4þ T-cell count (cells/mL)

TABLE 9 U.S. Department of Health and Human Services Guidelines for the Initiation of Antiretroviral Therapy in Chronically HIV-1-Infected Patients

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adherence to drug regimens [213–215]. Although optimal strategies for achieving the therapeutic goals have not yet been fully delineated, efforts to improve patient adherence to therapy are clearly important [23,125]. For the purpose of this contribution we now review the 2002 guidelines for initiation of treatment for adolescents and adults, issued by the U.S. Department of Health and Human Services (DHHS) [23]. Treatment recommendations are based both on clinical studies and on expert opinions. Recommendations for patients with chronic infections are discussed ﬁrst (see Table 9): Therapy may be considered for asymptomatic individuals with a viral load below 55,000 copies/mL and CD4þ T-cell counts above 350 cells/mL. For this patient group, many experts would defer therapy while observing the patient more closely. However, antiretroviral treatment is frequently recommended by experts for individuals with conﬁrmed plasma HIV RNA levels above 55,000 copies/mL irrespective of CD4þ T-cell count, recognizing that the 3-year risk of developing AIDS in this patient group is greater than 30%. Treatment should generally be offered for patients with CD4þ T-cell counts below 350 mL1. Some serious illness, especially active tuberculosis and bacterial pneumonia, may occur when the CD4þ T-cell count is above 200 mL1. Furthermore, some laboratory markers show slower rates of favorable response when antiretroviral therapy is delayed until the 200 cells/mL threshold is reached [125]. However, some controversy exists as to whether individuals with viral loads below 10,000 copies/mL beneﬁt from therapy, and there are no deﬁnitive data that deﬁne at which level above 200 cells/mL therapy should be initiated. In the case of individuals with CD4þ T-cell counts below 200 cells/ mL, treatment should always be recommended, irrespective of the viral load and the clinical status. These individuals have a high 3year risk of developing AIDS, and clinical beneﬁt has clearly been demonstrated for this patient group. Therapy is recommended for all patients with established symptomatic HIV infection. However, acute treatment of a serious opportunistic infection may take precedence over initiation of antiretroviral therapy [124,125]. In situations of adverse drug interactions (e.g., rifampin and protease inhibitors) it may be wise to defer treatment until the opportunistic infection is controlled.

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Primary HIV infection is deﬁned as the period from initial infection to complete seroconversion [9,92]. Many patients experience some symptoms of the acute retroviral syndrome (Table 6) and are thus candidates for early therapy [23]. The rationale for early treatment of primary infection is to decrease the severity of the acute disease, diminish the number of infected cells, maintain or restore HIV-speciﬁc immune responses, possibly reduce the rate of viral mutation due to suppression of viral replication, and possibly lower the viral ‘‘set-point’’ to improve the subsequent course of disease (see Fig. 6 and Table 7) [9,23,25,124]. Early intervention in primary infection can lead to restoration or protection of HIV-speciﬁc immune responses, and preliminary data suggest that treatment for primary HIV infection with combination therapy has a beneﬁcial effect on laboratory markers of disease progression [25,107–109,111,208,209,216]. However, the perceived beneﬁts of therapy are based primarily on theoretical considerations, and clinical beneﬁts from early intervention have not been deﬁnitively established [23,25,125]. Therefore, potential beneﬁts should be weighed against potential risks, and individualized decisions need to be made. 4.2

Initial Therapy

Presently three classes of antiretroviral drugs are approved for treatment of HIV infection targeting two viral enzymes: protease and reverse transcriptase (Table 8) (Fig. 5). Whereas presently approved protease inhibitors (PIs or PRIs) are similar in their mode of action (see Chap. 15), inhibitors of reverse transcriptase represent two functional entities: substrate analogs [nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs)] (Chap. 13) and allosteric inhibitors [designated non-nucleoside reverse transcriptase inhibitors (NNRTIs)] (Chap. 14). Drugs from all three classes may be used in current regimens of combination therapy (Table 10) [23,125,194]. There are no deﬁnitive data regarding superiority of one acceptably potent regimen over another, and recommendations for speciﬁc combinations of individual drugs cannot be generally made. Choice of the regimen, however, should be individualized based on the strength of supporting data and on regimen potency, tolerability, adverse effect proﬁle, likely drug–drug interactions, convenience and adherence likelihood, potential for alternative treatment options if the initial regimen fails, and, possibly, baseline resistance testing results [23,125,185–187]. Each possible regimen has advantages and disadvantages (Table 10). Initial regimens of two NRTIs and one or two PIs, or of two NRTIs and one NNRTI, are recommended. Regimens of three NRTIs offer

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TABLE 10 Advantages and Disadvantages of Antiretroviral Regimens for Initial Treatmenta Regimen

Advantages

Recommended regimens PI þ 2 NRTIs Documented efﬁciency; longest experience for viral suppression; preserves NNRTI regimens for use in treatment failure. 2 PIsb þ 2 NRTIs High efﬁciency; increased drug levels; long experience for viral suppression NNRTI þ 2 NRTIs Convenient dosing; defers protease inhibitors 3 NRTIs Convenient dosing; defers PI and NNRTI

Regimens under evaluation PI þ NRTI þ NNRTI High potency TDF þ other regimens

High potency

Disadvantages

Complex regimens; long-term toxicity; drug–drug interactions

Long-term toxicity; drug–drug interactions; limited experience with ﬁxed combination LPV/rb Limited long-term data; resistance conferred by few mutations Lower potency than PI þ 2 NRTIs in patients with high baseline viral loads; limited long-term data Complex regimen; multiple drug toxicity Limited experience

a

See text for details. See Table 8 for abbreviations. Low-dose ritonavir is given as an inhibitor of Cyp 450 in order to increase drug levels of the ﬁrst PI (see Sec. 4.6). In this regimen, low-dose ritonavir is designated by ‘‘. . . /r’’ such as LPV/r. Source: Adapted from Refs. 23, 124, and 125.

b

advantages, but there is concern about their potency in patients with high baseline RNA levels and more long-term data are needed. In addition, regimens including drugs from all three classes and regimens including tenofovir, enfuvirtide or experimental compounds (see Chap. 16) are also being assessed. In all cases, issues of adherence, adverse effects, and drug–drug interactions are important considerations (see below).

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407

Effects and Beneﬁts of Antiretroviral Therapy

When effective combination regimens of three or more drugs are given to HIV-infected patients, plasma virus decays in phases (Fig. 8) [115– 118,176,204]. An initial rapid about 100-fold exponential decline (ﬁrst phase) is followed by a slower exponential decline (second phase). The slope of the decline depends on the efﬁcacy of the therapy, with faster declines corresponding to more potent regimens. Virologically efﬁcient regimens generally reduce viral load by more than 90% within 4–8 weeks of therapy. Plasma HIV RNA levels should continue to decline over the following weeks, in most individuals to below 50 copies/mL (present detection limit; see Chap. 12) within 16–20 weeks after initiation of therapy [117,125,212,217,218]. The precipitous decline in the ﬁrst phase mainly corresponds to inhibition of virus production from acutely infected activated CD4þ T lymphocytes [115–117,119,120]. Virus originating from longer-lived populations of productively infected cells (such as macrophages), from tissue reservoirs, or from latently infected cells

FIGURE 8 Decay of circulating virus following initiation of effective antiretroviral therapy. (Adapted from Refs. 53 and 117.)

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following activation also affects the slope of the ﬁrst phase to some extent and is responsible for the slower decay represented by the second phase [95,115,117,118,204,219]. Estimates from mathematical models of viral dynamics suggest that some latently infected cell types such as resting CD4þ memory T cells may have a half-life of up to several decades [9,95,115,180]. Clearly, the pool of latently infected cells is not affected by present antiretroviral agents, and albeit at low levels it represents a continuous source of virus production [156,178,179,181,197,198]. In many patients treated with HAART, viral loads remain below 50 copies/mL for months or years. Viremia may, however, occasionally and transiently increase (so-called ‘‘blips’’), and in another subset of patients, low levels of plasma viremia may be constantly observed [23,125,197,220]. Discontinuation of therapy usually leads to relapse of viral load to pretherapy levels within 1 or 2 months, even after years of maximal suppression of viral replication [25,200,201,203]. Suppression of viral replication with antiretroviral therapy is clearly associated with an improvement of immune function and with a better clinical outcome as evidenced by reduced morbidity and mortality of HIV-infected individuals. Before the availability of effective antiretroviral therapy, AIDS advanced to being the leading cause of death among persons 25–45 years of age in the United States (Fig. 9). With the availability of HAART in the mid-1990s, both the incidence of

FIGURE 9 Trends in numbers of deaths of persons 25–45 years old due to leading causes 1987–2000, in the United States. *Data for 2000 are preliminary. (Data from Centers for Disease Control and Prevention, Atlanta, Georgia.)

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AIDS and the number of AIDS-related deaths signiﬁcantly declined in industrialized countries (Fig. 9) [2,8,12,137,139,141,221,222]. Moreover, despite the increasing cost of antiretroviral drugs, treatment of patients with these agents has been shown to be cost-effective from a public health point of view [139,223–225]. 4.4

Side Effects

Potent antiretroviral therapy comes at a cost, because dosing regimens are complex and the compounds have side effects [9,23,124,125]. Due to their pharmacokinetic proﬁles, most antiretroviral agents presently in use require two or three doses per day at ﬁxed intervals, frequently with more than one tablet (high pill burden). Resorption of some compounds is dependent on food intake. In some cases convenience and pharmacokinetic problems were solved by new formulations (Fortovase2), by coformulations of two or three compounds (Combivir2, Trizivir2), or by exploiting cytochrome P450 inhibitory properties (Kaletra2) (see below). A number of antiretroviral drugs may lead to serious subjective complaints, especially to gastrointestinal intolerance (nausea, vomiting, ﬂatulence, diarrhea, stomatitis) and symptoms of the central nervous system (headache, insomnia, dizziness, abnormal dreams, etc). Some side effects are class-speciﬁc. For example, long-term administration of NRTIs may lead to pancreatitis, peripheral polyneuropathy, lactic acidosis, and hepatic steatosis. These symptoms are the result of mitochondrial toxicity caused by interference with mitochondrial DNA replication through inhibition of mitochondrial DNA polymerase gamma. The relative potency of nucleoside analogs in this regard is highest for ddC followed by ddI, d4T, 3TC, ZDV, and ABC. In contrast, the ﬁrst-generation nucleotide analog tenofovir does not seem to cause signiﬁcant mitochondrial toxicity. Non-nucleoside reverse transcriptase inhibitors may lead to an increase in hepatic transaminase levels and frequently cause skin rash (which usually resolves after 2–3 weeks of treatment). Efavirenz readily crosses the blood-brain barrier, leading to central nervous system symptoms (including dizziness, somnolence, insomnia, abnormal dreams, confusion, impaired concentration) that can be severe. Treatment with protease inhibitors frequently leads to abnormalities of lipid metabolism (increased triglyceride and cholesterol levels) and hyperglycemia (insulin resistance), which may lead to premature cardiovascular disease, cerebrovascular disease, pancreatitis, and cholelithiasis. Changes in body fat redistribution, sometimes referred to as ‘‘lipodystrophy syndrome’’ are frequently observed in patients receiving

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HAART [9,23,226]. The physical changes occur gradually and are generally not apparent until months after the initiation of HAART. Clinical ﬁndings include central obesity, peripheral fat wasting, and lipomas. Pathological changes may include visceral and dorsocervical fat accumulation, breast enlargement, extremity wasting, and facial thinning. All these changes can be very disﬁguring and have led to treatment interruption in a number of cases. The mechanisms leading to lipodystrophy are still unclear. An increased incidence of lipodystrophy has been associated with the use of protease inhibitors but may also occur with NRTI therapy or in the absence of antiretroviral treatment. Especially in asymptomatic patients, side effects and dosing regimens may have a stronger impact on the quality of life than symptoms caused by the HIV infection itself, ultimately leading to poor adherence to the drug regimen. Poor adherence, however, is associated with incomplete suppression of viral replication and leads to drug resistance. 4.5

Resistance

Drug failure has been broadly deﬁned as inadequate viral suppression and is also termed virological failure (deﬁned as conﬁrmed detectable HIV RNA) [23,124,185,186,192,193]. The main reasons for virological failure are resistance to antiretroviral agents or poor adherence to the drug regimen. Resistance to antiretroviral agents is a frequent problem in the treatment of HIV-infected patients, and therefore an understanding of the underlying principles is crucial for decision making for antiretroviral therapeutic regimens. Following virological failure, virus resistance is determined by using laboratory tests. Two strategies for evaluation of resistance are available: genotypic analysis and phenotypic tests (see Chap. 12) [185,186]. Genotypic tests rely on the detection of mutations in the virus genome that are known to confer resistance to antiretroviral drugs. Phenotypic tests use molecular biological techniques to transfer resistance-conferring mutations to otherwise drug-sensitive laboratory strains of HIV, subsequently allowing analysis of viral sensitivity to the drug in the test tube. HIV has an extraordinary ability to mutate, with about one mutation occurring in every replication cycle. Emergence of mutations is strictly dependent on viral replication [176]. With about 10–100 billion virions produced per day in infected individuals (see Sec. 3.1.2), the high mutation rate of HIV-1, and its genome size, mutations would on average occur in every position in the genome multiple times each day (if distributed evenly over the genome), and a sizable fraction of

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all possible double mutations would also occur each day [118,176]. Although many virions would not ﬁnd a target cell and many mutations would have no effect or would be deleterious for the virus, some variants would by chance confer resistance to antiretroviral drugs. Viral variants with an advantage for replication in the presence of the drug would accumulate and might become predominant in the patient. Replication of this variant would invariably yield new mutations, conferring resistance to additional antirectroviral drugs and ultimately resulting in selection of multidrug-resistant viruses [192]. For all treatment regimens it is therefore imperative that maximal suppression of viral replication be achieved, because only this will prevent or delay the evolution of resistant viruses. Conversely, suboptimal drug concentrations with inadequate suppression of viral replication favor the emergence of resistant viruses [23,124,125]. At least in a laboratory, resistance can be overcome with higher drug concentrations. In patients, however, very high drug concentrations may not be achievable. So there is a need to develop compounds with higher potency and higher safety margins, permitting treatment regimens using high doses that are more likely to achieve maximal inhibition of viral replication, even in the face of moderately resistance conferring mutations. At present three classes of antiretroviral compounds are available. Resistance against a drug within one class is frequently (but not always) associated with resistance against other members of the same class (see Chaps. 13–15) [37]. Therefore, combination therapy using agents from more than one class is thought to be more potent and less prone to development of resistance than combinations with agents from a single class [9,23,125,194,195]. Moreover, drugs developed against targets other than reverse transcriptase and protease are likely to increase therapeutic effectiveness and to overcome resistance (see Chap. 16) [227]. Interestingly, some resistance-conferring mutations to one drug were observed to increase sensitivity to other drugs (see Chap. 14). Also of note, drugresistant viruses can have an impaired biological ﬁtness compared to wild-type viruses and may be less pathogenic. This observation raises important questions as to whether to continue therapy in the presence of resistance [228–231]. Currently there are several drugs in clinical development against targets other than RT and PR (Chap. 16), and there is great hope for an increase of therapeutic effectiveness of existing regimens and for overcoming at least some problems of resistance [227].

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Pharmacokinetics

In addition to antiretroviral therapy, HIV-infected individuals may be treated for various other conditions and receive prophylactic therapy for prevention of opportunistic infections [9,23]. Thus, patients are frequently exposed to a variety of drugs, and drug–drug interactions and food–drug interactions can occur, affecting both the effectiveness and toxicity of the regimens used [232]. Food–drug interactions mainly affect absorption, whereas drug–drug interactions can affect both absorption and, more commonly, elimination. Many drugs are eliminated from the body through common pathways involving so-called cytochrome P450 (Cyp 450) enzymes [233]. Some drugs (e.g., protease inhibitors, especially ritonavir) have the ability to inhibit this enzyme, leading to delayed clearance of drugs from the circulation that are normally eliminated by this pathway. Alternatively, some drugs (such as rifampin) induce the activity of Cyp 450 enzymes, accelerating clearance of other compounds eliminated by this pathway. Independent of the mechanism involved, delayed clearance or higher rates of absorption may lead to elevated serum levels of some drugs, increasing drug toxicity. Accelerated clearance or reduced absorption may lead to lower serum levels and reduced drug efﬁciency. Suboptimal levels of antiretroviral drugs lead to accelerated selection of resistant viruses. In antiretroviral therapy, drug–drug and drug–food interactions are a matter of serious concern [9,23,124,125,232]. However, drug–drug interactions may also be expoited to the beneﬁt of the patient. For example, ritonavir efﬁciently inhibits Cyp 450 3A4, and it was noted that ritonavir can improve the pharmacokinetic proﬁle of other protease inhibitors, resulting in higher trough levels and increased efﬁciency of these compounds in vivo [23,125]. A ﬁxed combination between lopinavir and ritonavir (Kaletra) has shown promising results in clinical trials [234]. The HIV pandemic has been matched by an unprecedented research effort in all areas of HIV disease, leading to an explosion of information, and this trend continues at a rapid pace. Information on the newest developments and on various topics of HIV including therapy is available on the World Wide Web (Table 11).

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Table 11 Selected Sites on the World Wide Web That Provide Information About Various Aspects of HIV/AIDS http://www.unaids.org

http://www.eurohiv.org

http://www.cdc.gov http://www.cdc.gov/nchs http://www.cdcnpin.org

http://www.global-campaign.org/ http://www.hivatis.org

http://www.iasusa.org

http://www.fda.gov/oashi/aids/ hiv.html http://www.actis.org http://hiv-web.lanl.gov

http://hivdb.stanford.edu http://www.niaid.nih.gov/daids/ default.htm

http://www.virology.net/ATV dict.html

Joint United Nations Program on HIV/AIDS (UNAIDS); worldwide epidemiology and information on the HIV/AIDS pandemic European Center for Epidemiological Monitoring of HIV and AIDS U.S. Centers for Disease Control and Prevention (CDC) U.S. National Center for Health Statistics U.S. Centers for Disease Control and Prevention; information on prevention of HIV infection Information on HIV prevention Guidelines for treatment recommendations of the Department of Health and Human Services (DHHS), U.S.A. International AIDS Society—USA; information and treatment guidelines, drug resistance mutations U.S. Food and Drug Administration (USFDA) AIDS clinical trials information service, U.S.A. Los Alamos National Laboratory, U.S.A.: HIV sequence database, virus resistance database, algorithms for sequence analysis, vaccine trial database, immunology database HIV and RT sequence database and drug resistance mutations U.S. National Institute for Allergy and Infectious Disease, Division of Acquired Immunodeﬁciency Syndrome Basic information about AIDS and virology in general

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ACKNOWLEDGMENTS We thank Hans-Georg Kra¨usslich for providing ﬁgures and Guy Hewlett for critical reading of the manuscript.

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12 Diagnosis and Management of HIV Infection Using Immunoassays and Molecular Technologies Rainer Ziermann, Charlene E. Bush-Donovan, and David A. Hendricks Bayer HealthCare Diagnostics, Berkeley, California, U.S.A.

1

INTRODUCTION

Human immunodeﬁciency virus (HIV) is having a devastating effect, with more than 40 million people infected worldwide. Because HIV results in lifelong incurable disease, a number of assays for diagnosing and monitoring this virus in infected individuals have been developed and reﬁned in the past 15 years. HIV infection is usually diagnosed and subsequently conﬁrmed by detection of antibodies to the virus using immunoassay technology. Quantitative molecular assays for HIV RNA viral load measurements have become the standard of care in the management of HIV infection. These assays provide information for timing the initiation of therapy, monitoring the efﬁcacy of therapy, and deriving prognostic value. In the event of treatment failure or in light of relevant information about the transmission of potentially drug-resistant virus, the patient may be tested for drug resistance–associated mutations to aid in selecting the most appropriate antiretroviral drug regimen. 433

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In this chapter we present the state of the art for immunoassays and quantitative molecular assays used in the diagnosis and management of HIV infection. The sections of the chapter are organized so as to parallel the sequence of assays likely to be encountered by a patient, beginning with HIV immunoassays and followed by viral load and drug resistance assays. However, recent discoveries of the potential beneﬁcial effects of antiviral treatment in the acute phase of infection (i.e., before antibodies are present) leading to a long-term nonprogressor status should be borne in mind [1–4]. It appears that viral suppression by drug treatment during the acute phase preserves those T cells that are HIV-speciﬁc and able to react to HIV by proliferation (rather than being killed by the virus), thereby allowing the individual to achieve better control of viremia at later stages. If these observations are supported by more clinical data, assays that detect the virus very early (i.e., from days to 2 weeks) after infection could become valuable tools in managing potentially HIVinfected individuals. Although we focus on assays for HIV-1 infection, we include those assays that are available for analyzing HIV-2 infection. We describe the development of and molecular basis for each type of molecular assay. We also present the current status of commercially available assays. 2

HIV IMMUNOASSAYS

The two main questions in HIV diagnostics are whether a person is infected and, if infected, how actively the virus is replicating. Several immunological and virological methods have been developed to diagnose and to monitor the progression of HIV infection. In this section we present the development and current status of HIV immunoassays, including screening and conﬁrmatory assays to detect antibodies to HIV, rapid and home HIV antibody tests, HIV Western blot tests, indirect immunoﬂuorescence assays, line immunoassay, and alternative testing strategies. 2.1

Assays to Detect Antibodies to HIV

Most exposed individuals will produce HIV-speciﬁc antibodies within a few weeks to a few months after viral infection. The time to seropositivity depends on the infectious dose, the transmission route, the sensitivity of the antibody test, and a variety of host-related factors. Using ﬁrstgeneration tests, seroconversion is estimated to occur on average 45 days after infection [5]. In a study based on the use of a third-generation antigen sandwich test, seroconversion occurred, on average, 21 days after

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infection [6]. A serological testing strategy and algorithm have been developed, using the third-generation assays, to identify an individual who is in the early period of infection before peak antibody production has occurred [7]. This testing strategy involves the sequential testing of serum or plasma using a screening ELISA according to the manufacturer’s instructions, followed by a second testing of the serum or plasma using the same ELISA but under less sensitive conditions. An algorithm is used to interpret the sensitive/less sensitive data and to identify a person as having a very early infection. This assay can provide information about HIV incidence and infection duration. The ultrasensitive fourth-generation assay that simultaneously detects HIV antibody and antigen has been incorporated into some newer testing algorithms [8]. Longitudinal serological testing of infected individuals over long periods of time has revealed that the major viral antigens to which antibodies are produced have remained fairly consistent throughout the population over time. The timing and intensity of an individual’s antibody response to a speciﬁc HIV antigen, however, may vary from individual to individual. The genome of HIV is known to code for three structural polyproteins and seven regulatory accessory proteins. The structural proteins and polyproteins of HIV (Fig. 1) are the targets of the circulating antibodies directed against the virus. The targeted structural proteins of HIV may include the envelope (Env) proteins (gp 120, gp 41, and the precursor gp160 glycoprotein), the polymerase (Pol) proteins (p66, p31, and p10), and the core (Gag) proteins (p18, p24, p7, and the precursor p55 protein). Antibodies against p24 and against gp160 and gp41 are among the ﬁrst HIV-speciﬁc immunoglobulins detected after infection. Tests to detect antibody to HIV can be deﬁned as screening or conﬁrmatory. HIV screening tests are designed to detect all potentially infected individuals. Conﬁrmatory tests determine the actual presence of speciﬁc anti-HIV antibodies and can identify individuals who are not infected but who have a false-positive screening test result. The enzyme-linked immunosorbent assay (ELISA) is the standard screening test for HIV infection. As designed, ELISAs are highly sensitive (to detect infection), whereas conﬁrmatory tests are highly speciﬁc (to detect false-positives). The Western blot is the standard conﬁrmatory test for HIV infection. As of April 2002, there were 24 HIV diagnostic kits for detection of HIV antibody licensed by the FDA (Table 1). Current information about licensed or approved HIV tests may be obtained at the Center for Biologics Evaluation & Research website at http://www.fda.gov/cber/products/testkits.htm.

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FIGURE 1

2.2

Schematic diagram of the components of the HIV-1 virion.

HIV Antibody Screening Tests

ELISAs are the most commonly used tests to screen for HIV infection. These tests have relatively simple formats that lend themselves to automation and batch testing and have inherent high sensitivity. The HIV ELISAs were developed in a series of stages or generations [9]. The FDA ﬁrst approved a screening test for the detection of antibodies to HIV in serum and plasma in March 1985. This ﬁrst-generation ELISA was based on an indirect binding format and used a puriﬁed viral lysate as the target antigen. In this format, the target antigen was bound to a solid support (microtiter plate wells or macroscopic beads) and the patient test serum was added and allowed to react with the antigen-coated support. After a wash step to remove unbound serum components, a secondary anti-human antibody with a bound enzyme conjugate was added. If the patient serum contained antibodies to HIV, the secondary antibody bound the Fc portion of the patient’s anti-HIV antibody. Following an additional wash step to remove unbound secondary antibody, substrate was added. The substrate produced a color reaction catalyzed by the bound enzyme conjugate, and color was detected by a spectrophoto-

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TABLE 1

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Companies Providing Licensed/Approved HIV Immunoassays

Company information

Licensed/Approved HIV immunoassays

Abbott Laboratories Abbott Park, IL, U.S.A. http://www.abbottdiagnostics.com

Abbott HIVAG-1 Monoclonal Abbott HIVAG-1 HIV AB HIV-1 EIA (donor) HIV AB HIV-1 EIA (non-donor) Abbott HIVAB HIV-1/HIV-2 (rDNA) EIA

Bio-Rad Laboratories Blood Virus Division Redmond, WA, U.S.A. http://www.bio-rad.com

Genetic Genetic Genetic Genetic Genetic Genetic

Calypte Biomedical Corp. Berkeley, CA, U.S.A. http://www.calypte.com

Cambridge Biotech HIV-1 Western Blot Kit (donor) Cambridge Biotech HIV-1 Western Blot Kit (non-donor) HIV-1 Urine EIA

Coulter Corporation Mi ami, FL, U.S.A. http://www.coulter.com

Coulter HIV-1 p24 Ag Assay; HIV-1 p24 Antigen ELISA Test System Coulter HIV-1 p24 Ag Assay

Epitope, Inc. Beaverton, OR, U.S.A. http://www.epitope.com

OraSure HIV-1 Western Blot Kit

Home Access Health Corp. Hoffman Estates, IL, U.S.A. http://www.homeaccess.com

Home Access HIV-1 Test System

formerly Murex Diagnostics, Inc. now Abbott Laboratories Abbott Park, IL, U.S.A. http://www.abbottdiagnostics.com

Murex SUDS HIV-1 Test

formerly Organon Teknika Corp. now bioMe´rieux Marcy-’Etoile, France http://www.biomerieux.com

Vironostika HIV-1 Microelisa System (donor) Vironostika HIV-1 Microelisa System (non-donor) Oral Fluid Vironostika HIV-1 Microelisa System

Systems Systems Systems Systems Systems Systems

rLAV EIA (donor) rLAV EIA (non-donor) HIV-1 Western Blot (donor) HIV-1 Western Blot (non-donor) HIV-1/HIV-2 Peptide EIA HIV-2 EIA

formerly Waldheim Pharmazeutika Fluorognost HIV-1 IFA (donor) now Sanochemia AG Fluorognost HIV-1 IFA (non-donor) Vienna, Austria http://www.ﬂuorognost.com

meter. Using known positive and negative serum samples that were run as controls, a standard curve for the color reaction was generated, and the optical density (OD) values of the patient serum were compared to those of the positive and negative controls. A cutoff value for the lowest OD still regarded as a positive signal was calculated on the basis of a statistical comparison of the intensity of a panel of positive control samples with that of a panel of negative control samples. Samples with an OD cutoff value greater than 1.0 were considered antibody-positive.

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Samples with an OD reading skirting the cutoff range were problematic and were considered indeterminate. All sera with positive and indeterminate results would be repeated and further evaluated by a conﬁrmatory test. The second-generation HIV ELISAs were introduced in the late 1980s and differed from the ﬁrst-generation assays through the introduction of a new source of target antigens. In these secondgeneration assays the target antigens were produced through recombinant DNA technology and/or synthetic peptide antigens that supplemented or replaced the crude viral lysates used in the ﬁrst-generation assays. These antigen preparations were considerably more pure than the earlier ones and allowed for more controlled binding of antigen to the solid support. For the ﬁrst time these assays combined both HIV-1- and HIV-2-speciﬁc antigens. A potential limitation of these recombinant antigen-based assays, however, is their inability to represent a full complement of HIV antigenic sites. The virus with which a patient is infected may exhibit reactive proteins that vary from the antigens used in the test. Consequently, the patient’s antibodies may not bind efﬁciently to the recombinant or synthetic antigens used in the test, and a falsenegative result will be recorded. This problem was found to be of clinical signiﬁcance in the early 1990s when some of these assays failed to detect infection by a new subtype 0 strain of HIV [10]. Overall, these secondgeneration assays have provided signiﬁcantly improved sensitivity over ﬁrst-generation conventional ELISAs. The third-generation double antigen sandwich ELISA method was introduced in the early 1990s. In this method, the HIV-speciﬁc antibody in the patient sample is ‘‘sandwiched’’ between two antigen molecules. One antigen is immobilized on the solid support (well or bead), and one antigen is conjugated with enzyme used to detect the antigen–antibody complex. The addition of substrate results in color development that is proportional to the amount of antibody in the patient’s sample. This format was developed to allow for the simultaneous detection of all isotypes to HIV-1 and HIV-2 in the clinical sample, including IgG, IgM, and IgA. The third-generation format detects HIV seroconversion on average 21 days after infection and approximately 5 days earlier than second-generation assays [11]. Moreover, these assays demonstrate superior sensitivities and speciﬁcities compared to second-generation assays, ranging from 96.9% to 100% and from 89.9% to 99.9%, respectively [12]. Third-generation-plus assays also detect HIV-1 group O [9]. Early data from a new third-generation-plus assay, ADVIA Centaur HIV-1 þ ‘‘O’’/HIV-2, under development by Bayer Corporation, demonstrates superior sensitivity (>99.9%) and speciﬁcity

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(>99.9%) compared to some third-generation assays. Additionally, because this assay employs two substrate addition steps, there is enhanced seroconversion sensitivity compared to third-generation assays [13]. The next generation HIV screening ELISA was developed in the late 1990s to further reduce the window period between time of HIV infection and diagnosis. These new fourth-generation screening assays combine the detection of anti-HIV antibodies (HIV-1, HIV-2, and group O) with the simultaneous detection of HIV p24 antigen. This simultaneous combination of HIV antibody and p24 antigen testing has further reduced the diagnostic window period in most patients to about 14 days, which is approximately 1 week less than third-generation assays [8,14]. It should be understood that the detection limit of these fourth-generation assays is dependent upon the sensitivity of the p24 antigen test component. Typically, the fourth-generation p24 antigen test component has been less sensitive (20 to >100 pg of p24 antigen/mL) than single p24 antigen assays (3.5–10 pg of p24 antigen/mL). A new fourthgeneration assay, VIDAS HIV DUO Ultra from bioMe´rieux (Marcy’Etoile, France), has been designed with an HIV p24 antigen detection limit equal to single p24 antigen assays. These fourth-generation assays have a reported sensitivity of 99.5–100% and speciﬁcity of 98–100% [14]. There are several causes of false-positive and false-negative ELISA results. Apart from technical and sample-handling errors, the major cause of a false-positive result is the presence of antibodies in a patient’s sera that recognize common human leukocyte antigens (HLA-DR) and cross-react with cellular contaminants that are present in the viral antigen lysates. These cross-reactive antibodies are present in a patient’s sera as a result of exposure to fetal cells during pregnancy or transfusions. The use of recombinant and synthetic peptide antigens has reduced this problem and has signiﬁcantly improved test speciﬁcity [15]. There are several additional causes of false-positive test results, including Epstein-Barr virus infections, hepatic disease, recent vaccinations (inﬂuenza), passive immunoglobulin administration, pregnancy, hemophilia, hemodialysis, history of injection drug use, and malignancies [16]. The causes of false-negative ELISA results are also varied. In addition to technical and handling errors, the single major cause of a false-negative result is the performance of the test on a patient sample prior to HIV seroconversion. Additionally, HIV-infected individuals on immunosuppressive therapy and undergoing replacement transfusions can also test negative by ELISA [16].

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Rapid and Home HIV Antibody Tests

Rapid tests for detecting HIV-speciﬁc antibody are deﬁned as those tests that take less than 30 min to yield results and require minimal laboratory equipment. These tests are technically less complex and less expensive than the more conventional HIV antibody screening methods. When performed accurately, these tests have been reported to have wide utility, including applications for testing in emergency rooms, autopsy rooms, funeral homes, organ donation centers, and physician ofﬁces. These tests are particularly useful under ﬁeld conditions where laboratory facilities may be less than optimal. Rapid tests can be of variable formats, including agglutination, dot immunobinding assays, immunoglobulin capture, double antigen sandwich, and indirect binding [17,18]. Most rapid assays include a control for correct test performance. This control is composed of an anti-human antibody that binds any immunoglobulin in the patient sample and produces a positive result, thus indicating that all reagents were added correctly. Additionally, many of the rapid tests are formatted to provide separate results that allow for the distinction between HIV-1 and HIV-2 infection. The overall sensitivity of rapid assays has been reported to be less than that of most recent generation ELISA tests [19]. Therefore, the use of these tests usually involves an emergency situation, ﬁeldwork, or other conditions where a ‘‘stat’’ result is required. The FDA has approved the Home Access HIV-1 Test System from Home Access Health Corp. (Hoffman Estates, IL). This test kit includes an instruction booklet about HIV and the test process, a lancet to produce a ﬁnger prick, a test card blood blotter with a unique identiﬁcation number, and a Federal Express mailer to the Home Access Laboratory. Results are complete in 3 days, and the customer can obtain results over the telephone when the unique identiﬁcation number is provided. Home Access Laboratory personnel are also available to provide counseling and medical referrals. This test system has reported excellent sensitivity and speciﬁcity, primarily because the testing protocol and performance remain in the hands of skilled laboratory technicians [20].

2.4

HIV Western Blot Test

The Western blot is the most widely used conﬁrmatory test for the detection of speciﬁc antibodies to HIV. It is considered by most authorities to be the ‘‘gold standard’’ for verifying a diagnosis of HIV infection. A positive result both conﬁrms the presence of antibodies in the individual that are capable of speciﬁcally reacting with HIV and

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deﬁnes which speciﬁc viral components the individual’s immune system has recognized. The test format is a line immunoassay in which individual HIV antigens are separated and immobilized on a nitrocellulose test strip. The test strip is incubated with a 1:50 or 1:100 dilution of a patient’s sample or control. The strip is washed and incubated with enzyme-conjugated antihuman immunoglobulin, and bands are detected by subsequent incubation with substrate that produces an insoluble colored band on the strip. Colored bands are indicative of a speciﬁc antigen–antibody reaction. By comparison with controls, the speciﬁc band pattern is determined. The HIV antigen band pattern that is resolved on the nitrocellulose blot (top to bottom) is gp160, gp120, p66, p55, p51, gp41, p31, p24, p21, and p18. Many laboratories follow the Association of State and Territorial Public Health Laboratories and the Centers for Disease Control and Prevention (CDC) guidelines for interpretation, which require a positive band result for at least two of the following antigens: p24, gp41, gp120/ 160 [21]. Western blot results that cannot be classiﬁed as either negative or positive are grouped into a category called indeterminate results. Illustrations of various HIV Western blot band patterns are shown in Figure 2. Indeterminate Western blot results occur when the antibody pattern does not meet the full criteria for a positive result. These results occur in approximately 3–8% of ELISA positive specimens [22]. Some individuals with indeterminate results eventually seroconvert, some remain indeterminate for years, and some retest negative. The recommendations for retesting vary by organization. In general the recommendation is to retest highly suspicious Western blots in 2 weeks, and if the repeat test is still indeterminate the individual should be considered for retesting in a few weeks. Indeterminate Western blots have been described in individuals with systemic lupus erythematosus, underlying herpesvirus infections including herpes simplex virus type 1 and cytomegalovirus, and in individuals after inﬂuenza vaccination [16]. Frequently the cause of an indeterminate Western blot result remains unknown. The positive predictive value of the Western blot is at least 99% when it is used as a conﬁrmatory test in sequence with a positive antibody screening test. An algorithm for the diagnosis of HIV infection in adults is shown in Figure 3. 2.5

Modiﬁed Western Blot

In some clinical situations an individual may test positive for HIV antibodies by screening ELISA but be found to have an indeterminate

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FIGURE 2 Sketch of Western blot banding patterns representing HIV positive (A), indeterminate (B, C), and negative (D) proﬁles.

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FIGURE 3 Model algorithm using serological testing in the diagnosis of HIV infection.

Western blot, with an atypical banding pattern suggestive of crossreactivity with HIV-2. As discussed previously, most commercial HIV screening ELISA assays react with both HIV-1 and HIV-2 antibodies. If HIV-2 infection is suspected, on epidemiological grounds or because of a suspicious Western blot, then the CDC has recommended that more than one test be performed as a screening or conﬁrmatory test on these samples. FDA-licensed HIV-2 ELISA screening assays have been developed. However, currently there are no HIV-2-speciﬁc immunoblot tests approved by the FDA. Modiﬁed Western blot assays have been

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developed that can differentiate and identify infections by both HIV-1 and HIV-2. One such assay, QualiCode HIV-1/2 Kit from Immunetics (Cambridge, MA) incorporates HIV-1 viral lysate and synthetic HIV-2 Env peptides. A positive result for HIV-2 must include reaction to the HIV-2-speciﬁc antigens plus a reaction to HIV-1-speciﬁc antigens, which alone would not meet the accepted criterion for HIV-1 positivity. 2.6

Indirect Immunoﬂuorescence Assay

In the United States, indirect immunoﬂuorescence assay (IFA) technology is used in some laboratories as a screening test and/or as a conﬁrmatory test for HIV infection. This technique employs immortalized human T cells expressing HIV surface antigens that are ﬁxed to a microscope slide. Patient serum or plasma possibly containing HIV antibodies is incubated with the infected cells. The slide is washed, and antibody to human immunoglobulin conjugated with ﬂuorescent label is added. The slide is viewed under a ﬂuorescent microscope. If antibodies to HIV are present in the specimen, then a characteristic ﬂuorescence pattern is discerned in comparison to uninfected negative control cells or HIV-infected positive cells. This technology has been shown to be as sensitive as the ELISA for screening and superior to Western blot technology for discrimination between HIV-1 and HIV-2 infection [23]. In general, this technology has been reported to have sensitivity and speciﬁcity equal to that of Western blot technology for conﬁrmation of HIV-1 infection [24]. 2.7

Additional HIV Conﬁrmatory Assays

The radioimmunoprecipitation assay (RIPA) is an alternative technology to the Western blot for conﬁrmation of HIV infection [25]. This technique employs T cells that are cultured in the presence of HIV and radioactively labeled amino acids to produce an HIV virion with labeled proteins. The HIV is partially disrupted with detergents and incubated with patient serum that may contain antibodies to HIV. HIV-speciﬁc antibodies bind to the labeled viral antigens, forming an antigen– antibody complex. The antigen–antibody complexes are immunoprecipated by incubation with protein A–coated Sepharose that binds the Fc portion of the anti-HIV antibody present in the complex. The antigen– antibody immunoprecipitate is subjected to gel electrophoresis, which separates the individual HIV proteins by molecular weight. The gel is dried and is overlaid with X-ray ﬁlm. The resultant autoradiograph is analyzed against controls for the speciﬁc banding patterns. The banding pattern seen by LIA is similar to that produced by Western blotting.

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Because of its complexity and the use of radioactivity, this technique is mainly performed in research and reference laboratories. Because a patient’s serum may be preadsorbed with uninfected T cells, which could eliminate nonspeciﬁc reactivity to HIV antigens, this technique has most frequently been used to resolve indeterminate Western blot results. The INNO-LIA HIV Conﬁrmation Assay (Innogenetics, Ghent, Belgium) is another conﬁrmation test that has been used to perform typespeciﬁc serodiagnosis of HIV-1 (including group 0) and HIV-2 [26]. The assay design is based on a line immunoassay format that uses HIV-1 and HIV-2 antigens coated onto a nylon membrane strip that recognize antibodies to HIV-1 p17, p24, p31, gp41, and gp120 as well as HIV-2 gp36 and gp105. Automated reading and interpretative software have been developed for this assay. 2.8

Alternative HIV Antibody Testing Strategies

In addition to serum and plasma, other body ﬂuids such as urine, oral mucosal transudate, and saliva are known to contain HIV antibodies. Both screening and conﬁrmatory tests have been developed using these ﬂuids. Assays that employ urine and oral ﬂuid as the test sample have the advantage of requiring noninvasive sample collection and can be more easily performed in a doctor’s ofﬁce or in the ﬁeld where blooddrawing personnel or supplies may be less accessible. The FDA has approved tests for using both oral ﬂuids and urine for the detection of antibodies to HIV. The accuracy of tests using oral ﬂuids or urine is similar to that of the conventional HIV antibody tests, and the window of seroconversion limitations is the same [27]. 3 3.1

HIV-1 VIRAL LOAD ASSAYS Nucleic Acid-Based Assays for Quantiﬁcation of HIV

Quantiﬁcation of infectious agent(s) in individuals is a basic tenet in management of infectious diseases. In general, prognosis is better if the quantity of the agent is very low or not detectable. Decreases or increases in the quantity of the infectious agent during therapy indicate good or poor response, respectively. These principles served as the basis in the early 1990s for development of assays that provided measures of viral load (VL), the ‘‘quantity’’ of virus in the infected individual. Approaches to the direct quantiﬁcation of HIV included enumeration of virions by electron microscopy and measurement of virion-associated molecules such as proteins or nucleic acids. The new diagnostic tests ultimately targeted HIV viral genomes, because the number of RNA molecules per

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virion was known and they were virion-associated in plasma. These tests do not distinguish between HIV RNA in infectious and noninfectious virions. 3.2

Technologies for Quantiﬁcation of HIV RNA

Suppression of VL to very low levels or below detection during antiretroviral therapy was the impetus for development of ultrasensitive VL assays. Technologies employed in these assays require ‘‘ampliﬁcation’’ by either signal enhancement or the creation of additional copies of target sequences; the result is the generation of sufﬁcient signal for reproducible quantiﬁcation even at low VL. These approaches to ampliﬁcation are shown schematically in Figure 4. Signal ampliﬁcation is based on development of large hybridization complexes with many signal-generating moieties that accumulate on HIV RNA from the original specimen. Target ampliﬁcation results in the production of large amounts of either RNA or DNA molecules (i.e., amplicons) that are generated in enzyme-dependent reactions using sequences in the input HIV genome as the original template. Assays employing target ampliﬁcation are based on the assumption that the amount of amplicons generated is directly proportional to the quantity of HIV nucleic acid in the original specimen. 3.2.1

Versant1 HIV-1 RNA 3.0 Assay (bDNA)

The VERSANT1 HIV-1 RNA 3.0 Assay (bDNA), produced by Bayer Corporation, Diagnostics Division, uses signal enhancement. A schematic of this technology is shown in Figure 5. A 1.0 mL plasma specimen is centrifuged to concentrate virus. Addition of a lysis buffer results in the release of HIV-1 RNA from virions; the lysis buffer contains components designed to inactivate nucleases that are in the specimen. Multiple capture extenders (oligonucleotides) in the lysis buffer hybridize to multiple sequences in the pol gene of HIV-1 as well as to capture probes immobilized on the surfaces of 96-microwell plates, thus anchoring the HIV-1 genomic RNA to the plate. Sequential addition of target probes (complementary to the pol gene), preampliﬁer molecules (complementary to a portion of the target probes), ampliﬁer molecules (complementary to a portion of preampliﬁer molecules), and alkaline phosphatase–modiﬁed label probes (complementary to portions of the ampliﬁer molecules) results in the formation of large hybridization complexes on the input viral RNA. If each hybridization step exhibited 100% efﬁciency, each HIV-1 RNA molecule could be labeled with 10,080 alkaline phosphatase molecules. Following addition of dioxetane, the

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FIGURE 4 Signal versus target ampliﬁcation. Target ampliﬁcation technologies, including PCR, nucleic acid sequence-based ampliﬁcation (NASBA), and transcription-mediated ampliﬁcation (TMA), achieve ampliﬁcation by generating multiple copies of speciﬁc sequences in the target nucleic acid molecules. By contrast, signal ampliﬁcation by the branched DNA (bDNA) technology enhances the signal by sequential hybridization of probes to create complexes with many signal-generating moieties.

substrate of alkaline phosphatase, a steady-state chemiluminescent signal is produced in proportion to the number of HIV-1 RNA molecules in the specimen. The amount of HIV-1 RNA in the specimen is calculated by interpolation from a standard curve generated by signals produced from calibrators that contain inactivated cell-culture-grown HIV-1. 3.2.2

NucliSens1 HIV-1 QT Test

The NucliSens1 HIV-1 QT Test, available from bioMe´rieux (Marcy’Etoile, France) is based on nucleic acid sequence-based ampliﬁcation

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FIGURE 5 Principles of branched DNA technology (bDNA) in the VERSANT1 HIV-1 RNA Assay (bDNA). Virus is concentrated by centrifugation, and HIV-1 RNA is released by addition of lysis buffer that contains components that inactivate nucleases in the specimen. The HIV-1 RNA is anchored to the microwell plate by hybridization of capture extenders to sequences in the pol gene of HIV-1 and to capture probes on the surface of the microwell plate. Target probes hybridized to the HIV-1 RNA facilitate signal ampliﬁcation through sequential hybridization of synthetic preampliﬁer molecules, ampliﬁer molecules, and alkaline phosphatase–modiﬁed label probes. Addition of dioxetane substrate produces a steady-state chemiluminescent signal that is proportional to the number of HIV-1 RNA molecules, and the amount of HIV-1 RNA in the specimen is calculated using a standard curve.

(NASBA) technology, producing RNA amplicons through target ampliﬁcation (Fig. 6). A plasma specimen is mixed with a lysis buffer containing guanidinium isothiocyanate and Triton X-100, which results in the release of HIV-1 RNA from virions and inactivation of nucleases. Three internal calibrators of known low, medium, and high concentrations of RNA are added to lysed mixtures, and viral and calibrator RNAs

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FIGURE 6 Principles of nucleic acid sequence-based ampliﬁcation (NASBA) technology in the NucliSens1 HIV-1 QT Test. HIV-1 RNA is released from virions by addition of lysis buffer and then concentrated along with three internal calibrator RNAs onto silicon dioxide particles under high salt conditions. Single-stranded DNA molecules are generated by primer extension from four template RNAs, a gag-speciﬁc primer containing the T7 RNA polymerase recognition site, and avian myeloblastosis reverse transcriptase (AMV-RT). After degradation of the RNA template with RNase H, a second strand of DNA is generated with a second primer and AMV-RT, and T7 RNA polymerase catalyzes production of antisense RNA amplicons. Amplicons are captured separately, and the ratio of electrochemiluminscence from the HIV-1 RNA and the appropriate calibrator RNAs is used to determine the amount of HIV-1 RNA in the original specimen.

are concentrated under high salt conditions onto silicon dioxide particles according to the method of Boom et al. [28]. Internal calibrators differ from the wild-type RNA by only a small sequence. RNAs are eluted from the particles. Through primer extension using HIV-1 or calibrator RNAs as templates, a gag-speciﬁc primer

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containing the T7 RNA polymerase recognition site, and avian myeloblastosis virus reverse transcriptase (AMV-RT), a single-stranded DNA molecule is generated from each of the four types of RNA templates. The RNA template is degraded with RNase H, and the second strand of DNA is generated through primer extension with a second primer and AMV-RT. T7 RNA polymerase in the reaction mixtures catalyzes production of antisense RNA amplicons. These reactions, which occur simultaneously, are isothermal (at one temperature). Amplicons from each of the four template RNAs are captured separately, and electrochemiluminescence is generated. The quantity of HIV-1 RNA in the original specimen is determined from the ratio of electrochemiluminescence from the HIV-1 RNA and from the appropriate calibrator. 3.2.3

AMPLICOR HIV-1 MONITOR1 Test, Version 1.5

The AMPLICOR HIV-1 MONITOR1 Test, version 1.5, available from Roche Molecular Systems, Inc. (Pleasanton, CA, U.S.A.) employs the polymerase chain reaction (PCR) technology for target ampliﬁcation with generation of DNA amplicons (Fig. 7). Viral RNA is released by guanidinium isothiocyanate in a lysis reagent either directly from plasma specimens or from virus concentrated from plasma in a centrifugation step. Then HIV-1 RNA is concentrated by precipitation with alcohol. Reverse transcription (RT) and PCR ampliﬁcation from the HIV-1 RNA template are carried out using 50 -biotinylated primers to a sequence in the HIV-1 gag gene and thermostable recombinant enzyme Thermus thermophilus DNA polymerase (rTth pol). A quantiﬁcation standard (QS) with a known concentration of molecules is added with the lysis reagent, and amplicons are produced from it as well as from HIV-1 RNA during the assay. The QS consists of a noninfectious HIV-1 transcript containing primer binding sites identical to those on HIV-1 RNA and unique internal sequences that allow amplicons from the QS to be distinguished from amplicons from HIV-1 RNA. Amplicons from the QS and from HIV-1 RNA are denatured and captured onto microwell plate surfaces by hybridization to amplicon-speciﬁc probes on the well surfaces. An avidin–horseradish peroxidase conjugate is incubated on wells. A color complex is then formed in the presence of the enzyme, hydrogen peroxide, and substrate 3,30 ,5,50 -tetramethylbenzidine. Amplicons from QS and from HIV-1 RNA are captured to different wells using different probes on the microwell surfaces. Quantiﬁcation of HIV-1 RNA is determined by the ratio of color complexes formed from amplicons from the QS and from HIV-1 RNA.

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FIGURE 7 Principles of polymerase chain reaction (PCR) technology in the AMPLICOR HIV-1 MONITOR1 Test, version 1.5. HIV-1 RNA is released by addition of guanidinium isothiocyanate and precipitated with alcohol. Reverse transcription and PCR ampliﬁcation of the HIV-1 RNA template and a quantiﬁcation standard is carried out using 50 -biotinylated primers and a thermostable DNA polymerase. Amplicons from the HIV-1 RNA and quantiﬁcation standard are denatured and captured by hybridization to amplicon-speciﬁc probes on the surface of the microwell plate. Addition of an avidin–horseradish peroxidase conjugate results in the production of a color complex, and the ratio of color complexes produced by amplicons from the HIV-1 RNA and quantiﬁcation standard are used to determine the amount of HIV-1 RNA in the specimen.

3.2.4

LCx HIV RNA Quantitative Assay

The LCx HIV RNA Quantitative Assay from Abbott Laboratories (Abbott Park, IL) employs competitive reverse transcription PCR (target) ampliﬁcation between input HIV-1 RNA and an internal standard (IS) using primers to conserved sequences in the pol IN gene [29]. A schematic of this technology is shown in Figure 8. The IS RNA transcript, which contains sequences recognized by the HIV-speciﬁc primers and a unique internal sequence, is added to lysis buffer that is added to each specimen. The IS controls for possible inhibition of any of the steps of the assay. HIV-1 RNA and IS are coextracted using a modiﬁed Qiagen sample preparation kit. RT-PCR ampliﬁcation is carried out with puriﬁed

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FIGURE 8 Principles of competitive reverse transcriptase PCR technology in LCx HIV RNA quantitative assay. HIV-1 RNA and an internal standard (IS) RNA transcript are coextracted using a Qiagen sample preparation kit. Following reverse transcription and PCR ampliﬁcation, amplicons are hybridized with adamantane-labeled HIV-speciﬁc probes and dansyl-labeled IS-speciﬁc probes. Complexes are captured onto magnetic particles. Particles are incubated with alkaline phosphatase–labeled antibodies that recognize adamantane and with b-galactosidase-modiﬁed antibodies that recognize dansyl moieties on the IS-speciﬁc probes. Substrates for alkaline phosphatase and b-galactosidase are added. The amount of HIV-1 RNA in the specimen is determined by interpolation of the signal produced from the sample against a standard curve.

RNA as template, Thermus thermophilus DNA polymerase, and primers at a concentration that is rate-limiting. The IS is at a concentration of 750 copies per reaction. Following ampliﬁcation, an adamantane-labeled HIV-speciﬁc probe and a dansyl-labeled IS-speciﬁc probe are allowed to hybridize to amplicons, if present, from HIV-1 RNA and IS RNA,

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respectively. These complexes are detected in a microparticle enzyme immunoassay performed on an LCx Analyzer. In brief, amplicons are captured onto magnetic particles that are modiﬁed with anticarbazole antibodies that recognize the carbazole hapten on the reverse primer. The particles are incubated with anti-adamantane antibody linked to alkaline phosphatase and anti-dansyl antibody linked to b-galactosidase; the former antibody binds to complexes containing amplicons from HIV-1 RNA, and the latter binds to complexes with amplicons from the IS RNA. Substrates for alkaline phosphatase and b-galactosidase are added, and signal is generated in proportion to the amount of amplicons produced. The amount of HIV-1 RNA in a specimen is determined by interpolation of the signal produced from the sample against a standard curve made of external calibrators that are included in each assay run. 3.2.5

Gen-Probe HIV-1 Viral Load Assay

The Gen-Probe HIV-1 Viral Load assay, a prototype assay in research at Gen-Probe (San Diego, CA, U.S.A.), has been described by Emery et al. [30]. This assay employs target ampliﬁcation using transcriptionmediated ampliﬁcation (TMA) with the production of RNA amplicons (Fig. 9). Plasma specimen is mixed with a lysis buffer that results in the release of HIV-1 RNA from virions, and RNA is captured onto poly(dT)modiﬁed magnetic particles by means of a probe that has both oligo(dA) sequences and virus-speciﬁc sequences. RNA amplicons are produced from this captured RNA using reverse transcriptase and T7 RNA polymerase in TMA that is isothermal and results in a theoretical ampliﬁcation of 109-fold. In brief, a primer that hybridizes to a sequence in the HIV-1 genome and contains the sequence recognized by T7 RNA polymerase is extended with reverse transcriptase to produce an antisense DNA molecule that is part of an RNA–DNA hybrid. RNA in this RNA–DNA duplex is degraded by the RNase H activity of reverse transcriptase, and the complementary sense strand of the DNA is synthesized using a second primer and reverse transcriptase. Large numbers of RNA amplicons are produced from the double-stranded DNA molecule using T7 RNA polymerase. RNA amplicons are detected by addition of DNA probes with acridinium ester–modiﬁed labels. Probes that do not hybridize to RNA amplicons are degraded, and those that form hybrids with RNA amplicons yield chemiluminescence in a subsequent step of the assay. The amount of HIV-1 RNA in a specimen is determined by interpolation of chemiluminescence produced from the sample against a standard curve made of external calibrators that are included in each assay run.

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FIGURE 9 Principles of transcription-mediated ampliﬁcation (TMA) technology in HIV-1 viral load assay. HIV-1 RNA and internal control RNA are captured onto magnetic particles, and exponential ampliﬁcation of the target RNA with generation of RNA amplicons is achieved by using reverse transcriptase and T7 RNA polymerase. The reverse transcriptase creates a double-stranded DNA template that includes the T7 promoter sequence, and the T7 RNA polymerase transcribes RNA from the DNA template to produce antisense RNA amplicons. DNA probes modiﬁed with chemiluminescent labels are added, and only those DNA probes that hybridize to RNA amplicons are protected from degradation. The amount of HIV-1 RNA in the specimen is determined by interpolation of chemiluminescence produced from the samples against a standard curve.

3.2.6

Real-Time PCR

Another promising new technology for detection and quantiﬁcation of HIV is kinetic or ‘‘real-time’’ PCR. One of the disadvantages of conventional endpoint PCR methods is that reactants may be ratelimiting, and therefore the relationship between concentration of input

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target HIV RNA and the amount of amplicons produced is not linear. This shortcoming limits the dynamic ranges of endpoint assays. In contrast, real-time PCR is based on the detection of amplicons as they accumulate during the PCR reactions. A standard curve based on the time (i.e., PCR cycle) of appearance of amplicons from calibrators with known amounts of target HIV RNA is produced in each run. The amount of viral nucleic acid in a specimen is determined from the standard curve based on the time of appearance of amplicons from the specimen. There are several chemistries used for detection of amplicons in real-time PCR methods, which have recently been reviewed in detail [31]. Real-time PCR offers the advantage of a very wide dynamic range (e.g., covering 8 log10 copies/mL) and low interassay and intra-assay variability [31]. One of the chemistries that have been applied to real-time detection of HIV, the TaqMan technology, relies on the Taq polymerase’s 50 ! 30 endonuclease activity on speciﬁc oligonucleotide–target DNA duplexes (Fig. 10A). The TaqMan technology employs a probe labeled with a ‘‘reporter’’ ﬂuorophore and a ‘‘quencher’’ moiety; ﬂuorescence is not emitted as long as both moieties are on the probe. During extension from primers during the PCR reaction, the double-modiﬁed probes that are annealed to amplicons are susceptible to degradation by the Taq polymerase, thus releasing the reporter ﬂuorophore from inhibition by the quencher moiety. Production of amplicons is detected by an increase in ﬂuorescence. Methods using TaqMan PCR technology have been developed for detection of HIV-1 RNA for screening blood donations [32,33] as well as for quantiﬁcation of HIV-1 proviral DNA [34,35]. Quantitative TaqMan-based PCR methods also have been developed for HIV-2 RNA and proviral DNA [36,37]. Another chemistry that has been applied to real-time detection of HIV involves the use of molecular beacons, which are single-stranded oligonucleotide reporter probes that form a stem–loop structure. Molecular beacons have a ﬂuorophore covalently attached at one end and a quencher at the other. When free in solution, the stem–loop structure of the molecular beacon keeps the ﬂuorophore and quencher in close proximity, and ﬂuorescence is quenched by energy transfer. However, when hybridized to one of the amplicon strands, the molecular beacon undergoes a conformational change (i.e., disruption of the stem– loop structure) that allows the ﬂuorophore to ﬂuoresce (Fig. 10B). Quantitative real-time PCR methods using molecular beacons have been described for HIV-1 RNA [38] as well as for HIV-1 and HIV-2 proviral DNA [39]. A similar chemistry involving the use of scorpion probes has also been applied to real-time detection of HIV. Like molecular beacons,

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FIGURE 10 Principles of real-time PCR using (A) TaqMan technology, (B) molecular beacons, and (C) scorpion primers. In TaqMan technology, the 50 ! 30 endonuclease activity of DNA polymerase displaces and hydrolyzes the oligonucleotide probe, resulting in removal of the reporter ﬂuorophore from the quencher and production of ﬂuorescence. With molecular beacons, ﬂuorescence is produced upon separation of the ﬂuorophore from the quencher by hybridization of the molecular beacon to one of the amplicon strands. Similarly, ﬂuorescence is produced upon separation of the ﬂuorophore from the quencher upon hybridization of the scorpion probe to the amplicon.

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scorpion probes form a stem–loop structure that positions a ﬂuorophore and quencher in close proximity, thereby preventing ﬂuorescence when in solution. Upon hybridization of the scorpion probe to the amplicon, ﬂuorescence is released (Fig. 10C). A real-time quantitative assay using scorpion probes has been described for HIV-1 RNA and DNA [40]. 3.3

Clinical Applications for Assessment of Viral Load

During the 1990s, clinical data emerged from studies of VL that established a clear link between viral load and disease progression and transmission. One of the earliest of these was described by Mellors et al. [41,42], who evaluated the risk of progression to AIDS or death in Multicenter AIDS Cohort Study (MACS) patients who were evaluated twice a year with a median follow-up of AIDS-free patients of 9.6 years. These investigators found that people with a baseline VL of >30,000 copies/mL (as measured by bDNA assay) had a 12.8- or 18.1-fold greater risk of progressing to AIDS or death, respectively, within 6 years than did individuals who had VL <500 copies/mL. In addition, they found that baseline VL was a stronger predictor of disease progression than CD4 cell count; these factors together were more predictive of disease than either factor alone. Risk of perinatal transmission of HIV from infected mothers to infants was sixfold greater if mothers had detectable virus than if virus was undetectable [43]. Other studies have found similar relationships between VL and disease progression [44–49]. Although HIV-1 levels remain elevated for longer periods in infants than in adults after infection, steady-state VL in infants is also predictive of disease progression [50]. By the end of the 1990s, guidelines for the management of HIVinfected patients were established. Viral load results were shown to play a key role because they can be applied to decisions regarding initiation of therapy and management of patients on therapy. Guidelines for initiation and monitoring of therapy have been written and updated by the U.S. Department of Health and Human Services [51] and the International AIDS Society, U.S. Panel [52]. To establish baseline VL, the average of two measures made within 1–2 weeks of each other should be determined. Therapy is generally recommended for people who have a VL of 30,000–55,000 copies/mL regardless of the CD4 cell count. After the start of therapy, VL should be measured after 4 weeks, then every month until VL is suppressed to below detection by the ultra sensitive assays. VL should be determined every 2–3 months in the clinically stable patient. An increase in VL may indicate development of resistance

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to antiviral agents, drug intolerance, intercurrent infection, recent vaccination, biological variability, or poor patient compliance. A threefold change (decrease or increase) in VL is considered to be statistically signiﬁcant. Following VL in the management of HIV-1-infected patients is the standard of practice in resource-rich countries today. The goal of therapy is to suppress VL to below detection by ultrasensitive assays. Muir et al. [53] reported that suppression of virus to <50 copies/mL was not more predictive of longer term (i.e., 12 months) response to therapy than was suppression to <400 copies/mL. Kempf et al. [54], in contrast, showed that suppression of VL to <50 copies/mL was associated more frequently with longer term (48 week) viral suppression during highly active antiretroviral therapy (HAART) than was suppression to <400 copies/mL. Raboud et al. [55] reported that patients with VL <20 copies/mL were at lower risk of virological failure during therapy than were individuals with VL of 21–400 copies/ mL. In contrast, Havlir et al. [56] described intermittent levels of virus, between 50 and 200 copies/mL, in patients without virological failure (i.e., >200 copies/mL) on initial triple combination therapy. These ‘‘blips’’ in VL may result from assay variation, improper specimen handling, or less effective viral suppression. Changes in VL need to be conﬁrmed by testing a second specimen, especially if changes in therapy are being considered.

3.4

Performance Attributes of Assays: Relevance to Disease Management

To provide clinical utility, described previously, VL assays should produce accurate and reproducible quantiﬁcation values regardless of the level of HIV and the genetic make-up of the HIV RNA targets. All of the VL assays measure the amount of HIV RNA in plasma, a specimen selected primarily because it is easily accessible. However, it should be recognized that HIV is also present in other compartments of the body [57] and therefore the total body VL can only be inferred by quantiﬁcation of virus in plasma. Important performance characteristics of assays as they relate to clinical utility are summarized in Table 2. During the 1990s, there was some progress toward standardization of methods for assessment of assay performance [58–61]. Still, it is very difﬁcult to compare performance of assays from data from the scientiﬁc literature because different authors used different experimental approaches.

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TABLE 2 Performance Characteristics of Assays for HIV Viral Load Performance attribute Sensitivity

Accuracy

Precision

Speciﬁcity

Linearity

Equal quantiﬁcation of all genotypes

Description

Clinical signiﬁcance

Lowest level of HIV RNA that can be Important to detect virus at very low detected consistently (e.g., 595% levels to assess the degree of viral of the time). Deﬁned as limit of suppression during therapy. detection. Refers to the ability of the assay to Results from clinical trial with one determine the true value of assay can be applied in analyte. Very difﬁcult to establish management of patients with any ‘‘gold standard’’ for VL. Calibration of the assays. Treatment (IAS and DHHS) guidelines of assays to common standard describe a viral load threshold for should result in consistency of initiation of therapy. quantiﬁcation between assays. Standardization will result in management thresholds being the same for all assays. Capacity to achieve very similar viral Physician and patient need to quantiﬁcation values regardless of understand if viral load decreases the lab, day, operator, instrument, or increases are statistically or kit lot. signiﬁcant or within the variability expected from biological variability plus analytical variance. Determines the change in viral load Treatment guidelines describe that is statistically signiﬁcant. threefold viral load changes as statistically signiﬁcant. Quantiﬁcation values for HIV RNA HIV VL assays designed to be used obtained only in specimens that only in people who are known to really contain HIV. be HIV-positive (i.e., antibodypositive). High speciﬁcity gives physicians and patients conﬁdence that assay really assesses only HIV RNA. Refers to the degree to which the Important to know that changes in VL assay standard curve reﬂect actual changes in amount of approximates a straight line. Used virus (without contributions from to determine the linear range of the quantiﬁcation biases in speciﬁc assay. areas of the dynamic range). Linearity is a measure of how accurately the assay measures changes in VL throughout its dynamic range. Capacity of assay to accurately Because treatment guidelines depend measure VL regardless of on speciﬁc viral loads, assay must sequence variation in HIV RNA. accurately quantify virus regardless of HIV group or subtype. Some assays that underquantify certain subtypes have yielded VL values that are inconsistent with CD4 counts and/or the clinical status of the patient.

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Accuracy

To assess the capacity of an assay to obtain the true value for VL, a ‘‘gold standard’’ preparation containing a known amount of HIV is required. The ideal gold standard preparation should be quantiﬁed by a means independent of hybridization-based assays to avoid potential inaccuracy of quantiﬁcation by any of the assays. Such a gold standard has not been adopted. A panel of 30 viral stocks of various HIV subtypes was enumerated by particle count [62]. This panel has been used very effectively in studies that evaluated performance of assays with different HIV subtypes [63]. In the absence of a gold standard, use of a single (international) standard for calibration of assays should result in consistent quantiﬁcation of HIV RNA by the different assays. Because an international standard was not available at the time of development of the assays for VL, each manufacturer established its own system for calibration. The result was that VL measures, expressed as copies per milliliter (i.e., RNA genomes or copies per milliliter), initially differed considerably between the commercial assays. Today, the newer versions of assays yield quantiﬁcation values that differ on average by no more than threefold or 0.5 log10 copies/mL [59,60,64–69]. Management guidelines recommend the use of absolute quantiﬁcation values or given changes in VL in making treatment decisions. For example, a quantiﬁcation threshold of 30,000–55,000 copies/mL is recommended for the initiation of therapy [51,52]. The observation that results obtained from different assays differ by as much as 0.5 log10, a difference considered to be statistically signiﬁcant, argues against use of different assays for the management of any one patient today. Standardization of the commercial HIV-1 assays to one international standard [70] or, perhaps, to a universal external standard [71] could result in more similar quantiﬁcation values from different products. Implementation of international standardization would allow results from clinical trials to be used in the management of patients regardless of the assay used. Such a scheme also would obviate the need strictly to employ only one assay in the management of patients. 3.4.2

Sensitivity

All of the commercial VL assays exhibit high sensitivity, expressed as the limit of detection (LoD). The LoD is the lowest concentration of virus that can be distinguished from HIV-negative specimens 95% of the time. LoDs have been reported to be 50, 75, 176, and *50–150 copies/mL for the AMPLICOR HIV-1 MONITOR1 Test, version 1.5 [62,87], VERSANT1

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HIV-1 RNA 3.0 Assay (bDNA) [60], NucliSens1 HIV-1 QT Test [72], and the LCx HIV RNA Quantitative Assay [29], respectively. In the clinical setting, the goal of therapy is to suppress VL to below the LoD. It remains to be determined if the differences in LoDs between the commercial assays would result in different management of patients. 3.4.3

Reproducibility of Quantiﬁcation

A change in VL can be considered to be signiﬁcant only if it is greater than changes that could be attributed to biological and assay variability. Analysis-of-variance experimental designs can be used to assess sources of variability of quantiﬁcation results [59,60,71]. Today, a threefold change in VL (about 0.5 log10 change) is considered to be signiﬁcant [51,52,71]. Thus, if an initial VL measurement is 25,000 copies/mL (4.40 log10) and it increases to 75,000 copies/mL (4.86 log10) or decreases to 8,300 copies/mL (3.92 log10), the change is statistically signiﬁcant. Reproducibility of quantiﬁcation is similar between the assays today; however, the bDNA-based assay is considered to have somewhat better reproducibility than assays employing target ampliﬁcation technologies, especially at very low levels of virus [59,67,70,74]. The smallest change in VL that is clinically signiﬁcant is unclear. In general, if during therapy the VL is suppressed to below detection and it increases to 500–1000 copies/mL, consideration is given to the development of resistance and to changing therapy. Other factors such as absolute viral load, CD4 count, and clinical status are also considered with changes in VL in assessing clinical signiﬁcance and making decisions about changes in therapy. 3.4.4

Linearity of Quantiﬁcation

Assessment of linearity of quantiﬁcation of VL assays is really an assessment of how accurately an assay measures changes in VL. If an assay is perfectly linear, then it should register accurate quantiﬁcation values regardless of the concentration of virus. Linearity is most often estimated by testing a dilution series of a high-titer HIV-1 specimen in the assay. Because the approaches for assessment of linearity described in the literature are different, it is difﬁcult to compare relative linearity of the assays [30,59,69,75]. Erice et al. [59] described a rigorous method for evaluation of the linearity of the VERSANT1 HIV-1 RNA 3.0 Assay (bDNA). They showed that the difference between observed values and expected values for members of a dilution series from a high-titer specimen did not exceed 0.1 log10 in the linear range of the assay. Likewise, Gleaves et al. [60] reported that the deviations between observed values and the best-ﬁtting 458 line were less than 0.05 log10.

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Linear ranges reported by the manufacturers of the assays are shown in Table 3. 3.4.5

Equal Quantiﬁcation of Genetic Variants of HIV

Viral load assays were designed to quantify HIV in the host based on detection of conserved sequences, in general, in the pol or gag genes. The early versions of the assays contained probes that were designed primarily to detect subtype B virus, a virus commonly found in resource-rich countries where the assays were developed. HIV-1 has been classiﬁed into three groups: group M (main), group O (outlier), and group N [76–78]. Subtype or clade B is one of the groups (subtypes A–K) classiﬁed in group M viruses. However, only about 3% of HIV-1 infections worldwide are subtype B, and increasing numbers of non-B subtypes are being identiﬁed in Europe and the United States, where much of the testing to date has been carried out [79–83]. In addition, recombinants of HIV-1 between subtypes and between groups M and O have been reported; in fact, 20% of the completely sequenced HIV-1 genomes have been reported to have ‘‘mosaic’’ sequences [84,85]. Studies of quantiﬁcation of subtypes of group M and some group O isolates have been reported [30,63,69]. The challenge of all of these studies is to have available preparations of virus that have been assigned quantiﬁcation values by methods that do not use hybridization technologies, because the latter are dependent on recognition of genetic variants and are used in VL assays. Electron microscopy, p24 antigen concentration, and quantitative VL analysis have been employed to assign viral concentrations in some panels [62,63]. In general, the most recent versions of the assays show close quantitative correlations for the group M viruses, but, with the exception of the Gen-Probe HIV-1 Viral Load assay in development, they have shown much less efﬁcient recognition of group O viruses [30,63]. The Roche AMPLICOR HIV-1 Monitor, version 1.5 ultrasensitive assay has been reported to detect subtypes A, E, G, and H better than earlier versions of the product [62,86,87]. Use of synthetic HIV transcripts that can be very accurately quantiﬁed and characterized should be considered for assessment of equal quantiﬁcation of genetic variants [88,89]. Transcripts can be produced for each of the genetic variants. However, this approach suffers from the disadvantage that the assays target different regions of the viral genome that could not be incorporated into single synthetic transcripts. Thus, the same transcripts could not be tested in all of the assays.

1

*100/200–1,000,000

Bayer Corporation Diagnostics Division Tarrytown, NY, U.S.A. http://www.bayerdiag.com

bioMe´rieux Marcy-E´toile, France http://www.biomerieux.com

Abbott Laboratories Abbott Park, IL, U.S.A. http://www.abbottdiagnostics.com

*50–1,000,000 for 1.0 mL sample

125/150–5,000,000 for 0.2 mL sample

Roche Molecular Systems, Inc. Pleasanton, CA, U.S.A. http://www.roche-diagnostics.com

Company

400–750,000 standard 50–75,000 ultrasensitive

VERSANT1 HIV-1 RNA 3.0 Assay (bDNA) 75–500,000

NucliSens1 HIV-1 QT Test

LCx HIV RNA Quantitative Assay

Test, v1.5

Dynamic range

Dynamic Ranges of Commercially Available HIV-1 Viral Load Assays

AMPLICOR HIV-1 MONITOR

Assay

TABLE 3

[60]

[72]

[29]

[73]

Ref.

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HIV-1 RESISTANCE TESTING

The high replication and mutation rates of HIV-1 coupled with incomplete suppression of viral replication frequently lead to the development of drug resistance, subsequently resulting in treatment failure [90]. Typically, drug resistance is deﬁned as the ability of virus to replicate in the presence of drug, and treatment failure is deﬁned as one or more consecutive viral load measurements that score above the limit of detection of a highly sensitive viral load assay. The common emergence of resistant viruses has led to the development of assays that analyze such viruses. These assays are referred to as ‘‘drug resistance’’ or ‘‘drug susceptibility’’ assays. For each individual antiretroviral drug, resistant HIV-1 variants have been described as well as multiresistant strains that are resistant to several or, rarely, all of the currently approved drugs [91,92]. Resistant strains also have been demonstrated to be transmissible to newly infected patients [91,93,94]. The purpose of drug resistance assays is to help physicians select the most appropriate and active drug regimen for each patient. Resistance assays are increasingly being used to guide physicians in management of their patients, mainly by identifying any antiretroviral drug(s) to which a patient may no longer respond. A clinical beneﬁt of resistance testing has been demonstrated in a growing number of studies, including CPCRA 046 or GART [95], VIRADAPT [96,97], and VIRA3001 [98]. A summary of key studies that support the clinical utility of resistance testing of treatment-experienced patients has been published [99]. Drug susceptibility assays are generally divided into two major categories: genotypic and phenotypic. Genotypic assays predict resistance phenotypes by determining the underlying DNA sequence of HIV1 virus isolated from patients and deducing the corresponding amino acid sequences of the viral protease and reverse transcriptase enzymes. By comparison, phenotypic assays provide for a quantitative measurement of drug susceptibility by generating recombinant viruses that harbor the region of interest (protease and reverse transcriptase) of patient-isolated virus and propagating these viruses in culture in the presence of drugs. Currently available HIV-1 resistance assays and the companies that provide them are listed in Table 4. 4.1

Genotypic Assays

Most genotypic assays determine the underlying DNA sequence of HIV-1 isolated from patients. This information is then used to deduce the corresponding amino acid sequences of the viral protease and reverse

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TABLE 4

465

Commercially Available HIV-1 Resistance Assays

Company information

Trade names of HIV-1 resistance assays

Visible Genetics, Inc. Toronto, ON, Canada http://www.visgen.com

TRUGENE2 HIV-1 Genotyping Test

Applied Biosystems Foster City, CA, U.S.A. http://home.appliedbiosystems.com

ViroSeq2 HIV-1 Genotyping System

Tibotec-Virco NV Mechelen, Belgium http://www.tibotec-virco.com

GENChec2 VirtualPhenotype2 Antivirogram1

ViroLogic, Inc. South San Francisco, CA, U.S.A. http://www.virologic.com

GeneSeq2 HIV PhenoSense2 HIV PhenoSense2 HIV GT

Bayer Corporation Tarrytown, NY, U.S.A. http://www.bayerdiag.com

VERSANT1 HIV-1 Protease Resistance Assay (LiPA) VERSANT1 HIV-1 RT Resistance Assay (LiPA)

(Developed by Innogenetics Ghent, Belgium http://www.innogenetics.com) VIRalliance Paris, France http://www.viralliance.com

Phenoscript2

transcriptase enzymes, which are the target of all of the currently approved drugs. Based on this information, a resistance phenotype is predicted. Commonly, this is accomplished either through the use of rule-based algorithms, databases that link genotypic with phenotypic data, or through correlation of the genotypic data with clinical data. 4.1.1

Genotypic Assays Based on DNA Sequencing

Several genotypic assays based on DNA sequencing are available, all of which use similar methodology. Brieﬂy, virus is isolated and puriﬁed from patient plasma, often after ﬁrst concentrating the virus through a centrifugation step. Next, the puriﬁed RNA is reverse-transcribed to yield complementary DNA, and the region of interest is ampliﬁed by PCR. Most DNA sequence-based assays amplify all of the protease region and a region that corresponds to approximately 300 amino acids

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of the viral reverse transcriptase. The vast majority of known resistanceassociated mutations fall into these two regions, although there are some noteworthy exceptions. For example, some gag-pol polyprotein precursor cleavage site mutations upstream of the protease region that affect viral resistance to protease inhibitors [100–102] and, more recently, a mutation at position 318 of reverse transcriptase that is linked to high level resistance to the NNRTI class of drugs [103] have been described. An overview of the regions covered by the most commonly used commercial genotypic resistance assays is shown in Figure 11. One such assay, the TRUGENE2 HIV-1 Genotyping Test, available from Visible Genetics, Inc. (Toronto, Canada), recently obtained FDA approval. It is expected that the ViroSeq2 HIV-1 Genotyping System available from Applied Biosystems (Foster City, CA, U.S.A.) will be approved in the near future. Similar assays are offered by two clinical reference laboratories as a service: the GENChec2 from Tibotec-Virco NV (Mechelen, Belgium) and the GeneSeq2 HIV from ViroLogic, Inc. (South San Francisco, CA, U.S.A.). Many variations of genotypic assays based on DNA sequencing, so-called home brew assays, are performed in some reference and other smaller laboratories.

FIGURE 11 Nucleotide sequences covered by the most commonly used commercial genotypic HIV-1 resistance assays. Short (vertical) arrows below the polymerase gene indicate point mutations analyzed by LiPA.

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467

VERSANT1 HIV-1 Protease Resistance Assay (LiPA) and VERSANT1 HIV-1 RT Resistance Assay (LiPA)

A different approach to obtaining genotypic data underlies the principle of the line probe assay (LiPA) [104]. The VERSANT1 HIV-1 Protease Resistance Assay (LiPA) and the VERSANT1 HIV-1 RT Resistance Assay (LiPA), developed by Innogenetics (Ghent, Belgium) and distributed by Bayer Corporation, Diagnostics Division (Tarrytown, NY, U.S.A), are based on reverse hybridization. Reverse transcription and PCR ampliﬁcation are used to amplify the protease or reverse transcriptase regions of virus isolated from patients. The primers used for the ampliﬁcation step in this procedure are biotinylated. The ampliﬁed product is then used to hybridize to immobilized oligonucleotide probes that are covalently attached to a nitrocellulose strip. On these strips, bands comprising different oligonucleotides are complementary to wild-type and mutant sequences. When no mixtures are present, only one band for each particular codon (either wild-type or mutant) lights up, thus determining the genotype of the virus at particular positions. Clearly, with more codons represented on the strip, a more complete genotypic proﬁle can be deduced. The current strip versions give information on a total of eight codons on two separate strips for protease (corresponding to amino acids 30, 46, 48, 50, 54, 82, 84, 90) and for 11 reverse transcriptase codons on a single strip (corresponding to amino acids 41, 69, 70, 74, 75, 103, 106, 151, 181, 184, 215). These mutations are referred to as key mutations. The reverse transcriptase strip includes the key mutations linked to resistance to both nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). 4.1.3

VirtualPhenotype2

In addition to the above-described assays, Tibotec-Virco NV (Mechelen, Belgium) has introduced a commercially available genotypic assay, the VirtualPhenotype2, which links a patient’s virus genotype to information stored in an extensive database [105]. The sequence of the patient virus is compared with sequences of viruses that harbor similar mutations and for which genotypic and phenotypic data have been obtained. The comparison is performed individually for selected mutated codons against viruses in the database that harbor the same mutant codons. Because individual mutant codons are linked to reduced drug susceptibility proﬁles to certain drugs, an average resistance value is calculated for the patient virus based on data for all viruses in the database with the same mutation. The ﬁnal result is a ‘‘virtual’’

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phenotype, which is based on actual phenotypic results of previously tested viruses. A quantitative prediction of resistance is provided. It is important to keep in mind that the ‘‘virtual’’ phenotype is not based on experimental phenotypic data obtained for the patient virus, as in true phenotypic assays. However, good agreement between VirtualPhenotype results and actual phenotypic results has been reported [105], and the assay has been shown to be an independent predictor of response [106]. This assay gives more information than typical genotypic assays without the time and cost involved in obtaining a phenotype. 4.2

Phenotypic Assays

Phenotypic assays are all based on recombinant vector technology. Similar commercially available phenotypic assays have been developed by three companies—Tibotec-Virco NV, ViroLogic Inc., and VIRalliance (Paris, France). These assays are based on the generation of recombinant viruses that harbor the region of interest (protease and reverse transcriptase) of patient-isolated virus and propagation of these viruses in culture in the presence of drugs. Phenotypic assays allow for a quantitative measurement of drug susceptibility, although the effect of each drug is determined individually and no drug combinations can be analyzed. In addition to providing information on patient samples, phenotypic assays are being used in drug development efforts [107]. 4.2.1

Antivirogram1

Antivirogram1 is a phenotypic assay available from Tibotec-Virco NV. In this assay, extracted RNA is reverse-transcribed and ampliﬁed, and the linear amplicons together with a linearized HIV-1 viral genome carrying a deletion in the polymerase gene are introduced in a CD4þ cell line by electroporation. In the transfected cells, intracellular homologous recombination will regenerate infectious HIV-1, a process that takes 5–10 days. Virus is cultured in microtiter well plates in the presence of serial dilutions of antiretroviral drugs. Virus replication is monitored, and IC50 and IC90 values are derived by comparison with a drug-sensitive wildtype strain. Due mainly to the culture requirement of the recombinant virus, the turnaround time of this assay is 2–4 weeks. This assay has been used for testing all major clades (A–G) of HIV-1. Interassay variability has been reported as 1.2–2.5-fold, and biologically relevant cutoffs for 14 drugs have been established [108]. Tibotec-Virco NV also offers therapeutic drug monitoring as a commercial product. The efﬁcacy of therapy may depend on individual variations in drug metabolism and drug–drug interactions. For instance,

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mechanisms have been identiﬁed that cause an elevated expulsion of nucleoside analogs from cells [109]. Ritonavir boosting of some protease inhibitors (indinavir, saquinavir, and amprenavir) is common, leading to elevated trough levels of these drugs [110]. Therefore, it is reasonable to expect that therapeutic drug monitoring might play a role in deﬁning individualized treatment regimens. Indeed, the effect of plasma protease inhibitor levels on virological response has been described in the VIRADAPT study [97,111]. 4.2.2

PhenoSense2 HIV

PhenoSense2 HIV is a phenotypic assay available from ViroLogic, Inc. (South San Francisco, CA). PhenoSense HIV is similar to Antivirogram in that it uses recombinant viruses that harbor the patient-derived virus’s region of interest. However, unlike Antivirogram, the recombinant viruses used in PhenoSense HIV are produced in vitro through conventional cloning techniques. The resulting recombinant viruses carry a deletion in the envelope region, substituted by a luciferase reporter gene, and are not replication-competent. Thus, only a single round of replication of pseudotyped virus will occur in transfected cells, which prevents the outgrowth of more ‘‘ﬁt’’ viral subpopulations in culture. The turnaround time for this assay is less than 2 weeks, primarily due to the single replication cycle nature of the assay. A recent abstract indicates that, comparable to Antivirogram, PhenoSense HIV is able to test all major subtypes of HIV-1 [112]. The reproducibility variation of the assay is reported to be less than 2.5-fold [113], which means that calculated values greater than 2.5-fold are indicative of reduced susceptibility whereas values less than 2.5-fold indicate increased susceptibility. Increased susceptibility or ‘‘hypersusceptibility’’ has been described for Amprenavir [114] and for the class of NNRTIs [115]. In addition to this technical cutoff value, clinically relevant cutoff values have been determined for abacavir as 4.5-fold [116] and for lopinavir as 10-fold [117]. More recently, clinical cutoff values of 1.7-fold were proposed for stavudine and didanosine [118]. A modiﬁed version of the PhenoSense HIV assay, designated RC assay, has been developed that can be used to obtain information on the replicative capacity, or ‘‘ﬁtness,’’ of the patient virus [119]. As a general rule, wild-type virus tends to be more ﬁt than drug-resistant virus [120,121]. Determination of viral ﬁtness may have clinical value because many patients are faced with highly resistant viruses and few or no effective treatment choices. It is unclear whether there may be clinical value in maintaining a resistant but ‘‘unﬁt’’ virus at comparatively lower viral load levels through continuation of a failing drug regimen as

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opposed to stopping treatment and allowing wild-type virus to rebound, frequently to high levels. These and other related questions are being addressed in several studies, most of which monitor not only viral load and drug susceptibility but also viral ﬁtness. ViroLogic, Inc. recently introduced a combination product, PhenoSense HIV GT, which provides both genotypic and phenotypic information. A further advancement of the PhenoSense HIV technology is the development of a phenotypic assay that measures drug susceptibility to inhibitors of viral entry. These constitute a new class of drugs, none of which has been approved, although one of them, T20, is currently being evaluated in late-stage clinical trials and is expected to be approved soon. The entry inhibitor assay is based on the same principle as the original PhenoSense HIV assay but uses different cell lines that express the receptor and coreceptors required for entry of HIV-1. This assay is currently being used in clinical drug trials, but it is not yet commercially available. 4.2.3

Phenoscript2

The major distinguishing feature of Phenoscript2, available from VIRalliance (Paris, France), is that the protease and reverse transcriptase regions are ampliﬁed as two separate fragments. Recombinant virus is produced by homologous recombination in cells cotransfected with the individual ampliﬁed fragments and a test plasmid in which the envelope region has been deleted. Because the resulting virus cannot replicate, this assay is limited to a single round of replication. Similar to the phenotypic assays Antivirogram and PhenoSense HIV, Phenoscript uses virus that is cultivated in microtiter well plates in the presence of drugs, and IC50 or IC90 values are calculated by comparison of the patient sample results with those of a drug-sensitive wild-type NL4-3 strain. This assay has been used in a large clinical study, the NARVAL (ANRS 088) trial [122]. 4.3

Assay Selection

Given the choice of assays available today, the question is, which assay should be used? There is no easy answer, because each assay has advantages and disadvantages and may address speciﬁc needs. In addition to features of practical interest to the clinical laboratory such as turnaround time, cost, and reimbursement issues, performance attributes to consider when deciding on a speciﬁc assay type include Sensitivity. What is the minimal viral load for which the assay has been validated?

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Speciﬁcity. Has the assay been tested for interfering substances, drugs, and other infectious agents? For instance, HBV and HCV coinfection rates are high among the HIV-1-positive population. Clade speciﬁcity. Does the assay pick up non-B subtypes? Most assays were developed based on clade B, which is most prevalent in developed countries. Ability to detect mixtures of quasi-species. Which percentage in a viral mixture can be reliably detected by the assay? 4.3.1

Advantages and Disadvantages of Genotypic Assays

Genotypic assays provide a shorter turnaround time than phenotypic assays because they do not require a cell culture step. Because of the less complex laboratory technology requirements, genotypic assays are less expensive than phenotypic assays. Typically, DNA sequencing assays range in price from US$300 to US$400 per sample. The total costs for obtaining a complete genotypic proﬁle using the LiPA technology range from US$150 to US$200 US per sample. One advantage of DNA sequencing over LiPA is that the entire genomic information of the region analyzed is obtained, which is useful for epidemiological studies. This additional information may be valuable in assessing the relevance of certain mutations when new drugs are introduced. Although DNA sequencing provides an enormous amount of information, proper analysis of this information and subsequent use of the resulting genotypic data to reach well-informed treatment recommendations can be difﬁcult. DNA sequencing assays beneﬁt tremendously from expert interpretation owing to the often complex genotypic proﬁles linked to the potentially synergistic interplay of multiple mutations. Another potential limitation is that most DNA sequencing assays pick up viral quasispecies only if they represent at least 20% of the mixture [123]. LiPA, on the other hand, has been reported to be more sensitive than DNA sequencing in picking up viral mixtures and is able to identify minor populations representing 2–5% of the population [104,124,125]. Although a potential challenge for the LiPA technology is that strips may require routine improvements by updating the codon menu in order to incorporate newly identiﬁed key mutations, LiPA is easy to use and results are relatively straightforward to interpret. 4.3.2

Advantages and Disadvantages of Phenotypic Assays

Whereas the result of genotypic assays is used to predict a phenotype, phenotypic assays will provide this information directly. Moreover, phenotypic assays are quantitative and can provide information on cross-resistance. These features make phenotypic assays a valuable tool

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in discerning mutational interactions. It appears that all three of the phenotypic assays discussed here are able to successfully test most HIV-1 subtypes. Although initial versions were cumbersome to perform, today’s versions of these assays are more rapid and reproducible. Phenotypic assays can be performed only on-site, and it is not to be expected that the technology can be modiﬁed sufﬁciently to allow for a kit type of product. Phenotypic assays are expensive, ranging from approximately US $700 to US $900 per sample. Owing to the complexity of these assays, it is unlikely that these ﬁgures will come down considerably in the near future. None of the assays is highly automated, which prohibits major cost savings. Although all phenotypic assays have deﬁned technical cutoff values, it is less clear how these values correlate with clinical response. The companies that provide phenotypic assays are attempting to deﬁne more meaningful clinical cutoff values. However, to do so requires extensive clinical data. Because most patients are treated with three or more drugs in their regimens, it may be difﬁcult and will probably be prohibitively expensive to obtain such data for each of the currently approved drugs. 4.4

Challenges and Future Directions

Although the development of resistance-associated mutations is a major reason for treatment failure, nonadherence to the drug regimens is a critical problem as well. There is little beneﬁt of resistance testing to the patients if treatment failure is caused by nonadherence. Therefore, the further development of complementary pharmacokinetic tests such as assays for therapeutic drug monitoring or other methods that may yield information on patient adherence are necessary. Although current guidelines do not recommend resistance testing for treatment-naive, newly infected patients [99], an increase in the transmission rate of resistant virus, which has recently been reported [94,126] as well as transmission of non-B subtypes [127,128], may lead to a change in these recommendations. It is to be expected that there will be a demand for resistance assays with increased sensitivity to less than 1000 copies/mL. It is easy to predict that drug resistance testing will increasingly play an important role in the management of HIV-1-infected patients, and it is likely that soon such assays will become the standard of care. REFERENCES 1.

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13 Nucleoside/Nucleotide Inhibitors of HIV Reverse Transcriptase Erik De Clercq* Rega Institute for Medical Research, Catholic University of Leuven, Leuven, Belgium

1

INTRODUCTION

The 20 ,30 -dideoxynucleoside (ddN) analogs have become the cornerstone in the chemotherapy of HIV infections, because virtually all drug combination regimens that are currently used for AIDS treatment contain one or more ddN analogs. In these AIDS treatment regimes, the ddN analogs (also commonly referred to as NRTIs for nucleoside reverse transcriptase inhibitors) are combined with non-nucleoside reverse transcriptase inhibitors (NNRTIs) and/or protease inhibitors (PIs). The following NRTIs have been formally approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infections: zidovudine (ZDV), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), and abacavir (ABC). In the meantime, the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate has also been approved by both the FDA and their European * Professor Erik De Clercq holds the Professor P. De Somer Chair of Microbiology at the Katholieke Universiteit Leuven School of Medicine.

485

486

De Clercq

counterpart, the European Agency for the Evaluation of Medicinal Products (EMEA). Also, the combinations of ZDV with 3TC, and of ZDV with 3TC and ABC, have been formally approved, so that there are now nine preparations registered as NtRTIs or NRTIs (Table 1). Further details on the dates of approval, the trade names, dosage level, and principal side effects are listed in Table 1 [1]. Special attention should be given to the toxic side effects of the compounds (as listed in Table 1), because they may compromise tolerability and compliance. Also, poor adherence to the prescribed drug regimes, together with resistance development (see Sec. 9) may in turn compromise the successful outcome of the current antiretroviral treatment strategies. 2

REVERSE TRANSCRIPTASE

Reverse transcriptase, or RNA-dependent DNA polymerase (Fig. 1), is an enzyme that is intrinsically associated with the replication of retroviruses in general, HIV in particular. This enzyme is essentially responsible for three consecutive catalytic functions: (1) transcription of the viral (þ)RNA genome to ()DNA, thus leading to the formation of a (þ)RNA ()DNA hybrid; (2) degradation of the (þ)RNA by the ribonuclease H (H for hybrid) function of the enzyme; thus allowing (3) the remaining ()DNA to be duplicated to double-stranded (ds) (+)DNA by the DNA-dependent DNA polymerase function of the reverse transcriptase. The (+)DNA will then be incorporated as proviral DNA into the host cell chromosome by the so-called integrase, akin to the reverse transcriptase, a speciﬁc enzyme encoded by the viral genome. 3

DNA POLYMERIZATION REACTION

Building blocks for any DNA polymerization reaction, including the reverse transcriptase reaction, are dATP, dGTP, dCTP, and dTTP, collectively denoted as dNTPs. During the polymerization reaction, these 20 -deoxynucleoside 50 -triphosphates are hydrolyzed between their ﬁrst and second phosphate groups so as to release a pyrophosphate moiety and engage in a phosphodiester linkage between the remaining 50 -phosphate group and the 30 -hydroxyl function of the preceding nucleotide (as demonstrated in Figure 2 for dTTP as the building block). As a result, the DNA chain is elongated at its 30 -hydroxyl end by the consecutive addition of new building blocks. For this DNA chain elongation to proceed, it is essential that the building blocks be equipped with a free 30 -hydroxyl function.

June 1994

November 1995

September 1997

Zerit

Epivir

Combivir Ziagen Trizivir

Viread

Abacavir (ABC)

ZDV þ 3TC þ ABC

NtRTIs Tenofovir disoproxil fumarate

Source: Adapted from Ref. 1.

June 1992

Hivid

October 2001

November 2000

February 1999

October 1991

March 1987

Date of FDA approval

Videx

Retrovir

Trade name

Didanosine (ddI) (20 ,30 -dideoxyinosine) Zalcitabine (ddC) (20 ,30 -dideoxycytidine) Stavudine (d4T) (20 ,30 -dideoxy-20 , 30 -didehydrothymidine) Lamivudine (3TC) (20 ,30 -dideoxy-30 thiacytidine) ZDV þ 3TC

NRTIs Zidovudine (ZDV) [Azidothymidine (AZT)]

Generic name

Compound Principal side effects

—

300 mg qd

—

300 mg ZDV þ 150 mg See ZDV 3TC bid 300 mg bid Hypersensitivity, fever, nausea, rash 300 mg ZDV þ 150 mg See ZDV and ABC 3TC þ 300 ABC bid

150 mg bid

Bone marrow suppression: anemia and/or neutropenia 200 mg bid or 400 mg Pancreatitis, peripheral qd neuropathy 0.75 mg tid Peripheral neuropathy, pancreatitis 40 mg bid Peripheral neuropathy

200 mg tid or 300 mg bid

Dose

TABLE 1 Review of the Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Nucleotide Reverse Transcriptase Inhibitors (NtRTIs) That Are Currently Used in the Treatment of HIV Infections

Nucleoside/Nucleotide Inhibitors of HIV RT 487

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De Clercq

FIGURE 1 Schematic presentation of the HIV reverse transcriptase.

4

NUCLEOSIDIC REVERSE TRANSCRIPTASE INHIBITORS (NRTIS) IN CLINICAL USE

The structures of the six 20 ,30 -dideoxynucleoside (ddN) analogs that have been formally approved for clinical use in the treatment of HIV infections [2] are presented in Figure 3. Their in vitro (cell culture) anti-HIV properties were ﬁrst described, for ZDV, in 1985 by Mitsuya et al. [3]; for ddI, and ddC in 1986 by Mitsuya and Broder [4]; for d4T in 1987 by Baba et al. [5]; for 3TC in 1991 by Soudeyns et al. [6]; and for ABC in 1997 by Daluge et al. [7]. All six compounds have been shown to inhibit HIV-1

Nucleoside/Nucleotide Inhibitors of HIV RT

489

FIGURE 2 DNA building blocks (dATP, dGTP, dCTP, and dTTP) and DNA chain elongation, as demonstrated with dTTP as the building block.

and HIV-2 replication, albeit at varying concentrations depending on the nature of the cell culture system used and the compound tested [the pyrimidine derivatives (AZT, ddC, d4T, 3TC) being, as a rule, more potent than the purine derivatives (ddI, ABC)]. After the compounds were also shown to be effective in vivo in reducing the viral load in HIVinfected individuals, they were approved for clinical use—ﬁrst in monotherapy and subsequently in multiple drug therapy regimens. From ZDV, a thioether lipid-ZDV conjugate, fozivudine tidoxil (FZD) was developed and has already been the subject of phase II clinical trials [8]. FZD, which reportedly releases the 50 -monophosphate of ZDV after it has been taken up by the cells, would be as effective as and potentially better tolerated than ZDV. 5

NEW 20 ,30 -DIDEOXYNUCLEOSIDE ANALOGS THAT ARE IN CLINICAL DEVELOPMENT

In addition to the established ddN analogs (Fig. 1), there are at least three ddN analogs presently in clinical development (phase I/II clinical trials): emtricitabine (FTC) [9], 20 -deoxy-30 -oxa-40 -thiocytidine (dOTC) [10,11],

490

De Clercq

FIGURE 3 Nucleoside reverse transcriptase inhibitors (NRTIs): 20 ,30 -dideoxynucleoside (ddN) analogs that have been approved for clinical use.

and ()-b-D-2,6-diaminopurine dioxolane (DAPD, amdoxovir) [12]. The structural formulas of these compounds are depicted in Figure 4. DAPD is readily converted by adenosine deaminase to dioxolane guanine (DXG) and has proven active against HIV-1 strains that are resistant to ZDV and 3TC [13]. So has dOTC. Despite its structural similarity to 3TC, dOTC proved active against 3TC-resistant HIV-1 (M184V) in the SCIDhu Thy/Liv model,* albeit at a relatively high dosage level (400 mg kg1 day1) [10]. Other new nucleoside analogs that have been recently * SCID-hu mouse model: a surrogate animal model for HIV infection, based on severe combined immune deﬁciency mice transplanted with human tissue [thymus (Thy) and liver (Liv)].

Nucleoside/Nucleotide Inhibitors of HIV RT

491

FIGURE 4 Examples of ddN analogs that are in clinical development.

described as in vitro inhibitors of the replication of both drug-sensitive and drug-resistant HIV mutants include methylenecyclopropane nucleoside analogs and their phosphoro-L-alaninate diesters [14,15] and 40 -ethynyl nucleoside analogs, such as 40 -E-20 -deoxycytidine, 40 -E20 -deoxyadenosine, 40 -E-20 -deoxyguanosine, and 40 -E-20 -deoxyribofuranosyl-2,6-diaminopurine [16,17]. Given their activity against multidrugresistant HIV variants, further development of the 40 -E analogs as potential therapeutics may seem warranted. 6

ACYCLIC NUCLEOSIDE PHOSPHONATES

In contrast with all the aforementioned compounds (Figs. 3 and 4), which can be considered ‘‘nucleoside’’ analogs, the acyclic nucleoside phosphonates, i.e., adefovir (PMEA) and tenofovir (PMPA), are already equipped with a phosphonate group and could therefore be designated as ‘‘nucleotide’’ analogs (Fig. 5). The presence of the phosphonate group, and the two negative charges associated therewith, limits the oral bioavailability of the compounds, which explains why the appropriate prodrugs, i.e., the bispivaloyloxymethyl ester for PMEA and the bisisopropyloxycarbonyloxymethyl ester for PMPA, were designed, thus providing the oral prodrug forms adefovir dipivoxil and tenofovir disoproxil, respectively (Fig. 5). The former, adefovir dipivoxil, was originally developed for the treatment of HIV infections, was discontinued for this purpose, and was then further developed for the

492

De Clercq

FIGURE 5 Nucleotide reverse transcriptase inhibitors (NRTIs): acyclic nucleoside phosphonate (ANP) derivatives.

treatment of hepatitis B virus (HBV) infections (momentarily in phase III clinical trials). The latter, tenofovir disoproxil fumarate, proceeded through phase III clinical trials and was recently approved in both the United States and Europe for the treatment of HIV infections (in combination with other anti-HIV drugs). The in vitro anti-HIV properties of PMEA were ﬁrst mentioned in 1986 by De Clercq et al. [18], and those of PMPA in 1993 by Balzarini et al. [19]. The clinical potential of PMEA and PMPA has been reviewed repeatedly; see, e.g., Naesens et al. [20] and Fridland [21]. The safety and efﬁcacy of PMPA were apparent from the ﬁrst clinical studies conducted

Nucleoside/Nucleotide Inhibitors of HIV RT

493

with this compound [22]. Prophylactic and therapeutic beneﬁts following both long-term and short-term administration of PMPA were demonstrated in newborn macaques infected with simian immunodeﬁciency virus (SIV) [23,24]. These studies underscored the potential of PMPA in the prevention of mother-to-child transmission of HIV. 7

MECHANISM OF ANTI-HIV ACTION OF NRTIS

All nucleoside or nucleotide analogs that act as RT inhibitors do so by a similar mechanism: After intracellular phosphorylation to their triphosphate form, they interact, as competitive substrates or inhibitors with respect to the natural substrates (dNTPs), in the HIV reverse transcriptase reaction. If incorporated into the DNA product (Fig. 6), they lead to termination of DNA chain elongation, because they do not provide the

FIGURE 6 Mechanism of action of ddN analogs, as exempliﬁed for azidothymidine (AZT). dThd kinase ¼ 20 -deoxythymidine kinase; dTMP kinase ¼ 20 -deoxythymidine 50 -monophosphate kinase; NDP kinase ¼ nucleonucleoside 50 -diphosphate kinase.

494

De Clercq

30 -hydroxyl group needed for the attachment, through a phosphodiester linkage, of the next nucleotide. For the ddN analogs, three subsequent phosphorylations are required: a nucleoside kinase (i.e., thymidine kinase), a nucleoside 50 monophosphate kinase (i.e., thymidylate kinase), and nucleoside diphosphate kinase [25]. The ddN 50 -triphosphates can be incorporated as efﬁciently as the natural substrate, as shown for d4TTP [26]. Also, the new ddN analogs (such as DAPD) would act as potent alternative substrate inhibitors of HIV RT (for DAPD, after deamination to DXG and subsequent phosphorylation to DXG triphosphate) [13,27]. For the acyclic nucleotide analogs (PMEA, PMPA) to be converted to their active metabolites (Fig. 7), only two phosphorylations are required; these could be carried out in one step [through 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase] or two steps (through AMP kinase). The active metabolites, PMEApp and PMPApp, would then, again, act as potent alternative substrates (with respect to dATP) and chain terminators of HIV RT. Although HIV RT and other retroviral RTs lack 30 ?50 exonuclease activity, they can remove 30 -terminal chain-terminating residues from blocked DNA chains through either pyrophosphorolysis (the reversal of the DNA polymerization reaction) or a nucleotide (ATP)-dependent mechanism leading to production of dinucleoside polyphosphates [28,29]. In fact, HIV-1 RT mutants containing the AZT resistance mutations D67N, K70R, T215F, and K219Q are capable of removing AZT 50 -monophosphate from the blocked primer through the ATPdependent mechanism more efﬁciently than wild-type RT. Removal of d4T 50 -monophosphate would also be possible, but this removal is strongly inhibited by the next complementary dNTP [30]. Also, the M184V mutation, which confers high-level resistance to 3TC, would severely impair the removal of chain-terminating AZT and hence suppress the rescue of AZT-terminated DNA synthesis [31]. Chainterminating PMEA and PMPA residues should not be readily excised from the 30 -termini, because the phosphonate group is likely to resist phosphorolytic cleavage. 8

PRODRUGS OF 20 ,30 -DIDEOXYNUCLEOSIDE 50 -MONOPHOSPHATES

In the phosphorylation pathway leading to the formation of the active metabolites (ddNTPs) of the ddN analogs, the ﬁrst phosphorylation (nucleoside kinase) step is the most difﬁcult and thus rate-limiting. The acyclic nucleotides PMEA and PMPA, which are already equipped with

Nucleoside/Nucleotide Inhibitors of HIV RT

495

FIGURE 7 Mechanism of action of ANP derivatives, as shown for adefovir (PMEA).

a phosphonate group, circumvent this nucleoside kinase reaction. For ddN analogs to likewise bypass this nucleoside kinase, several 20 ,30 dideoxynucleotide (ddNMP) prodrugs have been constructed that, once they have been taken up by the cells, deliver ddNMP intracellularly.

496

De Clercq

Essentially three (i.e., bis-S-acetyl-2-thioethyl, aryloxyphosphoramidate, and cyclosaligenyl) approaches (Fig. 8) have been described to deliver ddNMPs such as ddAMP and d4TMP inside the host cells. Thus,

FIGURE 8 Bis-S-acetyl-2-thioethyl, aryloxyphosphoramidate, and cyclo-saligenyl pronucleotides.

Nucleoside/Nucleotide Inhibitors of HIV RT

497

the bis-S-acetyl-2-thioethyl phosphotriester of ddA [bis(SATE)ddAMP] was found to be 1000-fold more potent against HIV than the parent compound, apparently by preventing the action of adenosine deaminase, which otherwise would rapidly convert ddA to ddI [32]. This approach was recently extended to S-acyl-2-thioethyl aryl phosphotriester derivatives of AZT 50 -monophosphate [33] to bypass the AZT phosphorylation step. Similarly, aryloxyphosphoramidate derivatives of d4T [i.e., So324, a d4T 50 -monophosphate (d4TMP) prodrug containing a phenyl group and the methyl ester of alanine linked through a phosphoramidate linkage] were constructed [34–36]. Within the cells, d4TMP is released via the alaninyl d4TMP intermediate [37], which thus serves as an intracellular depot form of d4TMP. The phosphoramidate prodrug approach does not seem to work so well with AZT, where the main metabolite formed from the alanyl AZTMP intermediate is AZT rather than AZTMP [38], thus explaining why d4T phosphoramidate prodrugs, but not AZT phosphoramidate prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase– deﬁcient cell cultures. In resting monocytes or macrophages, the aryloxyphosphoramidate derivatives of d4T, d4A, and ddA provided an anti-HIV activity that was 25- to 625-fold higher than that of the parent compounds [39]. The thymidine kinase (in the case of d4T) and the adenosine deaminase (in the case of ddA) can also be bypassed by using the cyclosaligenyl approach [40,41]. Cyclo-saligenyl pronucleotides of d4T and ddA were found to deliver exclusively the nucleotides d4TMP and ddAMP under both intracellular and chemically simulated hydrolysis conditions [42,43]. Although attractive in cell culture conditions (in vitro), the therapeutic utility of the intracellular delivery principle of ddNMPs by the pronucleotide approaches outlined in Figure 8 remains to be demonstrated. 9

HIV NRTI RESISTANCE MUTATIONS

As could be expected from any selective antiviral agent aimed at a speciﬁc viral target, resistance to NNRTIs can arise through mutations in the HIV RT gene. These mutations are primarily clustered at positions 41, 65, 67, 69, 70, 74, 184, 210, 215, and 219, with additional mutations (Q151M or T69SSS/G/A) in the case of multi-NRTI resistance (Fig. 9). The amino acid mutations that have been most frequently found in vivo in association with antiretroviral drug resistance are M41L, D67N,

498

De Clercq

FIGURE 9 Reverse transcriptase mutations associated with reduced susceptibility to NRTIs and NtRTIs. Positions of the amino acid mutations in the reverse transcriptase are indicated by numbers, and the nature of the mutations is indicated by letter codes for the amino acids (see Abbreviations). Thus, as an example, M184V means that methionine at position 184 has been mutated to valine. (Adapted from http:// www.iasusa.org/resistance mutations/index.num, International AIDS Society—U.S., November 2002, Topics in HIV Medicine.)

K70R, L210W, T215Y/F, and K219Q/E for AZT; L74V, K65R, and M184V for ddI; K65R, T69D, and M184V for ddC; V75T for d4T; M184V/I for 3TC; and K65R, L74V, Y115F, and M184V for ABC. Low-level resistance to 3TC has been observed with the mutations E44D and/or V118I, in the absence of a concurrent M184V mutation [44]. Resistance mutations toward the ANPs, i.e., PMEA and PMPA, have been rarely observed in vivo; the mutations to be expected are K65R and K70E, but it is not clear whether the appearance of such mutants would be clinically relevant,

Nucleoside/Nucleotide Inhibitors of HIV RT

499

because they may have reduced replicative ability (as shown speciﬁcally for the K70E mutant in Ref. 45). To engender a signiﬁcant reduction in drug susceptibility [less than tenfold increase in 50% effective concentration (EC50)], multiple mutations are required, as has been assessed with recombinant HIV clones: M41L, D67N, K70R, and T215Y for AZT (>100-fold) [46]; V75I, F77L, F116Y, and Q151M for ddI (>100-fold) [47]; M41L, T69SSG, and T215Y for ddC (55-fold) [48]; K65R, S68G, F77L, Y115F, Q151M, and M184V for d4T (15-fold) and ABC (40-fold) [49]. For 3TC, however, one mutation (M184V) sufﬁces to generate full-ﬂedged (>100-fold) resistance. It has been demonstrated that genotypic resistance testing has a signiﬁcant beneﬁt on the virological response when choosing a therapeutic alternative [50]. Thus drugs suggested not to be included in a therapeutic regimen should be (if the following mutations have been diagnosed): d4T (V75T), AZT (M41L, D67N, K70R, L210W, T215Y/F, and K219E), 3TC (M184V), ABC (K65R, L74V, and M184V), ddI (K65R, L74V, and M184V), ddC (K65R, T69D, L74V, and M184V), and multinucleoside analogs (A62V, V75I, F77L, F116Y, and Q151M), according to Mene´ndezArias and Domingo [51].

10

SUMMARY

The nucleoside/nucleotide type of HIV reverse transcriptase (RT) inhibitors consists essentially of two classes of molecules: (1) 20 30 dideoxynucleoside (ddN) analogs (AZT, ddI, ddC, d4T, 3TC, and ABC), which need three phosphorylations, and (2) acyclic nucleoside phosphonate (ANP) analogs (PMEA and PMPA), which need only two phosphorylations, to be converted to their active metabolites (ddNTPs and ANP diphosphates, respectively). These active metabolites thus compete with respect to the natural substrates (dNTPs) as inhibitors or alternative substrates of the RT reaction. If incorporated into the DNA chain (as ddNMP or ANP, respectively), the compounds obligatorily act as DNA chain terminators, thus shutting off viral DNA synthesis. Resistance mutations can, in principle, arise for any of the NNRTIs, although, as a rule, it takes multiple mutations to generate multidrug resistance, except for 3TC, for which a single mutation (M184V) sufﬁces.

ACKNOWLEDGMENTS I thank Christiane Callebaut for her proﬁcient editorial assistance.

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ABBREVIATIONS Letter codes for amino acids: A (alanine), C (cysteine), D (aspartate), E (glutamate), F (phenylalanine), G (glycine), H (histidine), I (isoleucine), K (lysine), L (leucine), M (methionine), N (asparagine), P (proline), Q (glutamine), R (arginine), S (serine), T (threonine), V (valine), W (tryptophan), Y (tyrosine).

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14 Non-Nucleoside Inhibitors of HIV Reverse Transcriptase Matthias Go¨tte and Mark A. Wainberg McGill University, Montreal, and Jewish General Hospital, Montreal, Quebec, Canada

1

INTRODUCTION

Because of its key role in the retroviral life cycle, the reverse transcriptase (RT) enzyme of the human immunodeﬁciency virus (HIV) is an important target for the development of antiviral agents that suppress viral replication. Retroviral reverse transcription is a complex process carried out by the HIV-encoded RT enzyme that displays DNA polymerase activities on both DNA and RNA templates as well as an RNase H activity that degrades the transcribed genomic RNA. Both the polymerase and RNase H activities of HIV-1 RT are essentially required to convert the genomic RNA into a double-stranded DNA molecule that is later integrated into the host genome [1]. However, all currently approved RT inhibitors used in the clinic are directed against the polymerase active center [2]. Blocking of the RT-associated RNase H activity has until now been demonstrated only in vitro or in cell-free assays [3–5]. Two categories of drugs have been developed to antagonize the polymerase activity of HIV RT: nucleoside analog RT inhibitors (NRTIs) that compete with cellular dNTPs for binding and incorporation 505

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and non-nucleoside analog RT inhibitors (NNRTIs) that act as noncompetitive antagonists of the enzyme. In addition, phosphonoformic acid (PFA, foscarnet), a pyrophosphate analog, is capable of inhibiting a broad spectrum of DNA polymerases including HIV-1 RT, but its clinical use is hampered by several factors, including the lack of an orally bioavailable form [6]. Nucleoside analog RT inhibitors are administered to patients in precursor forms that are intracellularly phosphorylated to their active triphosphate form. This type of compound is modiﬁed at the 3’-position of the sugar moiety and lacks the hydroxyl group necessary for the nucleophilic attack at the a-phosphate of an incoming nucleotide. Once incorporated into the growing chain, these nucleoside analogs cause termination of DNA synthesis. Zidovudine (AZT), lamivudine (3TC), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), and abacavir (ABC) are approved chain terminators that are currently used in the clinic. Although the base moiety and the nature of the chemical modiﬁcation at the 30 -position of the sugar ring can differ among these compounds, they all lack the 30 -hydroxyl group as a common structural feature (Fig. 1A). In contrast, NNRTIs are a diverse set of compounds that bind to a hydrophobic pocket in close proximity to the active site. Kinetic studies conﬁrm that NNRTI inhibition does not affect nucleotide binding. In fact, dNTPs can be tightly bound, even if NNRTIs are present, but the rate of the polymerization step is strongly diminished [7,8]. Three NNRTIs are currently used in the clinic: nevirapine [9], delavirdine [10], and efavirenz [11] (Fig. 1B). Numerous derivatives or other compounds with similar characteristics have been described. Both NNRTIs and NRTIs have been shown to successfully diminish the plasma viral burden of HIV-1-infected hosts. However, protracted monotherapy with any of the currently available drugs, including compounds that inhibit the viral protease, was shown to lead ultimately to the development of drug resistance. The emergence of mutations in the RT or protease genes is, in most cases, associated with a rebound of HIV RNA plasma levels. In contrast, levels of plasma RNA can be signiﬁcantly reduced (below 20–50 copies/mL) when three or more drugs are administered in combination [12]. Triple therapy with drug regimens containing two NRTIs with a protease inhibitor, an NNRTI, or a third NRTI may provide comparable activity in antiretroviral-naive patients [13]. Unfortunately, although signiﬁcant suppression of viral replication may diminish the outgrowth of resistant variants, the emergence of breakthrough viruses cannot be prevented [14]. Thus, resistance to antiretroviral agents remains a major problem, and properties of available drugs must be discussed in the context of genetic

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FIGURE 1 Chemical structures of nucleoside and non-nucleoside analog RT inhibitors. (A) NRTIs lack the 30 -hydroxyl group that is located in the sugar moiety of natural nucleosides. (B) NNRTIs represent a chemically diverse set of compounds that bind close to the polymerase active site of the RT enzyme.

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alterations that change the susceptibility to these drugs. In the following, we review mechanisms involved in NNRTI-mediated inhibition of viral replication, the problem of drug resistance associated with this set of compounds, and the concerted activities of both classes of RT inhibitors in the context of their speciﬁc resistance patterns. NNRTIs that are currently used in the clinic, as well as compounds that are in development, are shown in Table 1 in relation to factors that restrict their clinical use, i.e., patterns of resistance as well as possible side effects.

TABLE 1 Available NNRTIs and Factors That Compromise Clinical Use Clinically approved NNRTIs Nevirapine (NVP) (Viramune1, Boehringer Ingelheim) Side effects: rash; Stevens-Johnson syndrome as a result of severe skin rashes (rare). Resistance-conferring mutationsa: A98G, L100I, K103N, V106A, V108I, Y181C/I, Y188C, G190E Delavirdine (DLV) (Rescriptor1, Agouron Pharmaceuticals) Side effects: As for NVP, the most frequent adverse effect seen with DLV is rash. Resistance-conferring mutationsa: L100I, K103N/T, V106A, Y181C/I, Y188H, G190E, E233V, P236L, L238T Efavirenz (EFV) (Sustiva1, Bristol-Myers Squibb) Side effects: The major side effects from EFV are on the central nervous system during the ﬁrst weeks of treatment. These effects include psychiatric symptoms such as dizziness, sleeplessness, intense dreams, and anxiety. Resistance-conferring mutationsa: L100I, K101E, K103N, V108I, V179D/E, Y181C, Y188L, G190E/A/S, P225H Some NNRTIs in development (þ)-Calanolide A, Sarawak MediChem Pharmaceuticals Capravirine (AG1549), Agouron Pharmaceuticals DPC083, Bristol-Myers Squibb MIV-150, Medivir and Chiron TMC120 and TMC125, Tibotec Virco Emivirine, Triangle Pharmaceuticals Resistance patterns and side effects associated with the NNRTIs have not yet been fully examined in clinical trials a

Positions associated with cross-resistance to all three clinically available NNRTIs.

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THE NNRTI-BINDING SITE

The ﬁrst high-resolution structure of HIV-1 RT was derived from cocrystals that contained the NNRTI nevirapine [15]. The model provided important information about the general architecture of the enzyme and the requirements for inhibitor binding. In mature HIV-1 viral particles, the RT enzyme exists as a heterodimer consisting of a p66 and a p51 subunit. The p51 is generated by protease-mediated cleavage of the p66 subunit. Both the polymerase and RNase H active sites are located in the p66 subunit, and despite the fact that the amino terminal sequences of the two subunits are identical, p51 does not directly contribute to the various catalytic activities. The YMDD* motif, which is highly conserved among DNA polymerases and involves amino acid residues 183–186 in HIV-1 RT, constitutes part of the polymerase active site in p66. The crystal structure indicates that equivalent residues in the p51 subunit are buried and do not form a catalytically active conformation [15]. Based on the same structure, the enzyme has been subdivided into several regions. These are ﬁngers, thumb, palm, and connection subdomains as well as the RNase H domain, found only at the carboxy terminus of p66. Crystal structures of HIV-1 RT complexed with DNA/RNA [16], or DNA/DNA primer/templates, in the presence [17] or absence of the incoming deoxynucleotide triphosphate (dNTP) [18], show a nucleic acid binding channel into which the duplex is wrapped by the thumb and ﬁngers of the large subunit (Fig. 2). Most interactions between RT and the duplex are seen in the vicinity of the polymerase active site that is located in the palm subdomain of the enzyme. Various crystal structure models of complexes between wild-type HIV-1 RT and different NNRTIs point to the existence of a common nonnucleoside inhibitor binding pocket (NNIBT), which is not seen in the unliganded form of RT [15,19–22]. The deep hydrophobic pocket lies between the palm and the base of the thumb in close proximity to the primer terminus. The aromatic side chains of residues Y181 and Y188 are involved in important stacking interactions with the bound NNRTI. A comparison with the structures of liganded and unliganded enzymes suggests that binding of the inhibitor caused severe rearrangements that include these two amino acids, as well as residues that constitute the

* Amino acid abbreviations and single-letter codes: Alanine, Ala, A; arginine, Arg, R; asparagine, Asn, N; aspartic acid, Asp, D; cysteine, Cys, C; glutamic acid, Glu, E; glutamine, Gln, Q; glycine, Gly, G; histidine, His, H; isoleucine, Ile, I; leucine, L; lysine, Lys, K; methionine, Met, M; phenylalanine, Phe, F; proline, Pro, P; serine, Ser, S; threonine, Thr, T; tryptophan, Trp, W; valine, Val, V.

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FIGURE 2 The crystal structure of HIV-1 RT bound to a DNA/DNA primer/ template substrate and an incoming nucleoside triphosphate. The large subunit of the heterodimer (p66) is shown in dark gray, and the small subunit (p51) is shown in light gray. The orientation on the right illustrates that the thumb and ﬁngers of p66 form a nucleic acid binding channel. The incoming dNTP is trapped by the ﬁngers. (From Ref. 17.)

polymerase active site [21]. It has been suggested that residues known to be crucial for catalysis may adopt conformations reminiscent of the geometry seen in the catalytically incompetent p51 subunit. In addition, the crystal structures of RT/NNRTI complexes point to structural changes in the primer grip, i.e., a motif that is implicated in the proper positioning of the primer terminus relative to the polymerase active site. These ﬁndings are in agreement with biochemical and clinical data that revealed some NNRTI resistance–conferring mutations in this region (see below). 3

MECHANISMS INVOLVED IN NNRTI RESISTANCE

It was shown that NNRTIs select for resistance in vivo and in cell culture experiments. Almost all resistance-conferring mutations associated with diminished susceptibility to NNRTIs are clustered around the hydrophobic binding pocket (Fig. 3). Most prominent mutations include the K103N, Y181C, and Y188C changes. Crystallographic data and kinetic studies suggest that amino acid substitutions in the NNIBP either affect optimal binding of the inhibitor or diminish the accessibility of the binding pocket.

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FIGURE 3 Location of amino acids associated with resistance to NNRTIs. The complex with DNA/DNA is shown in the presence of the incoming nucleoside triphosphate. Residues that have been associated with resistance to NNRTIs are highlighted. These amino acid substitutions are located in close proximity to the dNTP binding pocket.

Changes at positions 181 and 188 were shown to interfere with inhibitor binding [23–25]. A 500-fold decrease in afﬁnity has been observed for nevirapine and the Y181C mutant enzyme relative to wildtype RT [26]. The difference is likely attributable to increased rates of dissociation of the bound inhibitor. Positions 181 and 188 were found to be naturally altered in HIV-2 and SIV, which helps to explain their decreased susceptibility to many NNRTIs. In contrast, the K103N mutation confers resistance by acting as a ‘‘gatekeeper’’ that blocks the access of the NNRTI to the hydrophobic binding pocket. The crystal structure of the unliganded mutant points to extensive rearrangements in the NNIBT that include a network of novel hydrogen bonds [27]. In particular, some novel hydrogen bonds appear between the mutated residue 103 and the two aforementioned amino acids at positions 188 and 181. These and other novel interactions must be broken during diffusion and the binding of the inhibitor to its binding site. Kinetic data point to reductions in the on-rate of nevirapine binding while rates of inhibitor dissociation remained unaffected [28]; this is consistent with structures of the liganded mutant and wild-type enzymes that do not largely differ from each other [27].

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Mutations associated with resistance to NNRTIs involve numerous other positions, including residues that are located around the ‘‘primer grip’’ (residues 227, 229, 234, 235, and 236) or those that are located in close proximity to the active site (179, 181, 188, 189, 190). The effects of these mutations with respect to NNRTI susceptibility are exerted through the large p66 subunit. The E138K mutation, known to confer resistance to TSAO* and some derivatives of TIBO,{ is the only substitution that encodes resistance to NNRTIs contributed by the p51 subunit [29,30]. This is because E138 of p51 forms part of the NNIBP, whereas the same residue in the larger p66 subunit is distal from this position.

4

DEVELOPMENT OF NNRTI RESISTANCE

In vitro selection experiments revealed that nevirapine rapidly selects for resistant viruses containing the Y181C mutation [31]. The Y181C change confers high-level resistance to nevirapine and shows cross resistance to most other NNRTIs, with the exception of efavirenz. These observations are in good agreement with clinical data. Resistance to neverapine is associated with a broad spectrum of mutations that include changes at positions 103, 106, 108, 181, 188, and 190. The frequency with which NNRTI resistance–conferring mutations emerge can critically depend on the administered drug regimen. Drug regimens containing AZT and nevirapine may suppress the emergence of the Y181C mutation and favor the K103N change [57]. These observations are consistent with earlier ﬁndings that show an increase in zidovudine susceptibility in the presence of the 181C mutation [32]. However, a recent detailed genotypic analysis of patients who partially failed a drug combination that included zidovudine, didanosine, and nevirapine showed that NNRTI resistance mutations were most likely to occur at positions 181 and 190 [33]. The 181C mutation was seen concomitantly with mutations associated with resistance to zidovudine. Mutations were observed less frequently at positions 101, 103, 106, 108, and 188. Some mutations were often seen in combination (181C/190A), whereas others predominantly occurred alone (188L). The same study also pointed to signiﬁcant differences in the mutational patterns of early and late therapy isolates. The heterogeneity of these NNRTI mutational patterns suggests that * tert-Butyldimethylsilylspiroaminooxathioledioxidethymines. { Tetrahydroimidazobenzodiazepinone.

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resistance testing may be necessary to determine whether patients failing nevirapine-containing drug regimens might beneﬁt from efavirenz. The K103N mutation is most frequently found in patients failing efavirenz combination therapy [34]. The mutational pattern associated with resistance to efavirenz does not appear to be as heterogeneous as that described for nevirapine. The K103N substitution emerged in isolates of patients who received efavirenz plus a protease inhibitor (indinavir), efavirenz plus 3TC or d4T, or efavirenz plus 3TC and AZT. In many cases the V108I and P225H mutations were found in isolates that contained the K103N change. Changes at positions 100, 101, 188, and 190 were found less frequently, and the V106A, Y181C, and Y188C mutations were rarely observed. Selection experiments in cell culture are in only partial agreement with these ﬁndings. In vivo, certain amino acid substitutions are found predominantly when others are also present, whereas cell culture experiments revealed isolates with single mutations at positions 101, 108, 179, and 181 [35]. The second-generation NNRTI HBY 097 [58] selects in vivo for the K103N mutant [59,60] whereas in vitro selection experiments revealed changes at positions 179 and 190 [60]. A similar discrepancy between in vivo and in vitro selections has been reported in the context of delavirdine [36]. The K103N change and the Y181C mutation are the most prominent mutations that emerge in the context of delavirdine monotherapy. Very few patient isolates show the P236L mutation, and isolates containing this amino acid substitution concomitantly harbor a change at position 103. In contrast, in vitro selection experiments suggest frequent emergence of the 236L mutation. The rare emergence of the P236L mutation in patient isolates is associated with diminished viral replication capacity of this variant. Indeed, mutant enzymes containing this amino acid substitution showed altered properties of the RT-associated RNase H activity [37]. Residue 236 is located in close proximity to the highly conserved primer grip (residues 227–235) and may thus play an important role in substrate binding. Of note, some residues that constitute the primer grip are also part of the NNRTI binding pocket. The crystal structure of HIV-1 RT and the NNRTI UC781 points to important interactions between W229 and the inhibitor [38]. The W229F-containing mutant enzyme showed severely diminished rates of DNA synthesis, which correlated well with the diminished ﬁtness seen with corresponding viruses [39]. In vitro data suggested an important role of the 318 mutation in regard to NNRTI resistance [40]. These ﬁndings were conﬁrmed by recent clinical data that showed the 318F mutation in patient isolates with a prevalence of >1% [41]. This mutation is selected by delavirdine

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in vivo and is most frequently found in association with K103N or Y181C. The 318F mutation alone confers high-level resistance to delavirdine and increases resistance to both efavirenz and nevirapine in combination with other NNRTI resistance–conferring mutations.

5

NRTI AND NNRTI COMBINATION THERAPY

The effectiveness of combination therapy relies in part on the different complementary mechanisms involved in inhibition through two or three antiretroviral agents that may or may not belong to the same class. Enzyme kinetic data point to additive or synergistic effects in the context of some NRTI and NNRTI combinations that include AZT-TP/ nevirapine and AZT-TP/efavirenz [42,43]. The underlying biochemical mechanisms are not fully understood, although previous ﬁndings suggest that NNRTIs may affect the potency of NRTIs in multiple ways that do not involve only the rate of incorporation. The addition of AZT-MP or other chain-terminating nucleotides is not irreversible. Recent studies have indicated that HIV-1 RT is capable of removing the incorporated chain terminator in the presence of physiologically relevant concentrations of pyrophosphate (PPi) or the pyrophosphate donor ATP [44,45]. Thus, increased rates of phosphorolytic cleavage may diminish the efﬁciency of the chain-terminating nucleotide. Indeed, RT enzymes containing AZT resistance–conferring mutations were shown to be capable of unblocking the primer with high efﬁciency, which provides an important mechanism for HIV resistance to some NRTIs including AZT [44,45]. In contrast, decreased rates of phosphorolytic cleavage may increase the efﬁciency of chain-terminating nucleotides, and such diminished rates of primer unblocking have been described under various conditions. First, the thiocarboxanilide NNRTI UC781 severely diminishes the efﬁciency of the pyrophosphorolytic removal of AZT-MP in the context of both wild-type RT and AZTresistant enzymes [46]. These data help to explain the synergistic effects of AZT–UC781 combinations. Moreover, the combination of the two drugs was shown to resensitize formerly AZT-resistant viruses. Second, the M184V mutation in HIV-1 RT, known to confer high-level resistance to 3TC, is associated with hypersusceptibility to AZT [47]. Previous ﬁndings in cell-free assays showed that M184V literally blocks rescue of 3TC-terminated DNA synthesis and diminishes the rates of unblocking of AZT-terminated primer strands [48,49]. These biochemical data help to explain the increased susceptibility to AZT in the context of viruses containing the M184V mutation.

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Sustained antiretroviral effects of AZT have also been reported in association with mutations that confer resistance to pyrophosphate analogs, which is likewise linked to diminished rates of phosphorolytic cleavage. Other mutations associated with resistance to NRTIs or NNRTIs show very similar effects. The L74V that confers low-level resistance to the nucleoside analogs 3TC, ddI, and ABC can resensitize AZT in a mutational background associated with resistance to AZT [50]. Moreover, both NNRTI-associated mutations, the frequent Y181C change as well as the L100I mutations, have been associated with this phenotype [32,51]. These data may help to explain the diminished emergence of the Y181C mutation in the context of combination therapy with AZT and nevirapine. It has recently been demonstrated that NRTI resistance–conferring mutations may also cause increased susceptibilities to NNRTIs [52]. In many cases, patient isolates that contained the M184V mutation as well as frequent AZT resistance–conferring mutations showed increased susceptibilities to all three approved NNRTIs. NRTI resistance–conferring mutations were also shown to resensitize viruses containing the K103N mutation. These effects seem to be associated with mutations at position 41, 210, and 215, although speciﬁc mutational patterns that ultimately result in increased susceptibilities to NNRTIs were not identiﬁed. Of note, the same study pointed to a correlation between EFV hypersusceptibility and a signiﬁcantly improved virological suppression. However, it remains to be seen whether drug hypersusceptibility and resensitization effects were maintained over protracted periods of time. Such complex phenotypes may occur only transiently, as shown in the context of AZT–3TC combination therapy, and the acquisition of additional mutations may reverse the increase in drug susceptibility. Resistance to multiple drugs is becoming a growing problem. A frequent pattern of multidrug resistance (MDR) to literally each of the available NRTIs involves the Q151M mutation, which is often accompanied by changes at positions 62, 75, 77, and 116 [53]. The existence of such mutational patterns can sometimes severely limit remaining treatment options. The effects of NNRTIs and NNRTI resistance– conferring mutations in the context of decreased susceptibility to multiple NRTIs have recently been studied [54]. Patient isolates containing the Q151M mutation were cultured in the presence of HBY097 to analyze genotypic and phenotypic changes. Resistant viruses containing the K103N and L100I changes were rapidly selected. Two mutations were sufﬁcient to confer high-level resistance to all NNRTIs.

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However, the presence of NNRTI resistance–conferring mutations partially reversed phenotypic resistance to most NRTIs. Despite the ongoing problem of drug resistance, NNRTIs represent an important class of antiretroviral agents that can signiﬁcantly improve clinical outcome in individuals infected by HIV-1. Moreover, the use of NNRTIs is not restricted to therapy; rather, members of this family of compounds were shown to act effectively in preventing transmission of the virus. Some of the available NNRTIs fulﬁll criteria for use as microbicides, based on their ability to bind tightly to RT (e.g., UC781 and efaviranz), and a trial in Uganda demonstrated that a simple single-dose regimen of nevirapine can dramatically reduce mother-to-child transmission of HIV-1 [55]. These are exciting ﬁndings, despite subsequent observations indicating that the K103N mutation can later be detected in about one-third of the women who received this treatment [56]. REFERENCES 1.

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15 HIV Protease Inhibitors Richard Ogden Agouron Pharmaceuticals, Inc., A Pﬁzer Company, La Jolla, California, U.S.A.

1

INTRODUCTION

In the history of drug development, no molecular target has attracted more attention in a shorter period of time than the protease from human immunodeﬁciency virus 1 (HIV-1). The human immunodeﬁciency virus was the ﬁrst new pathogenic agent to be dissected at the structural and functional level by the tools of modern interdisciplinary science. Advances in recombinant DNA technology, rapid DNA sequence analysis, and detection and ampliﬁcation of viral nucleic acid from tissue samples were initially used to characterize and analyze the virus, a retrovirus that uses reverse transcriptase to copy its RNA genome, and its constituent gene products. For a review of the molecular biology of retroviruses, see Ref. [1]. One of those gene products, reverse transcriptase, was the target for the ﬁrst nucleoside-derived enzyme inhibitors that provided the initial hope of reducing the mortality due to AIDS. It became clear, however, from early clinical studies that combination therapy with more than one drug directed against reverse transcriptase provided superior clinical outcomes and that new antiretroviral agents with new mechanisms of action would also be needed to provide long-term 523

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suppression of viremia and consequent reductions in mortality and comorbidities [2]. This drug discovery challenge was met by both academic and corporate research on a global scale. The aspartyl protease encoded by HIV-1 quickly became an attractive target for drug discovery, and within 5 years of the puriﬁcation, characterization, and structure elucidation of the enzyme in 1988, tens of thousands of potential inhibitors had been synthesized and screened. Over 20 compounds had been tested in human subjects by 11 pharmaceutical and biotechnology companies. By 1996, treatment options and the availability of potent triple combination therapy for those in developed countries with HIV/AIDS changed signiﬁcantly when three protease inhibitors received marketing approval by the U.S. Food and Drug Administration following controlled clinical trials. These trials demonstrated their unprecedented capacity to suppress viral load, reduce disease progression, and prolong life. This chapter brieﬂy describes the properties and structure of the enzyme and its role in the viral life cycle. A discussion of the discovery of the marketed HIV protease inhibitors and their clinical development is followed by descriptions of the pharmacokinetic properties of the drugs, the emerging understanding of drug resistance as it relates to the protease inhibitors, and the short- and long-term toxicities observed over the 7 years they have been widely used. HIV-1 protease continues to be the template for the discovery and development of new agents, many of which are designed to have activity against drug-resistant HIV-1. These agents are described at the conclusion of the chapter. This overview is necessarily limited in scope. A more comprehensive account of many of the topics discussed brieﬂy in this chapter can be found elsewhere [3].

2 2.1

HIV PROTEASE: MECHANISM, ROLE IN THE VIRAL LIFE CYCLE, AND STRUCTURE Mechanism

Proteases in general can be classiﬁed according to the nature of the amino acid that provides the functionality necessary for cleavage of the peptide bond. Thus we have serine or cysteine proteases in which that active site amino acid reacts with the peptide backbone directly; metalloproteases, where ions indirectly activate the bond to be cleaved; and aspartic proteases, where the aspartate group can function as a general base, facilitating the hydrolysis of the scissile bond. Proteases have evolved to maintain certain structural homologies (in terms of both sequence and

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tertiary structure), and early examination of the sequence and inhibitor studies led to the hypothesis that HIV protease was related mechanistically to the aspartic protease family typiﬁed by pepsin [4,5]. Cellular aspartic proteases in this family are characterized by two lobes, each contributing an aspartic acid residue to the active site. It has been suggested that these enzymes evolved from a gene duplication event from a primordial aspartic protease gene [6]. The lengths of the aspartyl proteases from Rous sarcoma virus (RSV) and HIV were such that it was proposed that these viruses would be composed of two identical subunits each of which would contribute an aspartic acid residue to the active site [7]. This hypothesis was conﬁrmed by the crystal structure determination of RSV protease and HIV protease (for a review, see Ref. [8]). 2.2

Role in the Viral Life Cycle

HIV protease plays a key role in the production of infectious viral particles. The genome organization and translational modules of HIV are shown in Figure 1. The positive-sense viral RNA is organized into three

FIGURE 1 Genome and proteome organization for HIV. Structural (gag, pol, and env) genes are shaded; regulatory (tat and rev) and accessory (vif, nef, vpr, and vpu) genes are clear. Common to all retroviruses, the gag and pol genes are translated in the cytoplasm from newly synthesized unspliced viral RNA. Translation usually occurs through to a stop codon at the 3’ end of the gag gene, resulting in the structural polyprotein Pr55gag. About 5% of the time, ribosome frameshifting during translation of gag results in the synthesis of a fusion protein Pr160gag-pol. The frameshift site (fs) is located upstream of the Gag p6 protein such that a transframe polypeptide (TF) is incorporated into Gag-Pol in place of p6. Abbreviations: matrix (MA), capsid (CA), nucleocapsid (NC), protease (PR), reverse transcriptase (RT), integrase (IN). (From Ref. [9].)

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major coding elements—gag, pol, and env—in common with all other retroviral genomes. The gag and pol gene products are translated from a polycistronic mRNA. A translational stop codon leads to a 55 kDa Gag polyprotein (p55) that contains the structural proteins matrix (MA), capsid (CA), and nucleocapsid (NC) along with smaller peptides, which are all necessary for assembly of mature viral capsids. The pol gene encodes the viral enzymes protease, reverse transcriptase, and integrase and is translated as a result of a frameshift into a 160 kDa polyprotein precursor. The frequency of the frameshift regulates the proportions of structural and catalytic proteins needed to produce infectious virus. This process of replication is highly efﬁcient and is also capable of generating huge primary sequence diversity due to the lack of proofreading activities in reverse transcriptase. It has been estimated that on a daily basis on the order of 1 billion new viral particles are produced in the course of untreated infection in an individual [10–12]. Proteolytic processing of both p55 gag and gag-pol is performed during virus assembly by the HIV protease, which must also catalyze its autoexcision from gag-pol [9]. In addition, the env gene product (gp 160), is cleaved by cellular proteases to generate two glycosylated proteins, gp 120 and gp 41, which are associated with the outer viral envelope and are necessary for the early events of receptor attachment and fusion needed for virus entry into cells. Protease inhibitors have played a key role in understanding the role of protease in the viral life cycle. They do not, for example, block the appearance of integrated proviral DNA [13,14], and their activity in preventing production of virus from chronically infected cells suggested a postintegration role. In 1988, a key experiment involving a virus deleted in the protease gene showed that virus produced from this construct had an immature morphology and was noninfectious [15]. This phenotype was also observed by site-directed mutagenesis of Asp 25 and by inhibition with HIV protease inhibitors [16,17]. These experiments provided the target validation necessary for a major drug discovery and development campaign. 2.3

Structure

The HIV protease consists of two identical subunits of 99 amino acids associated in a twofold symmetrical fashion. A typical structure of the inhibited enzyme is shown in Figure 2. The active site of the enzyme is formed at the interface between the monomers and contains the two aspartic acid residues. HIV is a highly polymorphic virus, with both geographic (clade) diversity and intraclade quasi-species being well

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FIGURE 2 Ribbon diagram of HIV protease complexed with nelﬁnavir. The inhibitor structure and the two active site aspartic acids are shown in detail.

described. The polymorphisms represented in this diversity are largely found at sites on the periphery of the structure, with the active site residues important for substrate and drug binding largely conserved. As prevention and treatment initiatives move toward the developing world and non-clade B virus as well as toward drug-resistant clade B virus, it will be important to monitor the structural changes in this protein if it is to be used for drug discovery purposes. This is discussed further below. To date, several hundred structures have been solved for inhibited complexes of HIV protease, indicative of the value placed by scientists on the information gained thereby in the process of drug design. The early structures revealed the nature of the active site pockets into which substrate and inhibitor functional groups penetrate. Hydrogen bonds can frequently be seen mediating interactions between enzyme and inhibitor, and bridging water molecules are frequently seen as key components of inhibited structures (Fig. 3). The initial structural studies and early descriptions of inhibitor design have been well reviewed [18–21]. In summary, the structures of HIV protease compose probably the richest database telling the story of protein structure-based drug design.

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FIGURE 3 General scheme of active site interactions in HIV protease with a substrate-based inhibitor. Hydrogen bonds are drawn as dashed lines. (From Ref. [8].)

3

STRATEGIES FOR DRUG DESIGN

Drug discovery programs were initiated in many cases as a result of the pivotal experiments deﬁning the essential role of protease in producing infectious viral particles. There were no precedents for the discovery and development of protease inhibitors as antiviral agents, and, furthermore, previous attempts to design inhibitors of aspartyl proteases as drugs in other therapeutic areas (e.g., renin as a target for hypertension) had been long and tortuous. Many problems had been encountered in translating excellent peptide-based enzyme inhibitors into orally bioavailable analogs that could be appropriate for long-term therapy. It was just this challenge that would have to be met and overcome in designing HIV protease inhibitors. In contrast to the various available inhibitors of HIV reverse transcriptase, which were discovered directly by infected cell-based screens or elaborated from lead compounds discovered that way, none of the currently available or research drugs targeting protease have come from screening. The recognition that a new approach to lead compound

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discovery involving the translation of knowledge not only of enzyme mechanism but also of enzyme structure was already being recognized and supported at a small number of laboratories within both the pharmaceutical industry and academia. These laboratories in particular quickly seized the challenge represented by this new target to demonstrate the beneﬁts of the new approach. Two approaches dominated the early discovery efforts, with pragmatism often dictating a mix-and-match combination of the best of each. The inhibitors discovered in those early days could clearly be assigned to classes on the basis of whether they owed more to the substrate-based or to the structure-based approach. With the possible exception of saquinavir, the ﬁrst drug in this class to reach the market, all of the protease inhibitors in current use can claim to have beneﬁted in varying degrees from the structure-based approach.

3.1

Substrate-Based Approaches

The elucidation of the sequence of amino acids representing the cleavage sites within the substrate and the wealth of chemistry surrounding the incorporation of noncleavable dipeptide isosteres discovered in the various renin programs existing in the 1980s led to a profusion of HIV protease inhibitors with often good potency in the nanomolar range in in vitro enzyme inhibition assays. These had been assembled in modular fashion by using cognate substrate sequences in the ﬂanking positions with a variety of isosteres in place of the P1–P10 dipeptide. There was no expectation that these molecules would be active orally, but they served the purpose in many cases to provide structural information on the inhibited protease that would serve as a basis for subsequent structurebased approaches. An example of one such enzyme inhibitor is provided by AG1004, in which the naturally occurring statine moiety is incorporated via standard synthetic peptide chemistry into the sequence of the cleavage site Ser-Gln-Asn-Tyr-Pro (Fig. 4). An early clinical success for this approach was produced by the group at Roche, who by use of standard medicinal chemistry strategies to mimic and constrain the peptidic portion of the inhibitor in the P20 and P30 pockets were able to generate the ﬁrst approved drug in this class. It cannot be overemphasized that despite the low oral bioavilability of the initial formulation the safety and efﬁcacy of this drug at the time gave us the ﬁrst glimpse at how triple combination therapy could begin to impact the mortality and morbidity associated with HIV/AIDS.

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FIGURE 4 Structure of the binding site of HIV protease complexed with the peptide analog NH2-Ser-Gln-Asn-Sta-Ile-Val-Gln-CO2H, shown in dark gray, ﬁtted into the experimental electron density.

3.2

Structure-Based Approaches

The introduction of the disciplines of protein crystallography and computational chemistry as integral components of a drug discovery program was pioneered in the 1980s by new biotechnology companies in the United States such as Agouron and Vertex and had enthusiasts within major pharmaceutical companies such as Burroughs Wellcome, Abbott, and Merck. A few academic centers also recognized the value of

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bringing medicinal chemists and pharmacologists into professional contact with computational chemists and protein X-ray crystallographers, and early funding for these efforts was provided by the U.S. National Institutes of Health (NIH) to foster these academic–industrial alliances in the HIV area. A generic description of the iterative cycle involved in structurebased drug design is shown in Figure 5. Once the technical hurdles associated with quickly producing crystals of newly synthesized inhibitors bound to the enzyme were overcome, the step of structure determination was no longer rate-limiting in a project, and chemists appreciated the practical value of obtaining that bound structure and being able to correlate it (or not) with any change in potency. With input from a variety of computational techniques, the next molecule synthesized was often more potent. As important as this methodology was for informed improvement of a lead compound, it also helped break the mindset often imposed by overreliance on the substrate-based approach. Drugs ready for clinical trial did not, of course, result directly from the structure-based approach. The ability to make the necessary chemical adjustments to enhance solubility (formulation), metabolic stability, cost, and (an important factor) patent exclusivity as well as to possibly modify toxicities apparent in preclinical work could now, however, be addressed within the known spatial constraints provided by the structure of the active site.

FIGURE 5 Drug discovery and development pathway highlighting the iterative structure-based design cycle.

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HIV PROTEASE INHIBITOR DISCOVERY AND DEVELOPMENT

The six currently available drugs resulted from the application of the principles outlined in the previous section in simultaneous discovery and development programs conducted within Roche, Abbott, Merck, Agouron/Eli Lilly, and Vertex/Burroughs Wellcome. Other major pharmaceutical companies, for example, Searle, Upjohn, and ParkeDavis, also had programs, and an unprecedented degree of communication between scientists in these companies and within academia worldwide existed in the face of the growing catastrophe of global AIDS. Recognizing the interdependence of these competitive programs, the discovery efforts are described in chronological order of drug approval. A comprehensive account of the efforts within each group forms the core of a recently published book [3], and the more human and logistical aspects of this endeavor in one instance have been written about in the popular literature [22]. 4.1

Saquinavir (InviraseTM, FortovaseTM)

The HIV protease drug discovery effort at Hoffmann-La Roche was initiated in 1986 (see Chap. 2 of Ref. [3]). Following assay establishment in which cleavage of a heptapeptide substrate could be monitored spectrophotometrically and therefore could be used to assay inhibitors rapidly, the early leads tested were small peptides incorporating the hydroxyethylamine transition state mimetic ﬂanked by phenylalanine and proline side chains. In the absence of any structural data, the most potent tripeptide analog in the in vitro assay was selected for optimization by systematic exploration of functional groups at the Nand C-termini of the peptide. This represented a classic example of structure–activity-driven medicinal chemistry. The availability of a cellbased antiviral assay at this time conﬁrmed the structural activity within this series, suggesting good intracellular penetration, and thus the most potent homolog (Ro 31-8959, later to be named saquinavir) was selected for further evaluation. It was quickly established that saquinavir was effective at inhibiting the natural substrate, either gag or gag-pol, in a dose-dependent manner [23,24] and that it had no inhibition up to 10 mM of other human aspartic proteases such as renin or cathepsin D or of anticipated proteases with other mechanisms of action such as elastase or collagenase [23]. Exploring activity against a range of viral isolates and different cell lines is a key component of any discovery effort, particularly in the absence of any animal model of HIV infection suitable for routine drug

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screening. Saquinavir showed impressive potency against laboratory viral strains and clinical isolates, including those resistant to the reverse transcriptase inhibitor AZT (zidovudine), and when tested in T-cell and macrophage cell lines as well as primary cells [25,26]. The therapeutic index of the drug (the ratio of cell cytotoxicity to antiviral activity) was approximately 1000, indicating a potential margin of safety exceeding that of the available nucleoside reverse transcriptase inhibitors. Graphic understanding of the activity of saquinavir was shown by electron microscopy of saquinavir-treated chronically infected cells which showed production of immature viral particles [27] that were noninfectious [28]. A critical step in the preclinical evaluation of potential HIV drugs is an understanding of whether viral variants with reduced sensitivities to the agents can be selected in vitro by serial passage studies. In the latter part of the 1980s, it was becoming clear that resistance was emerging in patients treated with the early nucleoside analogs, who were experiencing viral load rebound or inadequate suppression of viremia. It was not at that time fully appreciated how mutable HIV could be in the presence of inhibitors, and it was thought that selecting viable protease inhibitor– resistant variants might take longer in vitro and might even not occur. This was because amino acid changes at the genetic level would be duplicated at the functional protein dimer level and perhaps because the substrate- and drug-binding site was located at the dimer interface, which might be structurally destabilized by selected changes leading to nonviable resistant isolates. Passage studies in vitro did, however, yield a strain with reduced sensitivity to saquinavir, although it was generated at a later time point in the experiment than in parallel studies involving nucleoside and non-nucleoside reverse transcriptase inhibitors [29]. Sequencing of the isolates at various time points demonstrated changes in amino acids ﬁrst at position 48 and subsequently also at position 90. The signiﬁcance of genotypic and phenotypic resistance to the class of protease inhibitors is discussed later. By 1991, following only minor toxicological ﬁndings in various animal species, the initial hard gel formulation of saquinavir, InviraseTM, was ready for clinical studies. Additivity of saquinavir in dual or triple combination with reverse transcriptase inhibitors had been demonstrated in vitro, suggesting the possible use of combination therapy to further reduce replication and the emergence of resistance. The initial studies, as with all drugs to this day, were, however, short dose range– ﬁnding studies to explore drug safety and in vivo efﬁcacy. The original formulation had limited oral bioavailability, and the compound showed extensive ﬁrst-pass liver metabolism. Food increased absorption, and the

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early efﬁcacy studies showed a positive dose–response relationship with respect to the surrogate markers of viral load reduction and CD4þ cell counts over a 24-week period when the compound was dosed at up to 600 mg (three pills) three times a day. Combination therapy with AZT as well as AZT plus ddC (Zalcitabine, another chain-terminating reverse transcriptase inhibitor) showed improved responses and led the way to the standard of triple combination therapy with agents active against two viral targets [30]. Tolerability was exceedingly good, and the drug was the ﬁrst in its class to receive approval from the U.S. regulatory authorities in December 1995. With emerging data on high-dose efﬁcacy studies with Invirase [31], a new formulation was developed (FortovaseTM). Studies were conducted to assess the efﬁcacy of the two formulations, and superiority was demonstrated in the triple drug arm containing Fortovase [32]. Fortovase was subsequently approved in November 1997. Both formulations have been extensively studied in combination with varying doses of ritonavir (an HIV protease inhibitor in its own right but also an inhibitor of protease inhibitor metabolism) and are now used in the clinic almost exclusively in this way on a twice-a-day or, experimentally, oncea-day dosing schedule [33]. 4.2

Ritonavir (NorvirTM) and Lopinavir/Ritonavir (KaletraTM)

The HIV protease inhibitor program at Abbott took as its premise that symmetry-based inhibitors of the enzyme might derive potency by matching the symmetry of the enzyme active site. This effort combined the traditional medicinal chemistry approach of systematically optimizing the inhibitors in a modular fashion with emerging structural studies deriving from the solution of the crystal structure of HIV protease by Wlodawer and colleagues, reported in 1997. A core unit mimicking two phenylalanine side chains spanning the customary hydroxyl group was modiﬁed and elaborated to optimize potency and to impact metabolic stability. The evolution of the inhibitors in the Abbott program and the property enhancements achieved are shown in Figure 6. The standard array of preclinical tests were conducted with ritonavir. Notably, it was found to be a potent inhibitor of cytochrome P450 isoform 3A4, which would perhaps become its most valuable property because it is via this pathway that most other protease inhibitors are metabolically degraded [34]. This property has led to its being used currently in low dose as a pharmacokinetic enhancer of other protease inhibitors [35], most recently in a single formulation with the protease inhibitor lopinavir. In passage studies in vitro to explore the selection of resistance, some mutations

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FIGURE 6 Evolution of structures of symmetry-based HIV protease inhibitors leading to the identiﬁcation of ritonavir and lopinavir (ABT-378). Obstacles to development that were addressed by subsequent lead compounds are shown between respective structures.

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were selected that were distinct from those observed with saquinavir, notably at positions 82 and 84, although the clinical consequences of this were unknown at the time [36,37]. The dose for initial efﬁcacy studies was picked by taking into account the attenuation of ritonavir activity in vitro in the presence of human serum. Twice-daily dosing of the drug at either 500 or 600 mg produced steady-state levels consistently above the EC50 value, and in dose-ranging phase II studies, a rapid decline in plasma viremia was observed [38,39]. The phase III studies that led to the approval of ritonavir were the ﬁrst to demonstrate that when added to existing therapy ritonavir signiﬁcantly reduced mortality and disease progression in patients with advanced HIV infection [40]. The HIV protease drug discovery effort continued at Abbott with the goal of discovering a second-generation molecule with reduced protein binding and high plasma trough levels. It had been noted that the mutation rate in vivo in patients failing ritonavir therapy was inversely correlated with the plasma trough, and it was thought that maintenance of high trough levels well above the EC50 might slow the emergence of resistance. The knowledge that even low doses of the drug ritonavir could dramatically enhance other members of the class led to the decision to pick a candidate for development that would interact favorably with ritonavir in this way and could potentially be coformulated with it. This property was found in the molecule ABT 378 (lopinavir), where a trough/EC50 ratio of approximately 75 could be observed in HIV-infected subjects when a serum-adjusted EC50 for wildtype virus was used for the calculation [41]. Lopinavir/ritonavir dosed as a 400/100 coformulated tablet has demonstrated impressive potency in combination therapy in both therapy-naive patients [42] and those resistant to existing protease inhibitors [43]. Resistance to the combination does emerge in patients failing therapy in those settings but appears to be impeded, as was intended. The clinical consequences of resistance to lopinavir/ritonavir with respect to subsequent therapeutic options within the class are currently unknown. 4.3

Indinavir (CrixivanTM)

The crystal structure of HIV protease was also seen as a key to rapid discovery and optimization of enzyme inhibitors in the program initiated at Merck. Having been in the aspartic protease arena previously with a drug discovery effort targeting renin, Merck’s ﬁrst steps were to develop a peptide-based assay suitable for enzymatic screening of the transition

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state mimetic peptide inhibitors already in existence and a cell-based assay for screening against virus. The existing molecules were then modiﬁed by standard medicinal chemistry principles to remove potency against renin and improve potency in the HIV assays. When these properties had met project criteria, attention turned to oral bioavailability and metabolic stability. Crystal structures of key compounds bound to the enzyme helped guide the medicinal chemistry effort. The most promising lead unfortunately demonstrated unacceptable hepatotoxicity in the dog, and this prompted the exploration of the hydroxyethylamine version of the series, because Roche had just recently published the ﬁndings with saquinavir that contained that transition state isostere. Modular replacement of the molecular entity from saquinavir onto the Merck scaffold followed by its modiﬁcation to optimize potency and solubility (as a reasonable surrogate for animal oral bioavailability) led to indinavir. The detailed description of the discovery of indinavir (Ref. [3], Chap. 4) is a prime example of the challenges faced by scientists in many disciplines in trying to optimize the several variables that must be dealt with in making up an agent for clinical trials. As with other members of the class, resistance to indinavir was observed in vitro and in patients failing therapy, with the by now familiar variants at positions 82 and 84 in the active site being selected [44]. The observation of in vitro cross-resistance within the class was also described for isolates resistant to indinavir, raising the possibility that clinical use of multiple agents in this class might be limited [45,46]. The early clinical studies with indinavir demonstrated a clear antiviral effect that was optimized with dosing in the absence of food every 8 hr. It was the pivotal study in naive patients in combination with the reverse transcriptase inhibitors AZT and 3TC [47] that set in many ways the gold standard for combination therapy and initiated the era of highly active antiretroviral therapy (HAART), which has been the standard of care ever since. Nowadays, indinavir is dosed almost exclusively in combination with low-dose ritonavir to remove the constraints of the strict dosing requirements of the drug [48]. In this way, twice-daily dosing can be readily achieved with subsequent beneﬁts to the patient. 4.4

Nelﬁnavir (ViraceptTM)

The immense cost associated with pursuing a full drug discovery and development program had deterred many small biotechnology companies that arose in the United States in the late 1970s and 1980s from competing with the established pharmaceutical industry in all but a few

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small and specialized markets. Many such companies had as their focus the discovery of enabling technology or diagnostic tools. The situation changed in the late 1980s with the U.S. National Institutes of Health (NIH) making grant money available for multicenter efforts involving academia and the biotech industry to pursue HIV as a target for drug discovery. Supported by one such grant, and in conjunction with a broader antiviral drug discovery partnership with Eli Lilly, Agouron Pharmaceuticals initiated a broad program of HIV drug discovery, using its core technology, protein structure–based drug design. Of the targets chosen, the protease seemed one of the most suitable for a program in which structural information could be fed back into the lead compound optimization process, especially because of the requirements for optimizing oral bioavailability in a class of compounds not noted for that feature. As the usual steps were being taken to generate the structural information, peptide-based inhibitors containing transition state isosteres were made that ultimately served to generate the ﬁrst in-house crystal structures of the inhibited enzyme. The publication of the saquinavir structure prompted scientists at Agouron and Lilly to generate a simple variant that served as a lead for one discovery effort (Fig. 7). In parallel, a more radical approach led to redesign of the molecule in the S1 and S2 pocket regions and served as a showcase for the efﬁciency of structurebased design. Eighty compounds and 11 crystal structures generated a preclinical candidate (10,000 times more potent than the lead) with good oral bioavailability in three species. It was abandoned in response to concerns about insufﬁcient antiviral activity in vitro. Optimization of the lead in a parallel project took the ﬁrst step of removing the P3 and P2 groups that were present in saquinavir and were thought perhaps to contribute to its low oral bioavailability. Initially bicyclic, then substituted monocyclic aromatic rings were substituted and, guided closely by structures; an unanticipated 1808 rotation in the active site of a compound in the series led to exploitation of a new hydrogen bonding interaction with the side chain of Asp 30. A signiﬁcant increase in antiviral potency was achieved. Finally, modular replacement of the Roche bicyclic moiety back onto this novel core led to nelﬁnavir. Resistance studies in vitro were conducted by standard serial passaging studies, and after 22 passages a unique mutation at position 30 was noted, the change of an aspartic acid to asparagine. Continuing the passages resulted in loss of the mutation at position 30 but acquisition of a change at position 84, seen with ritonavir and indinavir [49]. The consequences for protease inhibitor therapy were not fully appreciated until resistance was encountered during clinical studies [50].

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FIGURE 7 Structures of saquinavir and LY289612 with modiﬁcations leading to nelﬁnavir.

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Having passed the usual battery of preclinical tests to demonstrate oral availability, the decision to initiate clinical trials was made. Because Agouron lacked the resources needed to fund a full development program, a partnership was established with the pharmaceutical division of Japan Tobacco to proceed. The clinical development phase 1 safety and pharmacokinetic studies revealed the doses necessary, with food, for adequate exposure, and although twice-daily dosing could achieve such levels, the conservative approach of dosing three times daily was initially pursued. It was also noted that one of the principal metabolites of nelﬁnavir (designated M8) found in humans is an active antiviral agent. As with the development of indinavir, superiority of either of two dosages of nelﬁnavir was demonstrated compared to the combination of AZT and 3TC in therapy-naive patients [51]. Nelﬁnavir received approval for use in both adults and, with a pediatric formulation, in children in the United States in March 1997, just 39 months after being selected for development. Subsequent approval of twice daily dosing was obtained in 1999 after equivalent efﬁcacy was shown [52]. A study of dual antiretroviral therapy using nelﬁnavir and d4T (stavudine) was conducted with patients who had received prior therapy with reverse transcriptase inhibitors. Not surprisingly in hindsight, the potency of the regimen was insufﬁcient for long-term suppression. The subsequent virological response of patients failing this regimen to a second protease inhibitor regimen provided the ﬁrst evidence that distinct mutational pathways leading to resistance resulted in a clinically signiﬁcant lack of cross-resistance to other members of the same class [53–55]. This trial and these cohort data were the ﬁrst to demonstrate that the strategic sequencing of antiretroviral therapy could provide durable viral suppression in later regimens—an important consideration for practical purposes. 4.5

Amprenavir (AgeneraseTM)

At the time Vertex, another biotechnology company using protein structure–based drug design, entered the protease discovery ﬁeld in 1990, much was known about some of the shortfalls of the molecules just described. The effort was to focus on minimizing molecular weight, associated with hepatic clearance, and avoidance of functional groups that had been correlated with poor solubility, absorption, and urinary elimination. As is often the case, the combination of lipophilicity, solubility, and neutrality is challenging in a selective and potent inhibitor. The detailed three-dimensional structure of the active site

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provided the researchers with the context in which to vary the necessary parameters. The key functionality on which design efforts were focused was the dialkyl benzenesulfonamide, which became part of a standard hydroxyethylamine transition state isostere. Final selection of the second alkyl chain was made following oral bioavailability determinations in the rat. A research and development agreement made with Burroughs Wellcome enabled development of amprenavir to proceed, and the customary preclinical tests were performed. Notably, the in vitro serial passage studies revealed a unique resistance mutation at position 50 [56]. Early clinical experience with amprenavir established that a dose of 1200 mg given twice daily should be investigated in combination studies, and following such studies in combination with nucleoside reverse transcriptase inhibitors [57], amprenavir was approved in the United States. The pill burden associated with the current formulation of amprenavir (eight pills twice daily) has led to its being used at lower doses in combination with low doses of ritonavir as a pharmacokinetic booster [58]. A phosphate ester prodrug of amprenavir (fosamprenavir) that has improved oral bioavailability is currently being evaluated [59] and is expected to be approved in the near future. 5

CURRENT CLINICAL USE OF HIV PROTEASE INHIBITORS

Since the early days of HAART therapy, ushered in by the protease inhibitor class, the need to improve regimen convenience, tolerability, and cost has often dictated the choice of initial combination therapy. All the approved drugs, when taken as prescribed, demonstrate durable potency. The demands of long-term therapy and the emergence of side effects not seen during the accelerated approval process needed to save lives have often meant that the market popularity of any new agent lasts little longer than it takes for the next agent to appear. Accordingly, the need to improve patient adherence has led to the evaluation of dual protease inhibitor combinations that take advantage of beneﬁcial pharmacokinetic interactions. Converting three-times-daily dosing into twice daily dosing is now routine with combinations of ritonavir with saquinavir and indinavir. The extension of this into once daily dosing is being pursued experimentally with saquinavir and lopinavir (as the coformulated brand Kaletra), and these combinations are frequently used in experienced patients who have failed either initial nelﬁnavir therapy or initial therapy with combination reverse transcriptase inhibitors. The pharmacological and drug interactions of HIV protease inhibitors are many and complex. The increasing diversity of regimen combinations has often produced unanticipated two- and three-way

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interactions that make taking and prescribing drugs as much art as medicine. These positive and negative aspects of the protease inhibitor class are too complex to discuss in detail here, and the reader is referred to two chapters by Flexner in a recent book dealing solely with HIV protease inhibitors (Ref. [3], Chaps. 8 and 9). The interesting recent observations that members of this class may be actively eliminated from cells in a differential fashion [60] and that there may be a pharmacogenomic component to this as well as to metabolic degradation by the cytochrome P450 system have led to concern that the same pharmacokinetic parameters may not serve as surrogates for clinical efﬁcacy for every protease inhibitor. Nevertheless, the adoption of drug level monitoring for this class of drugs is generally seen as important, particularly in Europe, and a complete understanding of the relationship between drug prescribed and drug in the body at the intracellular site of action (incorporating the dual variables of adherence and pharmacokinetics) will be invaluable to prescribing clinicians. 6

RESISTANCE TO HIV-1 PROTEASE INHIBITORS

As described in the foregoing text, resistance to drug candidates in development was very much in the minds of the research teams during the preclinical stages of the projects. It was known that resistance to the ﬁrst nucleoside analogs was being seen in patients with incomplete or transient suppression. The highly dynamic nature of HIV replication and the T-cell regeneration combined with the error-prone RNA replication machinery involving reverse transcriptase presented the worst combination for preventing the virus from adapting to an environment of suboptimal inhibitor concentration by selection of resistant mutants. Following the deﬁnition of a clade B consensus sequence, post-therapy variations from that sequence are determined by sequencing an ampliﬁed population of the protease gene and part of the reverse transcriptase gene with samples collected on one or more failing drugs. This has become a standard way of describing a resistant genotype. Only recently have pre-therapy genotypes been obtained and the full extent of natural polymorphism and, alarmingly, transmission of resistant variants appreciated. As a result, there has been a general classiﬁcation of resistant variants selected by the protease inhibitor class as being either primary (generally active site–associated, common, and conferring phenotypic resistance in vitro) or secondary (generally removed from the active site, compensating for mutant enzyme catalytic inefﬁciency and viral ‘‘ﬁtness,’’ and perhaps polymorphic or less common in some deﬁnitions). The classiﬁcations are often ambiguous

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and are constantly evolving as in vitro data and published data from failing patients are merged. The latest composite chart for the approved inhibitors is shown in Figure 8. Clearly, resistance to the class is widespread. New drug candidates in the class invariably have, as a selection criterion for development, the ability to suppress the broadest possible range of clinical isolates. The advent of commercial genotyping and phenotyping services has generated interesting data on the consequences of mutations on intraclass cross-resistance. Not surprisingly, those protease inhibitors with unique and distinct mutation patterns such as nelﬁnavir (D30N) or amprenavir (150V) generate viruses that can be suppressed in vitro by all other members of the class [54]. Suppression of mutants carrying the

FIGURE 8 Compilations of mutations in the protease gene associated with resistance to protease inhibitors. Primary mutations are those most frequently seen that are associated with phenotypic resistance. Secondary mutations are those that either are less frequently observed and may be associated with phenotypic resistance or are observed as compensatory mutations that are not by themselves associated with phenotypic resistance. The data come from a combination of in vitro and clinical studies. A comprehensive recent summary can be found in Ref. [71].

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variants at positions 82, 84, 48, and 90, frequently in various combinations, do not exhibit the same degree of suppression in vitro by other inhibitors. The relationship between the elevation of phenotypic resistance and clinical outcome is not yet fully established for each member of the class, but two published examples appear to hold up in cohort data. The potency of lopinavir/ritonavir (combined with ﬁrst use of the non-nucleoside reverse transcriptase inhibitor efavirenz) in reducing plasma viremia in patients with prior exposure (and resistance) to a median of three prior protease inhibitors is high. The response to patients with genotypic failure to nelﬁnavir (associated primarily with the genotypic change D30N, and to a minor extent L90M [61]) has been demonstrated, both groups responding durably to a dual protease inhibitor regimen as the second choice in the class [53,55]. An intriguing new observation associated with resistance to the protease inhibitors has been the reduction in replicative capacity observed in an in vitro test of these mutants [62]. This has been associated most strongly with the D30N mutation characteristic of nelﬁnavir resistance. It has been tempting to speculate that this phenomenon may contribute to the discordant responses seen in about 20% of cohorts where an increase in viral load is associated not with CD4 cell decline but with stability or an increase in CD4 cell count [63]. It is too early to attribute this phenomenon clearly to any single variable or collection of variables, but the observation has prompted a reevaluation of the deﬁnitions of therapeutic success, particularly in patients with few remaining treatment options. 7

ADVERSE EVENTS RELATED TO PROTEASE INHIBITOR TREATMENT

The toxicities associated with protease inhibitors are a concern not only from the standpoints of patient safety and quality of life but also because they are the primary reason that drug therapy is discontinued. They have been reviewed recently in detail (Ref. [3], Chap. 11). The tolerability of long-term drug therapy signiﬁcantly affects a patient’s adherence to combination therapy, which is in turn critically important to reducing the likelihood of resistance to one or more components of the regimen and perhaps drug options not yet taken by virtue of cross-resistance. The unambiguous attribution of toxicities and adverse events to individual drugs is complicated by several factors. The frequent occurrence of adverse events owing to underlying HIV disease, the stage of the disease, and the use of multiple medications makes it difﬁcult to make cause-and-effect connections even in controlled clinical

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trials. In addition to each individual drug’s toxicities, the complex pharmacokinetic interactions often seen with protease inhibitors, particularly those such as ritonavir, which interacts with multiple members of the cytochrome P450 family, can often lead to new adverse events associated with the particular combination. Placebo-controlled studies are certainly useful in deﬁning differences in incidence rates but rarely extend beyond 48 weeks of careful data collection. Furthermore, many baseline parameters such as fasting lipid levels were not obtained until concerns were raised after several years of experience with the drug. It is almost impossible now to go back and ethically conduct trials that would isolate each drug in a placebo-controlled way. Accordingly, researchers have turned to large cohorts of patients treated over time where stratiﬁcation by baseline and treatment parameters still allows sufﬁcient numbers of adverse events to be used for assessment of relative risk associated with drug, disease state, gender, ethnicity, etc. Overall, the short-term safety proﬁle of protease inhibitors is good. Although there is considerable overlap in the toxicities of individual protease inhibitors, particularly with general intestinal events such as nausea and diarrhea, each drug has a distinct proﬁle that is important to consider when considering a particular patient. An important factor, excluded from consideration in early clinical trials, is the presence of coinfections such as hepatitis C, which can contribute to enhanced liver toxicity. According to information listed in drug package inserts, which classify toxicities according to incidence and severity on a ﬁve-point scale, nausea, vomiting, and diarrhea are the most common class effects, with amprenavir being the least likely to show an effect. Mild to moderate diarrhea is the only principal toxicity associated with nelﬁnavir, but as in the case of other drugs it can often be treated by dietary counseling or antimotility drugs. Indinavir use resulted in kidney stones being seen in a number of patients, with the recommendation that ﬂuid intake should be increased. Elevations in triglyceride and cholesterol levels were notably absent from early package inserts owing to an underappreciation of the issue. All members of the class produce such elevations, but the numbers are of concern principally for lopinavir/ritonavir and other ritonavir-boosted regimens. The longterm consequences of these elevations in these patients have not been established. One study reported a ﬁvefold increase in the risk of myocardial infarction in association with protease inhibitors [64], whereas others found no signiﬁcant association [65]. Even new agents in development are not without signiﬁcant toxicities, although increasingly an effort is made to screen out candidates on the basis of surrogate

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in vitro or animal studies. However, this is far from a precise science. Atazanavir, for example, which has the desirable property of unboosted daily dosing, shows a high proportion of patients with elevated bilirubin [66]. Changes in fat distribution are among the most striking abnormalities associated with but not proved to be caused by protease inhibitor therapy. Patients display either peripheral fat wasting (lipodystrophy or lipoatrophy), central fat accumulation, or a mixed syndrome. The fat wasting component is seen in the face, arms, and legs with associated subcutaneous fat loss, and fat accumulation is seen in the abdomen, breasts, or a dorsocervical and submandibular fat pad enlargement, the so-called buffalo hump. Measures of body composition including dual energy X-ray absorptiometry (DEXA) and whole body MRI have provided detailed information on the changes observed. Again, baseline measures of these parameters, not obtained in the earlier studies, are now routinely collected in clinical and postmarketing studies for many antiretroviral drugs in all classes. In general, for the vast majority of patients, the beneﬁts this class of drugs bring to the reduction in mortality and morbidity associated with HIV infection still outweigh the risks, although in recent years there has been a shift to using them more frequently in later therapeutic regimens. 8

HIV-1 PROTEASE INHIBITORS—THE FUTURE

Three drugs classiﬁed as protease inhibitors are generating considerable interest currently in the clinical research community. Two are at a stage where approval in the United States is widely anticipated within this year. The criteria for selecting new molecules in this class for full development have shifted considerably since the time the decisions described above for the approved agents were made. A better appreciation for the demands of long-term combination therapy have made all researchers look for dosing to be no more frequent than twice daily, for pill count to be as low as possible, and for modest or no inhibition of enzymes involved in oxidative metabolism that can have a profound impact on drug interactions necessitating dose adjustments or counter-indications of certain combinations. In addition, two strategies for suppression of resistant viremia have emerged that include either the design of agents with a small change in IC50 to a panel of common resistant variants (intrinsic lack of phenotypic cross-resistance) or the design of agents that can achieve sufﬁciently high plasma or cellular levels to overcome the elevated IC50. This latter can involve ritonavir boosting or intrinsically better absorption and elimination proﬁles.

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The ﬁrst new agent is a prodrug of amprenavir, fosamprenavir, which does not have any distinguishing antiviral properties because it is hydrolyzed in the body to amprenavir. It does, however, allow for a vastly superior formulation compared to the parent drug (two pills twice daily versus eight pills twice daily) and will certainly be expected to bring the beneﬁts of amprenavir, already seen in the salvage setting, at a time when it may be most needed. The new agent atazanavir being developed by Bristol Myers Squibb was discovered at Ciba Geigy and belongs to a class of peptide mimetic compounds incorporating an aza linkage in the backbone. Its distinguishing feature is the pharmacokinetic proﬁle, which allows for once-daily dosing without the necessity for low-dose ritonavir coadministration or coformulation. Clinical studies have indicated potency in triple drug regimens comparable with that of nelﬁnavir and also with that of the non-nucleoside reverse transcriptase inhibitor efavirenz. The principal causes for concern are elevated bilirubin in a substantial proportion of patients (which necessitated dose reduction in some trials) and the possible development of resistance due to partial adherence to a oncedaily regimen where drug levels in a population at trough may vary considerably more than with more frequent dosing. The selection of a resistant variant at position 50 (a different amino acid substitution to amprenavir) is also unique and may result in a variant with increased susceptibility to other protease inhibitors. Atazanavir can also ‘‘salvage,’’ in vitro, viruses that are resistant to other protease inhibitors, notably nelﬁnavir (86% of isolates tested [67]). Tipranavir was discovered at Pharmacia and is being developed by Boehringer Ingelheim. Its structure is unrelated to those of any of the other molecules in the class, and it has demonstrated impressive potency in vitro against a large panel of resistant clinical isolates. It is currently being explored in combination with the pharmacokinetic enhancer ritonavir at various doses [68] and is expected to move into registrational trials shortly. 9

CONCLUDING REMARKS

In the eight years since inhibitors of HIV protease ﬁrst became available to people living with HIV/AIDS, we have witnessed the increasingly sharp contrast between the impact that AIDS is having in the developed, as opposed to the developing, world. In developed countries, where access to health care, including the use of protease inhibitor–based combination therapy, is more straightforward, mortality and morbidity associated with HIV infection have declined for most [69]. In developing

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countries, where there are medical, social, educational, and ﬁnancial barriers to health care, infection rates remain high, and the future prosperity of entire countries seems in jeopardy. The recent UNAIDS report on the global epidemic provides a comprehensive summary [70]. The science behind the discovery of the inhibitors of HIV protease provides a textbook example of how structural biology in conjunction with medicinal chemistry can accelerate drug discovery. The availability of protease inhibitors drove the advent of combination therapy with three drugs that is the current standard of care. As cornerstone drugs of the ﬁrst HAART regimens, their promise as key contributors to longterm viral suppression and immune function restoration was and remains high. Protease inhibitor use adapted well to the realities of long-term combination therapy as hopes of a ‘‘cure’’ receded. All marketed protease inhibitors are prescribed now in simpler regimens than those originally approved and reduce some of the complications for patients. As with any long-term therapy, and as the threat of serious morbidity and death declined in the late 1990s in the developed world, the appearance of new toxicities and adverse events not anticipated in the accelerated approval process has become a focus. The sequential use of agents within this class has also emerged as a clinical reality despite early skepticism. Drugs such as nelﬁnavir, amprenavir, and lopinavir/ ritonavir select for or suppress resistant variants with clinically relevant consequences. Research and development involving new protease inhibitors continue at an intense pace. There are several agents at all stages of the process from early discovery to registration. Existing agents are being investigated in new ways, in particular as agents whose continuous selective pressure on the viral population, without suppression of viremia, may provide medium-term impairment of viral replication and maintenance of immune function. These new research initiatives with this class of agents will continue to contribute to the body of scientiﬁc research that is needed to contain the spread of HIV/AIDS and turn it into a truly manageable disease to live with.

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16 Emerging Therapies for HIV Infection Julie M. Strizki Schering–Plough Research Institute, Kenilworth, New Jersey, U.S.A.

1

INTRODUCTION

The introduction of HIV protease inhibitors in the mid-1990s made a tremendous impact on the success of antiretroviral therapy for many patients. The powerful combination of protease and reverse transcriptase inhibitors (known as highly active antiretroviral therapy, or HAART) can effectively suppress viral replication, improve CD4 counts, and dramatically reduce morbidity and mortality related to HIV infection [1]. Unfortunately, however, the success of this new combination therapy comes with a cost. Patients on HAART must deal with complicated dosing schedules, drug toxicities, and the emergence of viral resistance, all of which have contributed to problems with patient compliance and treatment failure [2]. It is estimated that approximately 30–40% of HIVinfected individuals on HAART harbor viral species that are resistant to one or more antiviral agents. Furthermore, despite long-term suppression of viremia with the new drug cocktails, viral eradication has not yet been achieved. Once therapy is interrupted, the latent pool of virus reactivates and viral titers quickly rise [3]. In fact, it is predicted that despite the success achieved with the current therapies, HIV persistence 555

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will be lifelong [4,5]. Therefore, it is imperative that new, more potent and less toxic drugs be developed not only to help those patients who are failing therapy but also to improve the quality of life for all infected individuals. In this chapter we discuss new antiviral targets and emerging therapeutic agents and treatment strategies under development (Table 1). These may provide additional ammunition to combat the existing problems of resistance and toxicity associated with the currently approved drugs.

2 2.1

NEW VIRAL TARGETS gp120

Currently, only two of the 14 virally encoded proteins have been successfully targeted for HIV therapy. These are the reverse transcriptase (RT) and protease enzymes. Another attractive target for antiviral intervention is the viral envelope glycoprotein gp120. This protein protrudes from the surface of the virion via a noncovalent association with the membrane-bound gp41 protein and is responsible for attachment of the virion to target cells. During infection, gp120 binds to the CD4 receptor on T cells or macrophages. This binding event induces a conformational change in gp120 that allows it to then interact with a secondary coreceptor molecule and initiate viral fusion and entry. Shortly after the interaction between gp120 and the CD4 receptor was identiﬁed, researchers exploited this cellular protein as a potential therapeutic agent. One group showed that a monomeric soluble form of CD4 (sCD4) could bind to HIV and act as a neutralizing agent to prevent infection in vitro [6]. In clinical trials, however, this agent was ineffective because the short half-life of the molecule (20 min) made it difﬁcult to sustain the levels required to achieve an antiviral effect. More recently, researchers at Progenics (Tarrytown, NY) made an improved, soluble, tetrameric form of CD4 with better in vitro and in vivo efﬁcacy and stability. This molecule was constructed by replacing the variable regions of IgG2 heavy and light chains with the gp120-binding domain of CD4 (Fig. 1) [7]. This chimeric CD4-lgG2 molecule, known as PRO-542, has a greatly improved in vivo half-life and better potency compared with monomeric sCD4 [8]. One advantage of this type of molecule is that it targets a highly conserved and essential element in the gp120 molecule and thus has broad neutralizing activity. However, because PRO-542 is a protein it will require intravenous or subcutaneous administration in

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order to achieve its antiviral effect. Despite this hurdle, clinical trials are under way, and early results are promising [8].

TABLE 1

New Viral and Cellular Targets for HIV Therapy

Target Viral targets gp120

gp41 Integrase

Capsid

Cellular targets Cyclophylin A

Agents

Mechanism of action

Development status

PRO-542, AR177, BRI2932, FP-21399, cyanovirin N, CCR5 peptides, BMS 806 T-20 (Fuzeon), T1249, 5-helix S-1360, diketo acid compounds, intracellular antibodies DIBA-I, CI-1012; azodicarbonamide (ADA)

Destabilization of gp120; inhibition of viral attachment/ fusion Inhibition of membrane fusion Inhibition of strand transfer reaction (small-molecule inhibitors) Displacement of zinc ions—disruption of nucleocapsid structure and function

PRO-542 is in phase I/II trials, others are preclinical.

SDS NIM811

Inhibition of virion Preclinical. uncoating and reverse transcription Inhibition of viral entry SCH-C is in phase I/II via CCR5 trials. AOPRANTES, TAK-779 not being advanced. Others are preclinical. Inhibition of entry/ AMD-3100 abandoned fusion for lack of efﬁcacy and toxicity. Early clinical trials for Inhibition of viral use as a attachment, may microbicide. also bind to gp120 Inhibition of protein Approved for cancer synthesis and viral and viral hepatitis; RNA degradation ongoing clinical trails for HIV. Inhibition of Preclinical. topoisomerase I activity and possibly reverse transcription Boost TH-1 immune Phase I/II trials as responses; increase intermittent therapy CD4 counts or as vaccine adjuvant.

CCR5

AOP-RANTES, SCH-C, TAK-779, PRO 140 peptide T, ribozymes

CXCR4

ALX40, T-22, AMD3100, ribozymes PRO 2000

CD4

Innate antiviral pathways (PKR and 20 50 OAS)

Interferon-a (PEGintron A, Pegasus)

Topoisomerase I

Topotecan

Immune modulators

IL-2, IL-12

Approved (T-20). S-1360 is in phase I/II trials.

Preclinical.

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FIGURE 1 Neutralization of HIV-1 by PRO 542 (CD4-IgG2). (Left) PRO 542 is a recombinant fusion molecule generated by replacing the variable fragment (Fv) regions of an IgG2, k antibody with the two outermost extracellular domains of the CD4 receptor, in essence a CD4–antibody hybrid. (Right) The CD4 portion of the molecule (shaded region) can bind to the gp120 protein on the virion surface and neutralize viral infectivity. Unlike monomeric CD4 proteins, this tetrameric CD4–IgG hybrid has potent neutralizing activity against a broad spectrum of primary HIV isolates and has much improved stability in vivo compared with earlier CD4-based proteins [8,111]. (Figure provided by William Olson.)

In addition to sCD4-based therapeutic agents, other peptides and compounds are being explored as potential gp120 inhibitors. These include sulfonated CCR5 peptides [9], a small oligonucleotide AR177 (Zintevir) [10], polyanionic compounds such as dextran sulfate, BRI2923 (phenyldicarboxylic acid) [11,12], and FP-21399 a bis-disulfonaphthalene [13]. In addition, cyanovirin N, a protein isolated from cyanobacterium, has been shown to speciﬁcally interact with the sugar moieties on gp120 [14–16]. All of these agents can bind to gp120 and act, at least in part, by disrupting the interaction between gp120 and CD4 and/or the coreceptor. Although these agents have respectable in vitro activity against HIV infection, none of them are orally available, and problems with delivery, pharmacokinetics, and production may limit their utility as front line therapies. An alternative application being explored for some of these large polymers and polypeptide gp120 inhibitors is prophylaxis in the form of

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topical microbicides. One such compound is PRO 2000, a high molecular weight naphthalene sulfonate polymer. Reportedly, this molecule can bind to CD4 and inhibit its interaction with gp120 [17]. This compound can effectively inhibit infection of a broad range of HIV strains and also has activity against HSV and other sexually transmitted pathogens [18]. In an SHIV monkey infection model, PRO 2000 protected about 50% of the animals from intravaginal infection [19]. The large molecular size of PRO 2000 minimizes its systemic absorption, thus reducing possible side effects or toxicity, and its stability can provide long-lasting activity, both of which are desirable properties for a topical microbicide. Because a safe and effective HIV vaccine has yet to be developed, the use of topical microbicides may provide some means of protection against HIV transmission in high-risk populations. However, large clinical trials will be required to demonstrate their efﬁcacy in real-life applications. Regardless of the fact that gp120 is an attractive target for antiviral intervention, orally available, small-molecule inhibitors of this protein have not yet been developed. In 1998, Kwong and coworkers published a breakthrough study deﬁning the three-dimensional crystallographic structure of the gp120 core [20]. One important discovery derived from this structure was the presence of a structurally conserved pocket within the molecule that is responsible for CD4 binding. This pocket appears to be an ideal target for small-molecule inhibition, although success to date has been limited. Several obstacles exist that may hinder the discovery of small-molecule inhibitors of gp120 function. These include the genetic heterogeneity of the envelope sequences, the cryptic location of the CD4 binding site, and the abundant glycosylation on the outer surfaces of the molecule, all of which may impede access of potential inhibitors to binding sites in the molecule. Despite these obstacles, one group from Bristol-Myers Squibb recently reported the discovery of a small molecule, BMS 806, that speciﬁcally binds to gp120 and can inhibit HIV infection [21]. Although this compound showed good antiviral activity against closely related clade B isolates of HIV, it was much less effective against viruses from other genetic clades. In addition, resistance to the compound required only a few amino acid substitutions and was generated relatively quickly in vitro. Despite these limitations, discovery of this molecule provides an important proof-of-concept by demonstrating that small-molecule inhibitors of gp120 can effectively inhibit HIV. It is hoped that this discovery will lead to the development of secondgeneration compounds with greater potency and broad-spectrum activity.

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2.2

Strizki

gp41

The primary function of the gp41 envelope protein is to mediate fusion of the viral and cellular membranes (Fig. 2). When gp 120 binds to CD4 and the chemokine receptor, a series of conformational changes take place in the envelope that lead to exposure of the fusion peptide domain of gp41,

FIGURE 2 Inhibition of HIV-1 fusion by gp41 peptides. (A) After HIV binds to CD4 and its coreceptor, a conformational change occurs in gp120 that allows the gp41 fusion peptide to be exposed and inserted into the target cell membrane. In the absence of an inhibitor, a second conformational change occurs that repositions the helical region 1 (HR1) and the helical region 2 (HR2) domains together, forming a six-helix bundle. Formation of this bundle draws the viral and cellular membranes together to initiate fusion. Fusion inhibitors such as T-20, T1249, and 5-helix are small peptides that act as decoys for either the HR1 or HR2 domains of gp41. They work by binding to the exposed complementary helical domain and prevent formation of the HR1–HR2 helical bundles, essentially freezing the fusion complex in an inactive state. (B) A schematic of the gp41 structure depicting the fusion domain on the N terminus and the two helical regions involved in bundle formation. T-20 mimics a region of the second helical domain (Figure prepared by Robert Doms.)

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which is then inserted into the target cell membrane. Then gp41 undergoes another conformational change that brings the N-terminal and C-terminal helical regions of the gp41 trimer together to form a sixhelix bundle structure (Fig. 2). Formation of this bundle helps to juxtapose the viral and cellular membranes and facilitates creation of a fusion pore through which the viral nucleocapsid can enter the cell. Researchers are still searching for small-molecule therapeutic agents capable of inhibiting the fusion activity of gp41, but this protein has been an elusive target for such inhibitors. However, a new class of peptide-based gp41 inhibitors has been discovered and has shown promise in early clinical trials. These agents, known as fusion inhibitors, work by targeting the intermediate conformational step in the fusion process. The initial discovery and subsequent development of this new class began as part of a search for peptide immunogens for vaccine trials. Researchers at Duke University were searching for immunogenic peptides that could effectively generate antibodies to conserved regions of the gp41 envelope protein. One such peptide, T-20, was found to have antiviral activity in vitro [22,23]. This 36 amino acid peptide was designed to mimic a conserved segment of the C-terminal helical region (HR2) of gp41 (Fig. 2). Following coreceptor binding, a conformational change occurs that triggers the insertion of the fusion peptide into the target cell membrane. During this step, the HR1 and HR2 regions of gp41 are transiently exposed prior to formation of the hairpin structure. Because T-20 mimics the HR2 domain, it is able to bind to the HR1 region during this intermediate step, thus preventing formation of the HR1 and HR2 helical bundles. The interaction of the peptide with gp41 forms a stable complex that freezes the gp41 in this intermediate conformation and prevents fusion from occurring. In clinical trials, T-20 has shown impressive antiviral activity and is safe and well tolerated [22]. Because this drug has a novel mechanism of action, it is effective against drugresistant isolates and can inhibit both CCR5 and CXCR4 tropic viruses [24,25] (see below). Continuing research on HIV fusion inhibitors has led to secondgeneration peptide inhibitors with greater potency and in vivo stability. One of these is T-1249, a chimeric peptide that partially overlaps with the T-20 sequence but was optimized to target a broader range of viral isolates [26]. This peptide is severalfold more potent than T-20 and, importantly, has activity against T-20-resistant isolates. Another peptide inhibitor being developed is 5-Helix [27]. This peptide works via a mechanism similar to that of T-20, except that the 5-Helix peptide mimics the HR1 region of gp41 and targets the HR2 domain. Although early clinical results for the T-20 and T-1249 peptides are promising,

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there are several disadvantages to their use. As with all peptide-based therapeutics, T-20 is not orally available and must be administered by injection. In addition, the high manufacturing costs and large-scale production problems associated with peptides must be overcome if these inhibitors are to be used in a large patient population. Despite these obstacles, this novel class of inhibitors presents a new option for patients failing therapy because of drug-resistant isolates or HAARTassociated toxicity. 2.3

Integrase Inhibitors

Another viral protein that is actively being pursued as a target for antiviral intervention is the viral integrase enzyme. The integrase protein is encoded by the HIV pol gene region and is produced by proteolytic cleavage of the pol polyprotein, which also gives rise to the protease and RT enzymes. This protein directs the insertion of the viral genome into the host cell DNA and is essential for viral replication. Proviral integration is a multistep process (Fig. 3). First, the integrase enzyme forms a stable preintegration complex with the LTR sequences on the proviral DNA molecule. Next, the endonucleolytic activity of the enzyme cleaves the terminal dinucleotide from each 30 end of the molecule. Subsequently, a strand transfer reaction occurs that covalently links the ends of the proviral DNA with the target (cellular) DNA. Finally, gaps in the DNA strand are repaired by cellular enzymes. Drug screening assays targeting integrase have identiﬁed numerous inhibitors of in vitro integrase activity; however, many of these compounds fail to have antiviral activity in cell culture [28]. Recently, researchers at Merck described a new class of integrase inhibitors that block HIV replication in cell culture [29]. These inhibitors all share a diketo acid structural feature. In vitro characterization of these compounds revealed that they work by inhibiting the ﬁnal stage of proviral integration, the strand transfer reaction [30]. Interestingly, inhibitors that interfere with formation of the preintegration complex show activity in biochemical assays but fail to inhibit viral infection in cells [31,32]. However, the diketo compounds, which inhibit the strand transfer reaction, can prevent proviral integration and the spread of infection in cell culture [29,30]. Although the undesirable pharmacological properties of some diketo acid compounds have impeded their clinical development, progress is slowly being made. Researchers at Shionogi have identiﬁed a small orally bioavailable integrase inhibitor, S1360, that has good antiviral potency against clinical strains of HIV in vitro [33]. This compound is synergistic with other antiretroviral drugs

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FIGURE 3 Inhibition of viral integration. Three distinct steps are involved in integration of the viral cDNA. Step 1 involves assembly of the preintegration complex that comprises the viral cDNA, the integrase protein, and possibly other viral and cellular proteins. In step 2, the integrase protein cleaves the two 30 -terminal nucleotides from the viral LTRs. Finally, in step 3, integrase catalyzes the cleavage of the cellular DNA and transfer of the proviral DNA strands. Diketo compounds that act by inhibiting the strand transfer reaction have potent antiviral activity in cell cultures.

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and has acceptable pharmacokinetic and safety proﬁles. However, clinical trials will be required to establish a proof-of-principle that integrase inhibitors can be efﬁcacious in vivo as part of an antiviral treatment regimen.

2.4

Capsid

The HIV particle is composed of several structural proteins encoded by the gag region. These include the nucleocapsid proteins p6 and p9, which associate with the viral RNA p24, which forms the inner conical protein shell surrounding the RNA, and p17, a myristylated protein that associates with the inner plasma membrane and facilitates envelope incorporation and virion budding. These proteins are all vital to virion integrity and infectivity. Disruption of capsid protein structure or function can have effects on multiple steps of the virus life cycle, including uncoating, reverse transcription, and virion assembly. Following attachment, fusion, and entry, uncoating of the viral RNA must occur for infection to proceed. This step is believed to involve the cellular protein cyclophylin A, which is known to bind to the capsid and is incorporated into virions [34,35]. Mutations in the capsid that prevent binding of cyclophylin A can reduce virion infectivity. Furthermore, inhibitors of cyclophylin A, such as cyclosporin A (Neoral), can inhibit viral replication in vitro, presumably by preventing uncoating of the viral RNA [36,37]. Because cyclosporin A is a potent immunosuppressive agent, it is not an ideal candidate for HIV therapy; however, other non immunosuppressive analogs of cyclosporin A such as SDS NIM811 that disrupt the capsid–cyclophylin A interaction could potentially be developed as antiviral agents [38]. Following the uncoating step, the nucleocapsid proteins that remain associated with the viral RNA are believed to be required for both reverse transcription and proviral integration. Inhibitors that disrupt the capsid protein structure, RNA binding activity, or ability to multimerize could also act as potential antiviral agents. The HIV nucleocapsid protein contains a highly conserved structural zinc ﬁnger element that is required for proper conformation and function. This zinc ﬁnger motif is an attractive target for new antiviral drugs because disruption of this structure can inhibit reverse transcription and render virions noninfectious [39–42]. To date, several zinc ﬁnger inhibitors have been described. These include azodicarbonamide (ADA), dithiobenzamide (DIBA-1), and CI-1012 [40,43–45]. Although all have shown antiviral activity in vitro, none have been thoroughly tested in the clinic.

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CELLULAR RECEPTOR TARGETS

The role of the CD4 molecule in HIV attachment to target cells has long been recognized [46]. However, expression of CD4 cannot by itself permit viral fusion and entry to occur [47]. It has long been speculated that other cellular factors were involved in the fusion and entry process. In 1996 several groups made a series of landmark discoveries that identiﬁed two cellular cofactors required for HIV entry into cells. The discovery of these coreceptor molecules opened the door on a whole new chapter in HIV research. The ﬁrst receptor identiﬁed by Berger and colleagues was a seventransmembrane G-protein-coupled receptor termed ‘‘fusin’’ and later renamed CXCR4 [48]. Expression of this receptor in CD4-positive cells made the cells permissive to infection by T-tropic viral isolates but not macrophage-tropic viruses. Shortly after this ﬁnding, several other groups reported the discovery of a second coreceptor, CCR5, that was responsible for infection of macrophage-tropic viruses [49–51]. These receptors belong to a seven-transmembrane G-protein-coupled receptor superfamily and are known as chemokine receptors [52,53]. The innate function of these receptors is to receive and transmit chemotactic signals that mediate recruitment of cells to an inﬂammatory stimulus. During acute infection and in the early stages of disease, viral isolates that use the CCR5 receptor (R5-tropic) predominate. As HIV disease progresses, the virus can evolve to use the CXCR4 receptor for infection, but this occurs in only about 40–50% of patients [54]. After the identiﬁcation of the CXCR4 and CCR5 receptors, scientiﬁc evidence quickly accumulated to suggest that these cellular coreceptors could be targeted for antiviral therapy. The ﬁrst evidence supporting this idea came from a study by Cocchi et al. [55], who demonstrated that the natural chemokine ligands of CCR5—MIP-1a, MIP-1b, and RANTES— could block HIV infection in vitro. In addition, Liu et al. [56] reported the discovery of a naturally occurring 32 amino acid deletion in the CCR5 gene of several individuals known to have been exposed to HIV by sexual partners but who themselves remained uninfected. Cells from these individuals were found to be devoid of CCR5 surface expression and were unable to be infected with macrophage-tropic or R5-tropic viruses, although they were susceptible to infection with viruses that used the CXCR4 coreceptor (X4-tropic). Because these individuals who lacked CCR5 had no apparent immunological deﬁciencies, CCR5 became an attractive target for antiviral intervention. Furthermore, a study by Dean et al. [57] showed that HIV-infected individuals heterozygous for the delta-32 allele showed signiﬁcantly slower disease progression than

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individuals carrying both wild-type CCR5 genes, further validating CCR5 as a target for therapy (Fig. 4). 3.1

CCR5 Inhibitors

The ﬁrst CCR5 inhibitors described were synthetic derivatives of the bchemokines. One of these is aminooxypentane-RANTES, a derivative of the CCR5 ligand RANTES that is modiﬁed on the N terminus by addition of an aminooxypentane group [58]. This compound binds to CCR5 with greater afﬁnity than the natural RANTES ligand and is more potent against viral infection in vitro, possibly because it strongly induces downregulation of the receptor from the cell surface [59,60].

FIGURE 4 Coreceptor inhibitors. A novel approach to HIV therapy is to target cellular proteins required for viral infection. The cellular chemokine receptors CCR5 and CXCR4 are important for viral attachment and fusion. Small molecules and antibodies directed against these coreceptors inhibit viral replication by preventing entry of the virus into the target cell. Theoretically, drugs targeting cellular rather than viral targets will be less likely to induce the emergence of resistance; however, resistance can be achieved in vitro. (From Ref. [112].)

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This molecule and a related molecule, NNY-RANTES, have been proven to have antiviral activity in animal infection models; however, their agonist activity and the need for parenteral administration are undesirable properties for therapeutic use. In addition to modiﬁed chemokines and peptide derivatives, small-molecule antagonists of CCR5 with potent antiviral activity have been developed by several groups. The ﬁrst of these to be described was TAK 779, a receptor antagonist that potently inhibits CCR5-tropic strains of HIV in primary cells but has no effect on CXCR4 isolates [61]. Although this compound showed promise in vitro, it has poor bioavailability and therefore has been abandoned as a therapeutic agent. A second small molecule that is now being advanced in the clinic is SCH-C [62]. This compound has similar properties to TAK 779 in that it is a speciﬁc antagonist of CCR5, and it has potent in vitro antiviral activity. SCH-C has good oral bioavailability and shows promising antiviral activity in animals [62]. This compound has entered early-stage clinical trials for safety and efﬁcacy. In addition to the small-molecule antagonists, speciﬁc monoclonal antibodies against CCR5 are being developed as potential therapeutic agents. One example is PRO 140, a humanized monoclonal antibody that recognizes the second extracellular loop of CCR5 [63,64]. This antibody can inhibit replication of a broad range of viral isolates, is synergistic with other antiretroviral drugs, and has potent activity in a mouse model of infection. Although peptidic inhibitors such as monoclonal antibodies have the advantage of speciﬁcity and low toxicity, production and delivery issues are a signiﬁcant challenge to overcome. One potential problem faced by all the inhibitors targeting CCR5 is the fact that HIV can evolve to use other coreceptors for infection, particularly CXCR4. Because emergence of CXCR4-using viruses during the course of infection is coincident with an increase in viral load and a decrease in CD4 cell counts, there is concern that antagonism of CCR5 receptors will drive viral evolution toward an X4-tropic phenotype. In vitro evidence suggests that HIV can develop resistance to agents that block viral infection by CCR5 but that this process does not involve coreceptor switching. However, one study performed in a mouse model found that treatment of SCID/Hu mice with an NNY-RANTES derivative resulted in a viral phenotypic switch from R5- to X4-tropic in at least a few of the mice [65]. In addition to potential changes in viral phenotype, the consequences of long-term CCR5 antagonism in a patient population are unknown. Closely monitored clinical trials will be required to determine the safety, efﬁcacy, and in vivo resistance patterns of these CCR5 antagonists.

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CXCR4 Inhibitors

The second major coreceptor used by HIV is CXCR4. This receptor is widely expressed and can be found not only on T cells and macrophages but also on a wide variety of cells, including neurons, microglia, astrocytes, endothelial cells, and colonic epithelium [48,66–69]. The only known natural ligand for the CXCR4 receptor is stromal cell derived factor 1 (SDF-1) [70,71]. This CXC chemokine is produced by a large number of cell types. Because of the ubiquitous expression of this receptor on nontarget cells, the potential for side effects induced by antagonism is greater than with CCR5, which has a more restricted expression pattern. CXCR4 and SDF-1 knock-out studies in mice have demonstrated that this receptor and its ligand are essential for embryonic development because mice lacking CXCR4 have multiple developmental defects and die before birth [72]. Despite these hurdles, several CXCR4 antagonists are being explored for HIV therapy. Because the extracellular surface of the CXCR4 molecule has a strong negative charge, many of the inhibitors that bind to this receptor are positively charged molecules. The ﬁrst CXCR4 inhibitor to be used in clinical trials was a charged nine amino acid peptide ALX40-4C, although at the time of the study its mechanism of action was unknown. Subsequent mechanistic studies showed that ALX40-4C speciﬁcally binds to the second extracellular loop of CXCR4 and can block infection of viruses that use this receptor for entry [73]. Unfortunately, this compound had no measurable efﬁcacy in vivo and was therefore abandoned in the clinic. Importantly, in this study ALX40-4C was well tolerated in the majority of patients, suggesting that the use of CXCR4 antagonists may be safe in adult populations. Another series of peptides being developed to target CXCR4 are T-22 and its derivatives T-134 and T140 [74]. Again these have potent in vitro activity, but issues of delivery and production may impede clinical development. The ﬁrst small-molecule antagonist of CXCR4 to be described was AMD 3100 [75,76]. Despite its limited oral bioavailability, an injectable formulation of this compound entered clinical trials. Disappointingly, like ALX40-4C, AMD 3100 failed to demonstrate signiﬁcant reductions in overall viral load in infected patients and was found to have undesirable toxicity [77]. It is unclear why AMD 3100 failed to show efﬁcacy in vivo, but factors such as pharmacokinetics, potency, or the presence of R5tropic viruses may have all contributed to its lack of clinical effect. Still, researchers have not abandoned CXCR4 as a target and are developing orally bioavailable derivatives of the AMD 3100 molecule, such as AMD

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7049, as well as other inhibitors that can target multiple coreceptors (AMD 3451 and distamycin analogs) [26,78,79]. 4

GENE THERAPY APPROACHES

One of the more innovative but challenging approaches to antiretroviral therapy has been the use of gene therapy technology to deliver antiviral proteins or peptides directly into susceptible or infected cells. Although most of these agents are still in the conceptual phase, some of the approaches being explored are quite interesting. An advantage that is offered by gene therapy vectors is the ability to deliver peptides, proteins, or oligonucleotides efﬁciently into target cells and, depending upon the vector used, into speciﬁc cell types. For example, retroviral particles can be produced that lack the HIV glycoproteins on the surface but instead express the cellular receptors CD4 and CCR5 on the surface of the virion [80]. These particles can bind to and fuse with HIV-infected cells via the viral envelope proteins expressed on the surface. Theoretically, they could be engineered to deliver toxic or antiviral genes speciﬁcally to infected cells or alternatively to progenitor cells that could then pass along these protective genes to daughter cells. Below, we describe some of the approaches being explored by investigators to develop novel antiviral agents. 4.1

Antisense Oligonucleotides

Antisense oligonucleotides are short complementary oligonucleotide molecules designed to target speciﬁc RNA elements and inhibit their function. When antisense oligonucleotides are transfected or transduced into cells, they seek out their complementary sequence and anneal tightly. Binding of the antisense sequence with its target prevents translation of the full-length mRNA, resulting in inhibition of protein synthesis or RNA function. This technology has been used to target such viral RNA elements as the HIV 5’ signal sequence, the rev responsive element (RRE), tat, and envelope genes [81–83]. In vitro, these antisense oligonucleotides can be quite effective inhibitors of viral replication. Other antisense vectors have been used to inhibit the cellular factors required for viral replication. One group has found that antisense oligodeoxyribonucleotides targeting the tRNA primer required for initiation of reverse transcription not only caused inhibition of DNA synthesis but also enhanced degradation of the viral RNA bound to the tRNA primer [84]. Another group has used antisense oligonucleotides to

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target the CXCR4 receptor mRNA and observed downregulation of the receptor and resistance of the cells to viral infection [85]. Clearly, this latter approach is less practical, because antiviral protection is dependent upon efﬁcient delivery and stable expression of the vector in a large number of cells, which may be difﬁcult to achieve. 4.2

Intracellular Antibodies

Antibodies are one of the immune system’s ﬁrst lines of defense against viral infection. Most neutralizing antibodies recognize structural proteins on the surface of the virion, prevent viral attachment, and facilitate viral clearance. In the past, the use of antibodies directed against nonstructural intracellular proteins (such as integrase) would not have been considered effective, because large antibody molecules do not readily penetrate cells. To overcome this obstacle, one group took an innovative approach by cloning the antigen-speciﬁc fragment from the variable region of an antiintegrase monoclonal antibody and expressing it in cells as a single chain. This single-chain variable fragment (SFv) binds to the integrase enzyme and inhibits HIV infection in cells [86,87]. Expressing this antibody fragment as a fusion protein with the viral vpr protein further expanded this approach. The vpr-SFv fusion protein is then incorporated into newly formed virions that are rendered noninfectious by the antibody fragment [88]. Like the antisense antivirals, the single-chain variable fragment approach has been attempted for chemokine receptors as well. Transduction of cells with an SV40 vector expressing a CXCR4-speciﬁc fragment resulted in downregulation of the receptor in cells and subsequent inhibition of infection by X4 isolates [89]. Theoretically, this technology can be expanded to include other intracellular viral targets such as rev, tat, and reverse transcriptase. 4.3

Ribozymes

Ribozymes are small catalytic RNA molecules that have the ability to cleave other RNA molecules. In general, ribozymes recognize their target RNA by annealing to a speciﬁc sequence on the target, and then the catalytic domain of the ribozyme cleaves the adjacent RNA sequence. In effect, destruction of the target mRNA leads to a decrease in the protein product of that message. Preclinical development of ribozyme vectors that target both viral and cellular coreceptor proteins include ribozymes against the viral LTR sequence and the CCR5 and CXCR4 receptors [81,90]. Functional knock-out of the CCR5 receptor by ribozymes has been pursued by several groups with some success [91–94]. When

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transfected into cells, the anti-CCR5 ribozyme effectively decreased cell surface expression of the CCR5 receptor, which in turn resulted in a decrease in viral infection. A similar approach was used for the CXCR4 receptor, except that it employed an RNA-cleaving DNA enzyme [95]. Although conceptually the delivery of antiviral agents via gene therapy vectors is an attractive alternative to more toxic and less speciﬁc small-molecule drugs, many obstacles stand in the way of their development. These include problems with efﬁcient and targeted delivery, low or unstable expression levels, and safety considerations. As the delivery vectors improve, this area of research may yield some very valuable and efﬁcacious new antiviral agents. 5

IMMUNE MODULATORS

One of the primary consequences of HIV infection is immune suppression that leads to opportunistic infections. Although HAART therapy has proven effective in curtailing viral replication, increasing CD4 counts, and restoring immune reactivity to recall antigens and neoantigens, virus-speciﬁc immunity is often not restored [96,97]. Researchers are investigating the use of immunomodulators or natural antiviral agents as alternatives or supplements to HAART to bolster the innate immune response against viral infection. Two of the most studied thus far are the cytokines IL-2 and IL-12. Both of these cytokines are known to stimulate TH-1 cell responses that are important for viral suppression. 5.1

IL-2

Cytokine IL-2 stimulates CD4 T-helper cells and induces activation and proliferation of these cells. In clinical trials, IL-2 can effectively increase CD4 cell counts and restore recall antigen responsiveness, although it has no apparent effect on viral load [98]. The clinical beneﬁts of IL-2 must be weighed against the signiﬁcant side effects associated with high-dose IL2 therapy. IL-2 can induce moderate to severe ﬂu-like symptoms in patients. This may limit its use for long-term continuous therapy. However, new protocols using intermittent or low-dose IL-2 therapy appear to be successful in maintaining CD4 cell counts and minimizing the undesirable side effects [99]. 5.2

IL-12

Cytokine IL-12 is known to be important for inducing and maintaining the cellular immune responses necessary for viral suppression. In HIV

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infection, IL-12 production can be suppressed, and exogenous addition of IL-12 to cells from infected patients can restore antiviral responses in vitro [100]. The ability of IL-12 to induce CD8 cell activity has led to its use as an adjuvant for vaccination trials [100]. Coadministration of the immunogen with IL-12 can enhance the generation of viral-speciﬁc CTL, which is important for combating infection. Although early studies show promise in the use of IL-12 as a vaccine adjuvant, much additional work will be needed to optimize the timing and dosing of IL-12 and vaccine in order to maximize the immune response and minimize undesirable side effects [101]. 5.3

Interferon

Interferons (IFNs) are a family of small proteins produced by virusinfected cells and are so named for their ability to interfere with viral replication. Interferon secreted by an infected cell is a signal to neighboring cells that danger is near and they need to be prepared. When IFN-a binds to its receptor, it induces the upregulation of a series of proteins that activate multiple cellular pathways leading to the inhibition of protein synthesis and RNA degradation, thus impeding viral replication. In vitro, IFN-a has potent antiviral activity against HIV-1 replication [102]. However, early clinical trials using recombinant IFN-a were disappointing, with only modest antiviral activity observed in patients [103,104]. More recently, new formulations of IFN that use a high molecular weight polymer, polyethylene glycol (PEG), as a carrier for multiple IFN molecules revived interest in IFN for HIV therapy [105]. This new formulation signiﬁcantly increases the half-life of IFN in serum, making it possible to replace the multiple injections previously required with a single weekly injection. Initial clinical data show that this new PEG formulation has a clear antiviral effect in heavily drug experienced HIV-1 patients. Although this result is encouraging, additional clinical data will be required to further document the safety, efﬁcacy, and durability of long-term IFN dosing in patients 6

STRUCTURED TREATMENT INTERRUPTION

Many of the new agents described in this chapter are still in the preclinical or early clinical development stage and it may take many years for them to be approved for HIV therapy. For the numerous patients who are failing the current drug regimens, options for effective therapy are limited. Researchers are now looking for ways in which to

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strengthen the antiviral immune response and rebuild the immune system to lessen the dependence on antiviral drugs. During the course of most viral infections, the immune system plays a major role in controlling or eradicating viral replication. Sometimes this is accomplished with the help of antiviral drugs or immunomodulators, but ultimately the immune system ﬁnishes the job once the scales are tipped in favor of the host. However, for HIV infection this has not been the case. Despite the arsenal of antiviral drugs used to keep the virus at bay, the immune system is unable to completely eradicate it. The reason for this is not completely clear; however, the fact that HIV can hide latently in cellular reservoirs and that its targets are the very cells responsible for ﬁghting infection likely contribute to the problem of viral eradication. Because the prospect of viral eradication is not promising, at least with the current drug regimens, researchers have been looking for new ways to boost the immune system to control viral replication in the absence of drugs. Studies have shown that patients on prolonged HAART treatment tend to have decreased viral-speciﬁc CTL responses. This is presumably because of the lack of viral antigen present in the circulation. However, when these patients stopped therapy and viral replication rebounded, a concomitant increase was seen in the virus-speciﬁc CD8 CTL response [106,107]. This observation prompted researchers to test the hypothesis that periodic discontinuation of therapy could act as an autovaccination and boost the immune system, leading to immunological control of viremia in the absence of drugs. This approach of systematically stopping and restarting therapy has been termed structured treatment interruption, or STI (Fig. 5). To date, numerous different retrospective and prospective studies have been conducted in different patient populations using a variety of dosing schedules [108]. Overall, the results of these studies suggest that treatment interruption may be beneﬁcial for some patients, but a great deal of inconsistency between the studies makes the data difﬁcult to interpret. One common ﬁnding among the studies, however, is that viral rebound resulting from interruption of therapy can usually be controlled (to preinterruption levels) by reinitiation of therapy. The beneﬁt of treatment interruption may differ in different patient populations. For example, in patients who are on effective HAART and have good CD4 counts, interruption of therapy can serve to boost CD4 Tcell help and CTL responses and lead to decreased plasma RNA levels following each interruption cycle [109]. However, this happens in only a subset of the patients, while other patients gain no apparent beneﬁt from interruption or may even rebound to higher RNA levels following multiple STI cycles.

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FIGURE 5 Structured treatment interruption (STI) is a new treatment strategy designed to increase innate HIV-speciﬁc immunity and at the same time reduce accumulated drug-related toxicities. During treatment interruption (shaded areas), the virus quickly rebounds to pretreatment levels, typically within a matter of weeks. During this rebound period, it has been noted that viral-speciﬁc CTL immunity (---) increases. Theoretically, repeated cycles of therapy interruption will serve to boost the immune system by an autologous vaccination mechanism, eventually leading to a reduction in viral rebound (solid curve) and better immunological control in the absence of drugs. In practice, this strategy has shown some promise; however, most patients still require the use of antiretroviral drugs to control their infections.

Patients who are heavily treatment experienced and are failing therapy may beneﬁt in other ways from treatment interruption. It has been observed that in patients who have multidrug-resistant virus, cessation of therapy can lead to a rapid switch from resistant to wild-type virus in the plasma [110]. This wild-type virus generally replicates more efﬁciently than the resistant virus. Often emergence of wild-type virus correlates with more rapid CD4 cell decline and higher viral loads but is sensitive to drugs. Reinitiation of therapy is usually effective at least initially, but because most patients continue to harbor resistant virus, viral control is often lost over time. Therefore, the utility of STI in this population still needs to be assessed more carefully in future trials. 7

SUMMARY

Over the last decade tremendous progress has been made in the ﬁght against HIV infection. For those patients fortunate enough to have access

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to treatment, the rates of morbidity and mortality have reduced drastically. In spite of this progress, problems of drug toxicity and viral resistance have brought new challenges to the future of HIV research. In this chapter we have highlighted some of the new targets and therapies emerging for the treatment of HIV infection. As evidenced by the large number of potential new viral and cellular targets and treatment strategies in development, the future for HIV therapy looks bright. Undoubtedly, many of the experimental compounds described here will require years of further development to reach approval or may not be approved at all, but they provide steppingstones that pave the way for more effective and less toxic drugs to treat HIV infection.

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17 Human Cytomegalovirus: Diagnosis, Pathophysiology, and Treatment Hermann Einsele and Gerhard Jahn Universita¨tsklinikum Tu¨bingen, Tu¨bingen, Germany

1

INTRODUCTION

Infection with cytomegalovirus (CMV) is common and varies geographically, with 40–100% seroprevalence in healthy adults, whereas associated disease is a relatively exceptional event. In newborns it causes a congenital syndrome that may at times be fatal. In normal immunocompetent subjects, it is a recognized cause of CMV mononucleosis. But among various groups of the immunosuppressed such as the immature neonate, the recipients of organ transplants, and patients with acquired immunodeﬁciency syndrome (AIDS), CMV causes the most signiﬁcant disease syndromes. Infection and diseases caused by CMV are becoming susceptible to some means of prevention and therapy. 2

THE VIRUS

Cytomegalovirus (CMV) strain AD169 was initially isolated in ﬁbroblast cell cultures in 1956. It was sequenced and published in its entirety in 587

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1990 and consists of four equimolar isomers produced by inversion of either the unique long (UL) or unique short (US) regions [1]. These UL and US regions are bounded by terminal repeat (TR) and inverted repeat (IR) regions [2]. Predicted open reading frames are numbered sequentially within each region and annotated using the abbreviations p for protein, gp for glycoprotein, and pp for phosphoprotein, followed by any common nonsystematic name; e.g., gp UL55 (gB) is the 55th open reading frame in the UL region and encodes a glycoprotein known as glycoprotein B. The original report describes 208 open reading frames in strain AD169, which, after allowance for known splicing events, were predicted to produce 203 proteins, 189 of which were unique and the rest were present in two copies in the repeat regions [1]. In 1996 it was reported that clinical strains of CMV contained a series of genes not found in the AD169 or Towne strains [3]. The details are complex (reviewed in Ref. [4]), but essentially 22 additional genes are present in wild-type strains. None of these genes has a homolog in other herpesviruses, and most are predicted to be type 1 glycoproteins. At least one has been shown to have interesting biological activity; gp UL146 is an a-chemokine, the ﬁrst such molecule described in a viral genome [5]. Thus, overall, CMV clinical strains have 225 genes. 3

LABORATORY DIAGNOSIS

The diagnosis of CMV infection in healthy or immunosuppressed children and adults requires laboratory conﬁrmation and cannot be made on clinical grounds alone. The ﬁrst useful laboratory test was reported by Fetterman [6], who found large inclusion-bearing cells in urine sediment. This test is diagnostic when positive but may be falsely negative in patients with viruria. It is less valuable after the newborn period. The laboratory diagnosis of CMV infection depends on either demonstration of the virus or viral components or demonstration of a serological rise. The sensitivity and precision of both approaches is generally good. More recent technical developments include the use of monoclonal antibodies to immediate early antigens to detect infected cells in tissue specimens [7]. Such antigens may be directly demonstrated in tissues by enzyme-linked or ﬂuorescence methods or after short incubation of specimens of tissues or body ﬂuids in ‘‘shell vials’’ [7,8]. Detection of antigenemia in circulating neutrophils has been shown to be a sensitive and clinically useful method of detecting viremia. Monoclonal antibodies against a CMV matrix protein, pp65, are used [9]. Polymerase chain reaction (PCR) methods, usually using primers in the part of the genome

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coding for immediate-early antigen, have also been used [10] as well as labeled, cloned, viral nucleic acid probes to detect virus DNA or RNA in specimens by nucleic acid hybridization [11,12]. Hybridization methods have been applied to clinical specimens such as buffy coats or tissue specimens. 3.1

Cultivation of Cytomegalovirus

Human CMV cannot be readily grown in any experimental animal [13]. It is, however, easily cultured on human ﬁbroblast cultures. The one drawback is the time needed to develop cytopathology. Ordinarily this may take 1–4 weeks, but the time required has also been shortened to 48 hr by the use of cytospin and monoclonal antibody to detect cytopathology before it becomes visible (‘‘shell vial’’ method) [8]. The typical cytopathology of CMV is usually sufﬁciently characteristic for identiﬁcation in the laboratory without further serological conﬁrmation. Cytomegalovirus can be readily isolated from urine, mouth swabs, buffy coat, cervical swabs, or other tissues obtained from biopsies or at postmortem examination. Virus is demonstrable in patients even if circulating neutralizing antibodies are present. Ordinarily, CMV is not detectable in normal adults. The exceptions are females who may carry the virus in the cervix and males, particularly homosexual males, who may carry CMV in semen. The presence of CMV in the throat, urine, and blood by culture is usually abnormal. It is, however, important to note additional circumstances under which CMV may be chronically carried. Anyone who is recovering from an acute infection may carry CMV in the urine, throat, and occasionally blood for months. Patients with congenital or perinatal infection and immunosuppressed patients with transplants or human immunodeﬁciency virus (HIV) infection and AIDS are often chronic virus carriers for years. Isolation of virus from such patients requires careful clinical interpretation. 3.2

Serology of Cytomegalovirus

Like herpes simplex virus, strains of CMV have enough genomic variation that they may be ‘‘ﬁngerprinted’’ after digestion with restriction endonucleases. However, there is enough DNA homology among different strains to suggest that only one serotype of human CMV exists for diagnostic purposes. The problem is more complex in understanding immunity to CMV, because serological distinctions on the basis of neutralization or other biologically meaningful measures may be present. The complement ﬁxation (CF) antigen of AD169 is more

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broadly reactive than are antigens of other strains and has been extensively used in epidemiological and clinical studies. The CF test itself is no longer widely used because of its relative lack of sensitivity. Compared with the CF test, the indirect ﬂuorescent antibody (IFA) and anticomplement immunoﬂuorescent (ACIF) tests are more sensitive [14]. In primary infections, titers by IFA and ACIF tests are higher and become positive earlier than the corresponding CF titers, at times by as much as 1–2 months. The ACIF test is superior because there is less nonspeciﬁc ﬂuorescence [14]. Many other tests are on the market, largely because ﬂuorescence microscopy is cumbersome. These are the indirect hemagglutination test, radioimmunoassay, latex agglutination, automated immunoﬂuorescence, and various versions of the enzyme-linked immunosorbent assay (ELISA). For many years now, the ELISA for IgG and IgM detection has been the most widely used assay for CMV antibody detection. A serological diagnosis of infection requires either elevation of an antibody titer or conversion from antibody-negative to antibody-positive. Samples of serum before and after the ongoing infection are essential, especially in pregnant women, to discriminate primary versus secondary infection. When they are not available, the presence of IgM antibody against CMV is a useful but not completely reliable indication of an acute infection.

3.3 3.3.1

Molecular Methods Nucleic Acid Ampliﬁcation

The widespread application of polymerase chain reaction (PCR) technology for the detection of viral nucleic acid (DNA or RNA) is the most important innovation in laboratory diagnosis and clinical management of post-transplant CMV infection. ‘‘Home-brew’’ or in-house CMV PCR testing with conventional thermocycling instruments that are programmed for nucleic acid ampliﬁcation for 40–45 cycles followed by gel electrophoresis of the PCR-ampliﬁed products and probe hybridization techniques is used by many centers. The assay is laborintensive, and the turnaround time is not signiﬁcantly reduced compared to that for shell vial culture or antigenemia assay. The home-brew PCR assay is highly sensitive but has a low predictive value, and the results are reported qualitatively as either positive or negative [15,16] or quantitatively [17]. There is a wide variability of the techniques used by different centers (e.g., differences in primers, concentrations of reagents, and cycling parameters), so the results are not widely reproducible among different laboratories and centers [15–18].

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The recent availability of automated PCR instruments that involve rapid thermocycling formats with the capacity for real-time quantiﬁcation of viral genomes has signiﬁcantly changed the practice of CMV diagnosis. The Cobas Amplicor CMV Monitor assay (Roche Diagnostics, Pleasanton, Ca) was designed to detect CMV DNA by amplifying a segment of the CMV DNA polymerase gene UL54 within a turnaround time of approximately 3–4 hr [19]. The assay is standardized, offering the advantage of reproducibility of results among many centers. With the LightCycler system (Roche Molecular Biochemicals, Indianapolis, In), results can be obtained rapidly (within 30–40 min); the system offers automation of PCR by precise air-controlled temperature cycling and provides continuous monitoring of amplicon development by a ﬂuorometer (ﬂuorescence resonance energy transfer) in a closed system [20]. When clinical samples collected during 19 episodes of CMV infection were analyzed using the LightCycler system, the results obtained closely correlated with those obtained with the COBAS Amplicor CMV Monitor assay [20]. In addition, the LightCycler assay can analyze more samples per run (32 compared to 24) more quickly (240 min compared to 460 min) than the COBAS assay [20]; this may offer an advantage to high-volume laboratories that process a large number of clinical samples. The quantiﬁcation of CMV viral load has assisted clinicians in accurately diagnosing and managing post-transplant CMV disease [17,21]. Not all patients with CMV reactivation develop clinical disease. It is the degree and the rate of CMV replication that predict impending CMV disease and thus the need for speciﬁc treatment [17,22]. In addition, the level (virus load) of CMV DNA in blood specimens at the end of therapy predicts relapsing CMV infection [21]. Preliminary data using the COBAS Amplicor CMV Monitor assay indicates that the threshold of viral load (around 1000–5000 copies mL plasma in solid-organ transplant recipients and around 400 copies/mL plasma in hematopoietic stem cell transplant recipients) predicts the likelihood of CMV disease, if untreated [23]. However, there is a need for additional studies to validate the optimal threshold. The wide variability in the laboratory techniques of different centers for detection of CMV DNA [in such things as samples (source, processing, and target volume), reagents (primers, probes, and master mix concentrations), patient characteristics (solidorgan or hematopoietic stem cell transplant recipients), the use and level of immunosuppressive agents, the use of antiviral prophylaxis regimens, and the presence of viral coinfections (human herpesvirus [HHV]-6 and -7)] [19,24–30] could account for this lack of standard and deﬁned threshold value. However, it is generally accepted that higher CMV

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DNA copy levels [17,21] or an increasing trend in viral loads [28] predict clinical progression to disease or clinical relapse. The NucliSens assay (Organon Teknika Diagnostics, Boxtel, The Netherlands), an isothermal nucleic acid ampliﬁcation reaction assay, detects the presence of CMV late-mRNA pp67. The presence of mRNA pp67 indicates active viral replication, and its detection is a marker for active CMV infection [31–33]. Nevertheless, early experience suggests that this assay is less sensitive than DNA ampliﬁcation assays and antigenemia assays for detection of CMV infection. The lower sensitivity of the assay may result in failure to detect or predict CMV disease in all patients; in one study, the assay did not detect the mRNA transcripts in 4 of 11 patients who developed CMV disease [32]. 3.3.2

Nucleic Acid Hydridization

The Digene Hybrid Capture CMV DNA assay (Digene Corporation, Silver Spring, MD) is a rapid, qualitative, signal-ampliﬁed solution hybridization assay that uses RNA probes that bind to the DNA target followed by antibodies directed to RNA–DNA hybrids, as well as a sensitive chemoluminescence detection system. In a multicenter study that included solid-organ and hematopoietic stem cell transplant recipients [34], this assay was found to be more sensitive than cell culture assays and to have a sensitivity and speciﬁcity similar to those of the antigenemia assay. The Digene Hybrid Capture assay has fewer technical variables than the antigenemia test. However, because of the assay’s qualitative nature, the clinical signiﬁcance of a positive result is unclear, because the assay may be detecting subclinical CMV replication that may not evolve into clinical disease (i.e., high sensitivity and low speciﬁcity). The utility of the assay in predicting the occurrence of CMV disease and in monitoring the response to antiviral therapy is currently being investigated [34]. 4

ANTIVIRAL SUSCEPTIBILITY TESTING

The three antiviral drugs that are currently licensed for use in the prevention and treatment of CMV are ganciclovir (and its valine ester, valganciclovir), foscarnet, and cidofovir. Other experimental drugs may also be useful for treatment of CMV infections resistant to standard agents [35,161,162]. Ganciclovir is a prodrug that is monophosphorylated into ganciclovir 50 -monophoshate in CMV-infected cells by virusencoded phosphotransferase UL97, then di- and triphosphorylated by the host cellular kinases. The active triphosphorylated form of ganciclovir inhibits DNA polymerase by competing with deoxyguano-

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sine triphosphate, thereby terminating viral replication. Foscarnet is a pyrophosphate analog that directly inhibits viral DNA polymerase by interfering with the release of pyrophosphate during a substrate incorporation event. Cidofovir is a nucleotide analog that requires phosphorylation by the cellular enzymes to achieve its active form. Unlike ganciclovir, neither foscarnet nor cidofovir requires the virusencoded kinase for activation. Antiviral drug resistance in CMV is an emerging problem in transplant recipients [27,36]. Studies of transplant recipients and of patients infected with human immunodeﬁciency virus suggest that mutations in the viral DNA polymerase UL54 (target of antiviral drugs) and in the viral kinase UL97 (phosphotransferase; phosphorylates ganciclovir into active form) confer antiviral drug resistance in CMV. The currently available methods for antiviral susceptibility testing rely on the suppression of virus growth in the presence of serial concentrations of antiviral drugs (phenotypic assays) or the determination of speciﬁc mutations that have been shown to confer resistance (genotypic assays). 4.1

Phenotypic Assays

Phenotypic methods assess the concentration of the drug that inhibits virus replication. Typically, the level of virus is plotted against the concentration of the drug that causes 50% inhibition of the virus in cell cultures. The phenotypic methods that have been employed include plaque reduction assay (inhibition of viral replication), enzyme-linked immunosorbent assays (inhibition of protein synthesis), ﬂow cytometric ﬂuorescence-activated cell sorting, and DNA hybridization assays (inhibition of viral DNA synthesis) [37,38]. The plaque reduction assay is the standard method of antiviral susceptibility testing for CMV. The test is burdensome and lacks standardization; it requires the recovery of the virus in cell cultures followed by several passages to attain the necessary viral titers for the performance of the assay. Benchmark analysis of several strains of CMV has shown wide variability in results. Typically, these assays require at least 4 weeks to obtain results [39]. 4.2

Genotypic Assays

Signiﬁcant problems with the use of phenotypic assays and the recognition that speciﬁc mutations in the UL54 and UL97 genes of CMV are associated with antiviral drug resistance have led to the

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development of molecular methods for the detection of the CMV mutants. UL97 encodes for the phosphotransferase that is essential for the initial phosphorylation of ganciclovir into its active form. Accordingly, the functional consequence of these mutations is the inadequate intracellular phosphorylation of ganciclovir into the ganciclovir monophosphate form [36,40,41], thus leading to ganciclovir resistance. Since cidofovir and foscarnet do not require viral kinase, UL97 mutations do not confer resistance to these agents. Analysis of the UL97 sequences of phenotypically ganciclovir-resistant clinical strains demonstrated mutations and deletions in this region; several of the more common point mutations occur at codons 460 (V460, I460), 520 (Q520), and 591–607 (e.g., V594 and S595) [40,42,43]. These mutations can be detected by direct sequencing of the PCR products [44]. It has also been recently demonstrated that molecular ampliﬁcation of the portion of the UL97 gene encoding the C-terminal half of the enzyme followed by two sequencing reactions provided rapid identiﬁcation of all presently known sites of ganciclovir resistance in this gene [45]. Mutations in the UL54 CMV DNA polymerase gene, the main target of all three antiviral drugs, could result in the resistance to any or all of the three drugs. For example, mutations at codons 375–540 confer ganciclovir and cidofovir cross-resistance, mutations at codons 756–809 confer ganciclovir and foscarnet cross-resistance, and mutations at codons 981–987 appear to confer simultaneous mutations to the three drugs. Most UL54 mutations are accompanied by UL97 mutations; strains with double UL97 and UL54 mutations are believed to be highly resistant to ganciclovir with possible cross-resistance to cidofovir and/or foscarnet [46]. The rapid thermocycling used by the automated PCR methods may help us attain the goal of real-time antiviral susceptibility testing. For example, the melting curve analysis of the LightCycler assay can detect nucleotide differences in the ampliﬁed products and the probe (thus, mutations or deletions) by a shift in the peak melting curve [47]. Although the LightCycler assay may not detect the exact point mutation, it can serve as a screening method before gene sequencing can be performed. If these applications are conﬁrmed, it will be possible to analyze the susceptibility of CMV to various drugs within a few hours of specimen collection, compared to the current turnaround time of several weeks with the use of phenotypic methods.

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Clinical Applications of Antiviral Susceptibility Testing

Antiviral drug resistance in CMV is now an emerging concern in transplantation. Thus, antiviral susceptibility testing will be a common occurrence in the ﬁeld of transplantation during the upcoming years. Currently, the conventional cell culture–based (phenotypic) methods are not rapid or standardized enough to be of immediate clinical utility in CMV disease management. Thus, surrogate markers such as the failure of the viral load to decrease during antiviral treatment are used as indirect measures of antiviral resistance. Genotypic assays are easily performed with modern molecular methods such as PCR and sequencing of ampliﬁed products. Nevertheless, these methods need optimization and clinical validation. For example, there are mutations and deletions in UL97 and UL54 genes that do not correlate with phenotypic resistance. Extensive research should determine whether a speciﬁc mutation in the genome confers low-level or high level resistance or does not confer any resistance at all to the antiviral drugs.

5

PATHOGENESIS

The pathogenesis of CMV infection and disease is complex, and substantial data have been accumulated with regard to viral factors and the interactions between CMV and the immune system. In seropositive recipients of a stem cell graft, reactivation of latent endogenous virus appears to be the most likely cause of infection. The role of exogenous virus in seropositive patients is not well deﬁned, but coinfection with different strains can occur [48]. Whether infection with multiple CMV strains results in a higher overall incidence or greater severity of CMV disease or higher viral load is not known. In seronegative individuals, CMV is acquired from blood products or from transplanted organs [49]. Both host and viral factors appear to be responsible for the progression from infection to disease. Host factors that have been associated with the CMV disease after allogeneic stem cell transplantation are: patient’s age, posttransplant immunosuppression, development of acute graft-versus-host disease (GvHD) and its treatment, and total body irradiation [50,51]. Dissemination of CMV in the blood is an important factor in the pathogenesis of disease. Earlier studies established that culture-proven viremia is highly predictive for CMV disease, but simultaneous detection

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of viremia and development of disease occur in more than 50% of patients [52–54]. Using more sensitive techniques for detection of CMV in blood such as the antigenemia assay of PCR for CMV DNA, CMV can be detected in almost all patients with CMV disease. Because of the high sensitivity of these assays, CMV is also detectable in a substantial number of patients with asymptomatic infection who never progress to disease, resulting in a low positive predictive value [55,56]. However, patients with disease often have higher viral loads than those who remain asymptomatic [57,58]. Patients with higher viral load are also more likely to develop CMV disease both early and late (after day 100) after transplantation [59,60]. There is some evidence that strain differences play a role in the pathogenesis of CMV disease. Recent studies suggest that strain differences based on the CMV gB envelope protein [61] may contribute to the tropism and pathogenicity of CMV in both marrow transplant and AIDS patients. In one study of allogeneic marrow transplant recipients, CMV gB type I was associated with favorable outcome of CMV infection [62]. Studies in the murine model and in human transplant recipients suggest that HLA-restricted CMV-speciﬁc cytotoxic T-cell (CTL) responses play an important role in the elimination of active infection and protective immunity [63,64]. The virus–T cell interaction is mediated through several mechanisms, including the virus effects on HLA expression, cytokine production, and adherence molecules. In allogeneic marrow transplant recipients who develop CMV disease, both CMV-speciﬁc CD8þ CTL and CD4þ Th responses are usually undetectable [65,66]. There is also a direct correlation between absence of CMV-speciﬁc CTL and Th responses and high CMV viral load in allogeneic marrow transplant recipients [67]. In autologous peripheral blood and marrow transplant recipients, recovery of CD8þ CTL responses is associated with subsequent protection from CMV infection [68]. Another effect of the interaction of CMV with the immune system is the association between CMV and acute and chronic GvHD. It has been clearly documented that patients with acute GvD are at an increased risk for CMV disease [42,52]. Studies in animal models have suggested that CMV increases the risk for acute GvHD [69–71]. However, this has not been conclusively shown in humans [72]. Seroepidemiological studies have shown an association between CMV infection and chronic GvHD [73–75]. Recently So¨derberg et al. [76,77] found that patients with chronic GvHD who had experienced CMV disease in a very high frequency had

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cytotoxic antibodies to CD13, an antigen that is expressed on many target cells for chronic GvHD. 6 6.1

DISEASES CAUSED BY CMV Allograft Recipients: Direct Effects

Cytomegalovirus has long been recognized to cause a series of end-organ diseases collectively called ‘‘direct effects’’ or ‘‘CMV disease.’’ These are deﬁned according to criteria agreed upon at the International CMV Workshop in 1996 and updated regularly thereafter. Essentially, these require the patient to have characteristic symptoms, to have clinical signs in the affected organ, and to have CMV detected in biopsies from the same organ. The deﬁnition is stringent and is useful as an endpoint for early clinical trials of anti-CMV compounds. It is now clear that the direct effects of CMV result from replication of virus leading to high viral loads. The previously documented risk factors of donor seropositivity, recipient seronegativity, and the post-transplant appearance of viremia are all explained by high viral load. 6.2

Allograft Recipients: Indirect Effects

In addition, CMV is statistically associated with several clinical conditions collectively termed indirect effects. These include graft rejection, immunosuppression manifest as secondary fungal or bacterial infections, opportunistic neoplasms, accelerated atherosclerosis after heart transplant, and death. Clearly, each of these conditions is multifactorial, but the results of double-blind, randomized, placebo-controlled trials show that CMV makes an active contribution to their genesis. 6.3

AIDS Patients: Direct Effects

The same principles of CMV viremic dissemination and high viral load indicating high risk of CMV disease apply equally to AIDS patients. Nevertheless, it is remarkable that 85% of viremic spread localizes to the retina, in contrast to only 1% in transplant patients. Other CMV diseases in AIDS patients include enteritis, polyradiculopathy, and encephalopathy. 6.4

AIDS Patients: Indirect Effects

A high CMV viral load is associated statistically with an increased death rate, and this effect is independent of HIV viral load. Multiple mechanisms have been shown whereby CMV (or other herpesviruses)

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could facilitate the pathogenicity of HIV. It is thus interesting that the increased mortality associated with CMV can be reversed through administration of drugs acting against CMV. The full signiﬁcance of these observations remains to be deﬁned in the era of highly active antiretroviral therapy (HAART).

6.5

CMV and Pregnancy

Cytomegalovirus is the most important cause of perinatal, postnatal, and congenitally acquired infection. Congenital infection occurs by transplacental transfer of the virus in about 40% of primary infection and can lead to cytomegalic inclusion disease (CID) in about 10–20%. Primary sources for postnatal infection include breast milk as well as transfusionassociated transmission of the virus. Intrauterine CMV infection is the leading cause of congenital infection worldwide. By the end of the ﬁrst year, about 20% of all children shed the virus in the urine; the actual rate varies (10–40%) depending on sexual contacts, maternal seropositivity, and breastfeeding practice. There is evidence that CMV is transmitted during maternal viremia into the fetus via various cells in the placenta, including trophoblasts, macrophages, ﬁbroblasts, smooth muscle cells, and endothelial cells [78,79]. Transmission of CMV from mother to fetus is assumed to occur during all three trimesters. Severe symptoms following primary infection in pregnancy are greater when the infection occurs in the ﬁrst trimester. In cases of CID with fatal outcome, CMV can be detected in nearly all organs and in a broad spectrum of cell types, including endothelial cells, epithelial cells, smooth muscle cells, mesenchymal cells, hepatocytes, monocytes/macrophages, and granulocytes [80,81]. The lung, the pancreas, the kidneys, and the liver are usually the major target organs with a high number of CMV-infected cells. In contrast to the high transmission rate and the outcome in newborns, the transmission rate during recurrent infection is much lower and clinical symptoms less severe. The intrauterine transmission in the presence of antibodies has been attributed to reactivation as well as reinfection with exogenous virus. Endogenous reactivation seems to be more frequent than reinfection with a new virus, but reinfection is more important in terms of clinical symptoms in the newborn [82,83]. Newborns with cytomegalic inclusion disease may have various symptoms, as listed in Table 1. Congenital CMV infection is one of the most important causes of deafness in childhood worldwide. Sensorineural deafness occurs in up to about 60% of newborns with

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Findings in Symptomatic Congenital CMV Infection

Physical ﬁnding Intrauterine growth retardation Thrombocytopenic purpura (petechiae) Hepatosplenomegaly Jaundice Sensorineural hearing loss Microcephaly Retinitis

Laboratory ﬁnding Thrombocytopenia Elevated transaminase enzymes

symptomatic CMV infection and in up to 15% of children born with asymptomatic infection. Breastfeeding signiﬁcantly inﬂuences the epidemiology of postnatal CMV infection. Recently it was demonstrated that, depending on the seroprevalence, about 40% of seropositive mothers transmit the virus via breast milk to their babies. Nearly all seropositive mothers reactivate CMV during lactation [84,85]. In contrast to term infected infants, preterm infants weighing less than 1500 g (less than 32 weeks) can develop CMV-associated symptoms such as neutropenia, lymphocytosis, thrombocytopenia, and hepatosplenomegaly. About 13% of infected preterm infants had sepsis-like symptoms [84,85]. Breastfeeding as a source of postnatal CMV infection in preterm infants has been underestimated for a long time and may be associated with symptomatic infection. 7 7.1

PROPHYLAXIS Prevention of Primary CMV Infection

Patients who are CMV-seronegative before transplantation should, if possible, receive the transplant from a CMV-negative donor. This is, of course, often not possible because only a limited number of donors are available and the time to ﬁnd a donor is frequently critical. The risk for CMV transmission in CMV-seronegative patients with seronegative donors is mainly through blood products [49]. Today two options exist for reducing this risk of CMV transmission: the use of blood products from CMV-seronegative donors or the use of leukocyte-ﬁltered blood products. These two options were tested in a randomized trial and shown to be comparable [86]. If only a CMV-seropositive donor is

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available, the risk for transmission of CMV by the marrow product to the recipient is approximately 30% [87]. Thus, these patients should be considered at risk for CMV disease, and preventive strategies similar to those used in CMV-seropositive patients (i.e., antigenemia- of PCRguided antiviral therapy) should be used. Two studies have been performed with intravenous immunoglobulin (IVIG) as prophylaxis. Bowden et al. [88] showed a reduction in the rate of CMV infection but no reduction in CMV disease. In a similarly designed study performed by the Nordic BMT group, there was no reduction in CMV infection [89]. 7.2 7.2.1

Antiviral Chemotherapy in Allogeneic Stem Cell Transplant Acyclovir

The ﬁrst study to demonstrate a reduction in the reactivation of CMV in seropositive allogeneic recipients was the nonrandomized prospective study by Meyers et al. [90]. In this study, transplantation patients seropositive for CMV and HSV received acyclovir (500 mg/m2 IV every 8 hr from day 5 until day þ 30); patients seropositive for CMV served only as controls. CMV infection, CMV disease, and transplant survival were signiﬁcantly improved in high-dose acyclovir recipients. In a prospective double-blind study performed by the European Acyclovir for CMV Study Group, patients received either high-dose IV acyclovir as used by Meyers and colleagues followed by oral acyclovir (3200 mg/day) until day 210 after transplant, high-dose acyclovir from day 5 until day þ 30 followed by placebo, or HSV doses of acyclovir (1600 mg/day p.o.) followed by placebo [91]. Acyclovir signiﬁcantly reduced the probability of and delayed the onset of CMV infection. There was no difference in the incidence of CMV pneumonia or of all CMV diseases between the groups. Survival was improved among patients who received IV acyclovir followed by oral acyclovir compared with patients who received low-dose acyclovir followed by placebo. The role of high-dose acyclovir in current prevention strategies consisting of prophylactic or preemptive use of ganciclovir cannot be determined from these studies. A recent retrospective analysis suggests that there is no additional survival beneﬁt of giving high-dose acyclovir when ganciclovir was given either for prophylaxis of for antigenemia [92]. 7.2.2

Ganciclovir

There are two strategies for antiviral prophylaxis with ganciclovir. First, ganciclovir can be given to patients who have evidence of CMV infection

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after marrow transplantation, as indicated by a positive culture from blood, urine, throat, or bronchoalveolar lavage (BAL) ﬂuid; CMV antigenemia; or PCR positivity (i.e., ‘‘early treatment’’ or ‘‘preemptive therapy’’). The second strategy is the prophylactic administration of ganciclovir to all patients at risk based on the pretransplantation serological status regardless of post-transplantation excretion, antigenemia, or PCR positivity (i.e., ‘‘early prophylaxis’’ or ‘‘universal prophylaxis’’). Three randomized double-blind studies have been published that used an early prophylaxis strategy [93–95]. Winston et al. [94] randomized patients before transplantation to either ganciclovir or placebo, whereas Goodrich et al. [93] and Boeckh et al. [95] started prophylaxis at engraftment. Goodrich and colleagues compared prophylaxis with ganciclovir given for CMV excretion from blood, urine, or throat, whereas Boeckh and colleagues compared prophylaxis with preemptive therapy based on quantitative antigenemia. All three studies showed a signiﬁcant reduction of infection and/or disease. Indeed, two studies showed an almost complete elimination of CMV disease while ganciclovir was given [93,94]. However, severe neutropenia was a limiting factor in all three studies. There was no beneﬁt in overall survival in any of the studies. Salzberger et al. [96] analyzed risk factors for neutropenia in 278 patients who received ganciclovir at engraftment and found that early liver failure, renal insufﬁciency after engraftment, and a low marrow cellularity at day 28 are signiﬁcantly associated with the development of neutropenia. Five nonrandomized studies have been reported using a pretransplantation induction course of ganciclovir from day 8 to day 1 followed by lower maintenance doses of ganciclovir (i.e., 5 mg/kg three times a week starting at engraftment) [97–102]. Two of these studies, which were performed in unrelated marrow transplant recipients and recipients of T-cell-depleted marrow, respectively, showed unacceptably high rates of CMV disease [98,100]. Thus, this strategy may be unsafe in high-risk patients. Although early ganciclovir prophylaxis appears to be highly effective in preventing CMV disease, signiﬁcant disadvantages are associated with this strategy. Ganciclovir given at engraftment causes prolonged neutropenia, leading to more invasive bacterial and fungal infections [93,95,96]. In addition, a substantial number of patients not at risk for disease (i.e., 60–65%) will unnecessarily receive a potentially marrow-toxic drug. Both factors may contribute substantially to morbidity and ﬁnancial cost. An interference of ganciclovir with the recovery of CMV-speciﬁc immune responses has been described [103].

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Finally, there appears to be an increased risk of late-onset CMV disease (i.e., after day 100) when ganciclovir is given at engraftment, which may be as high as 17% [95]. 7.2.3

Foscarnet

The role of foscarnet for prevention of CMV disease remains undeﬁned because no controlled studies have been published. Two uncontrolled studies have been reported [103,104]. Reusser et al. [103] reported breakthrough CMV infection in 4 of 12 allograft recipients and none of the seven autograft recipients but no CMV disease, and renal toxicity occurred in 11 of 19 patients who received relatively low doses of foscarnet (40 mg/kg three times daily) from day 7 to day 30 followed by 60 mg kg1 day1 (from day 1 until day 75) Bacigalupo et al. [104] treated 11 allograft recipients (60 mg/kg three times daily from day 10 until day 15 followed by 90 mg/kg three times per week until day 100), ﬁve of whom developed CMV antigenemia and one of whom progressed to CMV disease. 7.3

Intravenous Immunoglobulin

The use of intravenous immunoglobulin (IVIG) or hyperimmune globulin for the prophylaxis of CMV infection and disease after allogeneic transplants remains controversial. Although the prophylactic use of IVIG is associated with virtually no toxicity, the regimens proposed are costly, and controlled studies assessing the effect in preventing CMV disease show conﬂicting results [49,88,89,106–109]. In addition, some studies showed a reduction of bacteremia, non-CMV interstitial pneumonia, and/or acute GvHD, whereas other studies did not report such a difference. An improvement of survival has not been reported in any of the studies. 7.4

Critique of Prophylaxis Strategies

An attractive feature of any effective prophylaxis strategy is that it is simple and does not require virology monitoring. Presently, intravenous ganciclovir prophylaxis appears to be the most effective way of preventing CMV disease. This strategy should be used in CMVseropositive allograft recipients when no virological monitoring by PCR or the antigenemia assay is available. However, ganciclovir prophylaxis has signiﬁcant disadvantages that can be overcome, in part, by PCR- or antigenemia-guided preemptive treatment strategies. Whether empiric reduction of the maintenance dose can reduce neutropenia has not been studied in a controlled fashion. There is,

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however, good evidence that such a strategy is not safe in high-risk patients, including those with severe graft-vs-host disease (GvHD) or after T-cell depletion [98,99]. High-dose acyclovir prophylaxis, although it has only a limited effect on CMV disease, is associated with a survival beneﬁt in low-risk patients (i.e., those with a low risk of acute GvHD) when no ganciclovir prophylaxis or antigenemia- or PCR-guided therapy is given [90,91,111]. However, there does not seem to be a survival advantage when either of these ganciclovir strategies is used [92]. Because of the high cost, the moderate effect on CMV, and the lack of data demonstrating that high-dose acyclovir adds additional beneﬁt when ganciclovir is given as prophylaxis at engraftment or for antigenemia or PCR positivity, most centers do not use acyclovir for prophylaxis of CMV disease. The reason that acyclovir and not ganciclovir prophylaxis has been associated with a survival beneﬁt in randomized trials is difﬁcult to determine because no comparative study has been done. There are several possible explanations: First, the toxic side effects of ganciclovir may lead to more fatal fungal infections that outweigh the reduction in CMV-related mortality [95]. Second, the risk proﬁle of the patient population may be important. Acyclovir studies have been performed in patients with a low incidence of severe acute GvHD [90,91,111]. In these patients the protection from fatal CMV disease provided by acyclovir may be sufﬁcient; possibly because of better CMV-speciﬁc immune reconstitution resulting from the only moderate inhibition of CMV reactivation by acyclovir and reduced use of high-dose corticosteroids. Third, the survival beneﬁt in acyclovir studies may have been due to a signiﬁcant reduction of other herpesviruses, such as HHV-6, during the preengraftment period [76]. Finally, both the design and sample size of the studies may explain results. In two of three ganciclovir studies, patients in the control group also received some ganciclovir, and the sample size in all studies was calculated to detect differences in the incidence of CMV disease rather than survival [93–95]. Study design plays an important role, as is illustrated by the fact that ganciclovir did show a survival beneﬁt when only high-risk patients (i.e., isolation from blood, urine, throat) were studied [112]. In this group of patients, the anti-CMV effect probably outweighed the neutropeniaassociated deaths. With regard to the use of IVIG, there are several factors that may explain the inconsistency of the results, including (1) use of nonspeciﬁc IVIG versus hyperimmune globulin; (2) differing doses, dose schedules, duration of administration after transplant, and preparation of the product; (3) mixed patient populations with differences in risk for CMV disease (i.e., inclusion of autologous and allogeneic patients with different CMV serological status); (4) varying

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supportive rare techniques; and (5) different GvHD prophylaxis regimens. Recently, the available studies were analyzed in three reviews that result in conﬂicting conclusions [113–115]. Furthermore, there is no randomized trial that evaluates IVIG when ganciclovir prophylaxis or preemptive treatment is given. Therefore most transplant centers do not use IVIG for prevention of CMV disease alter allogeneic transplant. 7.5

Autologous Stem Cell Transplant

There was no evidence that high-dose acyclovir had any impact on the incidence of disease in the study by Boeckh and colleagues [115]. There is also no evidence that IVIG is effective for prevention of CMV disease in autologous transplant recipients [117]. 8 8.1

PREEMPTIVE THERAPY Allogeneic Stem Cell Transplant Patients

A disadvantage with prophylaxis given at engraftment is that all patients with varying risks for CMV disease will receive the antiviral drug and will thereby be at risk for drug-related side effects. It would therefore be of value to identify the patients who have the highest risk for development of CMV disease. The preemptive therapy strategy is based on detection of CMV reactivation with a rapid diagnostic technique and then initiation of antiviral therapy. One advantage of this strategy is that only those patients who are judged to be at high risk for CMV disease will get treatment, which would reduce the risk of side effects and, potentially, the cost. The disadvantages with this strategy are the requirement to monitor patients and the possibility that some patients might develop CMV disease before the indicator test becomes positive. Thus, the requirements for use of a preemptive strategy are (1) availability of a reliable and rapid early diagnostic technique, (2) close surveillance (e.g., weekly) with the selected technique, and (3) selection of adequate samples for detection of virus. Several studies have shown that CMV viremia is predictive for the development of CMV disease [52,54,118]. However, the rapid or standard isolation techniques used in the early studies were not sensitive enough to allow initiation of antiviral therapy before CMV disease had developed in a signiﬁcant proportion of the patients. This was shown in a study by Goodrich and colleagues in which, although the risk for CMV disease was signiﬁcantly reduced in the preemptive therapy group, 12% of the patients developed CMV disease before antiviral therapy had been initiated [111]. Schmidt et al.

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[54] used BAL ﬂuid obtained from asymptomatic patients at day 35 after transplantation that was analyzed by rapid isolation and showed that preemptive therapy reduced the risk for progression to CMV pneumonia. However, this technique also failed to identify 13% of the patients who developed CMV pneumonia. More recent studies that included a high number of seropositive unrelated and HLA-mismatched recipients showed disease rates of up to 30% with shell vial–guided early treatment [93,119]. Today more sensitive techniques are available, such as the antigenemia assay and PCR. 8.2

PCR for CMV DNA

The polymerase chain reaction technique for detection of CMV DNA has been evaluated in several studies. PCR can detect CMV infection earlier than rapid isolation [118–124]. A direct comparison between PCR in leukocytes and plasma suggests that PCR in plasma is less sensitive [56,121]. Several laboratories are now evaluating quantitative PCR and also PCR for CMV RNA [122–125]. In a randomized trial the use of PCRbased ganciclovir treatment reduced the incidence of CMV disease and CMV-associated mortality compared with ganciclovir based on rapid isolation [119]. A comparison of PCR-based diagnosis with rapid isolation techniques showed PCR to allow signiﬁcantly earlier initiation of therapy and to reduce the risk for CMV disease [125]. Furthermore, when the samples that were positive by PCR were analyzed in a semiquantitative fashion, a higher amount of CMV DNA was associated with an increased risk for CMV disease [125]. This ﬁnding indicates that the preemptive therapy strategy could be developed even further and that the proportion of patients requiring antiviral therapy might be reduced without increasing the risk for breakthrough CMV disease. However, because even patients with low systemic CMV viral load can develop CMV disease after receiving allogeneic bone marrow transpant, this strategy might fail in patients who progress rapidly from low viral load to overt disease, such as those with severe acute GvHD [95]. 8.3

Antigenemia

The antigenemia assay is based on the detection of the CMV lower matrix protein pp65 in polymorphonuclear leukocytes by immunostaining with monoclonal antibodies [103,127–129]. Results are available within 5 hr, and the technique is more sensitive than rapid isolation. Furthermore, it is quantitative; patients who have a high number of antigenemia-positive leukocytes are at a higher risk for developing CMV disease [59]. Boeckh

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and colleagues, in a randomized, double-blind study, evaluated a preemptive therapy strategy based on CMV viral load measured by quantitative antigenemia. The study indicated that antigenemia-guided early treatment based on a certain level of antigenemia could be as efﬁcacious in preventing CMV disease by 1 year as ganciclovir prophylaxis [95]. However, ganciclovir prophylaxis was more effective in preventing CMV disease during the time it was given (the ﬁrst 100 days after bone marrow transplantation), but the risk for late CMV disease was higher in the ganciclovir prophylaxis group, equalizing the risk for CMV disease at 180 days after transplantation. Survival was similar at any time during the ﬁrst 400 days after receipt of the transplant. The higher incidence of CMV disease before day 100 was likely due to the delay of ganciclovir until levels of antigenemia of 52 positive cells per slide and discontinuation based on a negative test in patients with severe GvHD. Another risk-adapted strategy that combines both immunological and virological risk factors is to give a short course of ganciclovir to patients who receive high-dose steroids for treatment of acute GvHD or for CMV antigenemia [130]. In an uncontrolled trial that included HLAmatched related allogeneic transplant recipients, there was no case of CMV disease within the ﬁrst 180 days after transplantation using such an approach [130]. Whether this approach is useful in unrelated and HLAmismatched patients and whether it is superior to early treatment strategies based on virological marker only has not been studied in a randomized fashion. Either of two antiviral drugs can be used for preemptive therapy: ganciclovir and foscarnet. Ganciclovir has been used in most published studies. Foscarnet has been studied in two small uncontrolled studies that showed rates of effectiveness similar to that of ganciclovir [131–133]. The duration of preemptive therapy has varied greatly in published studies. In the early studies ganciclovir therapy, once started, continued until day 100 after transplantation, which gives a therapy duration of 6–8 weeks in most patients [54,113]. More recently, shorter periods of therapy have been used. For example, Einsele et al. [119] gave ganciclovir for a mean of 3 weeks, and Ljungman and colleagues [89] gave a mean of 2 weeks of therapy. Drawbacks with shorter courses of therapy are that treatment might have to be reinstituted in up to 30% of patients and occasional cases of CMV disease occur shortly after discontinuation of ganciclovir [55,95]. However, the advantages are lower cost, lower risk of side effects, and the possibility that short duration of therapy might allow better reconstitution of the speciﬁc immune response to CMV, thereby reducing the risk of late CMV disease [95].

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Either ganciclovir or foscarnet can be used for preemptive therapy. Reusser et al. [133] found no difference in efﬁcacy between the two drugs while the toxicity proﬁles differed. Ganciclovir caused more neutropenia and foscarnet more electrolyte disturbances, but there was no difference in renal toxicity. The combination of ganciclovir and foscarnet has been used with high efﬁcacy in patients with a high level of antigenemia without additional toxicity [135]. Cidofovir (3–5 mg/kg per week) might be an effective alternative as second-line preemptive therapy, resulting in a 66% success rate when instituted after failure of either ganciclovir or foscarnet [136]. Cidofovir is associated with signiﬁcant renal toxicity, and controlled prospective studies are necessary. A recent study of preemptive therapy showed a difference in effectiveness when used in patients who had undergone nonmyeloablative conditioning (7/10 patients) compared to those (0/7) who had received standard myeloablative conditioning [137]. The duration of preemptive therapy has varied in the published studies. The two most commonly used variations have been to continue therapy until day 100 after stem cell transplantation [138] or until the indicator test becomes negative, usually resulting in a shorter duration of therapy [134,139]. The drawback with shorter courses of therapy is that treatment might have to be restarted [140], but the advantages are lower cost, less risk of side effects, and potentially better reconstitution of the speciﬁc immune response to CMV.

9

TREATMENT OF ESTABLISHED DISEASE

The results of therapy in established CMV disease are still poor. Survival in CMV pneumonia remains at approximately 50% when the combination of ganciclovir and immunoglobulin is used [141–144]. Machado et al., however, recently published the results of an uncontrolled study of 139 allogeneic SCT patients showing that the advantage of adding immunoglobulin might be limited, with no improvement in survival in CMV pneumonia over ganciclovir therapy given alone [144]. For patients with CMV disease other than pneumonia, the addition of immunoglobulin does not seem to be beneﬁcial [145]. The combination of ganciclovir and foscarnet is suggested to offer a clinical beneﬁt in the treatment of CMV disease without a signiﬁcant increase in toxicity [134]. Recently, a retrospective survey reported that cidofovir could salvage nine of 16 patients with CMV pneumonia failing therapy with ganciclovir, foscarnet, or the combination [135,136].

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Antiviral Resistance

Resistance to ganciclovir is usually mediated through mutations in the UL97 gene. It has been more commonly reported in AIDS and in solidorgan transplant recipients than in stem cell transplant patients. It has been proposed that prolonged exposure to ganciclovir, a high viral load in the presence of suboptimal concentrations of antiviral drugs, and heavy immunosuppression predispose for the emergence of resistant strains [147,148]. Other therapeutic alternatives are then foscarnet or cidofovir. However, only increasing antigenemia early after initiation of antiviral therapy is usually not a sign of antiviral resistance and does not necessitate a change of therapy [149]. 9.2

Adoptive Immunotherapy

Despite that major advances have been reached in CMV management, several problems still exist, including the increased incidence of late CMV disease. The lack of speciﬁc immunity to CMV both regarding cytotoxic T-cell (CTL) response and helper T-cell response to CMV has been associated with a high risk for CMV disease [149–152]. Monitoring of speciﬁc immunity is therefore likely in the near future to become important for routine patient management. Fluorescent HLA-peptide tetramers containing immunodominant peptides from CMV have been used to monitor the presence of CMV cytotoxic T cells (CTL) in the stem cell graft [153] and their recovery post-transplantation [153,154]. When both recipient and donor were CMV-seropositive pretransplantation, recovery of CMV-speciﬁc CTL was rapid and reached up to 21% of all CD8þ cells [153]. Recovery was slower in recipients of unrelated transplants than after HLA-identical sibling grafts. CTL numbers increased after episodes of CMV reactivation but were suppressed by corticosteroid treatment. Recovery of CMV-speciﬁc CTL to a level greater than 10 6 106/L was associated with protection from CMV disease. Furthermore, the number of CMV-speciﬁc CD8þ T cells present in the graft pretransplantation correlated with the number of courses of preemptive ganciclovir administered post-transplantation [153]. In addition, peptide-speciﬁc intracellular cytokine staining was shown to allow documentation of CMV-speciﬁc CD4þ and CD8þ T-cell responses. For long-term control of CMV infection post-transplantation CMVspeciﬁc T helper cell responses seem to be essential [150,151,155]. Riddell and coworkers showed that speciﬁc CTL can be cloned in vitro and can be safely given to the patient, and that their activity can be detected during follow-up [155,156]. A phase II study has recently been completed but has not been published. Preliminary data indicate that the

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risk for CMV infection and disease was low and side effects were rare. New techniques such as the use of peptide pulsed dendritic cells and the tetramer technology have been developed that might allow an easier selection of CMV-speciﬁc T cells, and several laboratories are presently testing these strategies in early-phase clinical trials [157–160]. For example, transfer of CMV-speciﬁc CD4þ T cells was shown to be effective in inducing CMV-speciﬁc CD8þ T-cell responses and reduction in CMV DNA load [160].

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18 Epstein-Barr Virus: Pathogenesis and Treatment Nancy Raab-Traub and Shannon C. Kenney Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

1 1.1

EPSTEIN-BARR VIRUS INFECTION Biological Properties

The Epstein-Barr virus (EBV) is a highly successful human herpesvirus that infects more than 90% of the world’s population. Like other herpesviruses, EBV establishes a latent infection with lifelong persistence in the infected host [1]. The virus is usually transmitted by exposure to saliva and is believed to initially infect the epithelial cells of the oropharynx and posterior nasopharynx, as well as the parotid gland and duct or possibly B-lymphocytes in lymphoid-rich oral tissues such as the tonsils [2,3]. The virus establishes a persistent latent infection in bone marrow and peripheral blood B lymphocytes [4]. After infection, the virus is sporadically secreted into saliva [5]. The cellular source of the salivary virus is thought to be epithelial cells and possibly tonsillar lymphocytes [6]. EBV DNA has been detected using in situ hybridization in cells lining the parotid duct, and viral replicative mRNAs have been detected in sloughed oropharyngeal and cervical 623

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epithelial cells [2,3]. Viral DNA and evidence of viral replication have also been detected in tissue from the sides of the tongue [7]. A subset of peripheral blood lymphocytes, thought to be resting memory B cells, persistently contain EBV [8]. EBV-infected cells can be explanted into culture in vitro and established as permanent cell lines [9]. The majority of these infected cells do not produce virus; instead, the EBV genome persists as a multicopy episome [10]. In an occasional cell in some cell lines, the virus reactivates from a latent infection and replicates. The presence of infectious virus produced by such cells or virus that is present in throat washings can be determined by infecting primary B lymphocytes and assessing their ability to proliferate indeﬁnitely in vitro [11]. Growth transformation of B lymphocytes is induced by the coordinate expression of multiple viral gene products [1]. Expression of the growth-inducing viral functions also occurs in vivo [12]. The overgrowth of the transformed cells is controlled by virus-speciﬁc cytotoxic T cells that can be continuously detected in the peripheral blood of infected individuals [13]. This continued surveillance and protection from proliferating EBV-infected cells by cytotoxic T cells is essential to the control of EBV infection. This is most clearly demonstrated by the increased risk of development of EBV-infected lymphomas in allograft recipients with T-cell immunosuppression [14]. 1.2

Molecular Properties of EBV

Within the virion, the EBV genome is a double-stranded linear molecule that circularizes after entry into the cell via direct DNA repeat elements located at each end of the genome [15]. This circular viral genome is replicated by the host cell DNA polymerase. A convenient assay to distinguish circular and linear genomes is based on identiﬁcation of the terminal restriction enzyme fragments [16]. The EBV terminal restriction enzyme fragments are highly variable in size due to varying numbers of a repeated DNA sequence. Within tumors, a single terminal restriction enzyme fragment is detected, indicating that all of the EBV episomes are identical. This suggests that the tumor is clonal and represents a proliferation of a single EBV-infected cell [16]. This assay has been used for multiple tumors associated with EBV and has indicated that the majority are monoclonal with oligoclonal proliferation in some immunosuppressed patients [17]. Identiﬁcation of the EBV termini can also be used as a marker of permissive infection, because the terminal restriction enzyme fragments of linear virion DNA appear as ladder arrays of fragments on Southern blots [18].

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Expression During Latency

Epstein-Barr virus has at least four patterns of gene expression in latently infected cells [1]. In B lymphocytes transformed in vitro, at least 11 viral genes are expressed. These include six EBV nuclear antigens (EBNAs), three latent membrane proteins (LMP1, LMP2A, and LMP2B), and two noncoding RNAs. This state of latency is called latency III and is also found in lymphomas that develop in immunocompromised patients following organ transplantation. In many of the tumors that are associated with EBV, expression is more restricted (latency II), and only EBNA1, LMP1 and 2, and the EBERs are expressed. In latency I, which is found in Burkitt’s lymphoma, only EBNA1 and the EBERs are detected. In peripheral blood lymphocytes, only LMP2, the EBERs, and possibly EBNA1 are detected (latency 0). In latent infection, replication of the viral episome requires EBNA1, which binds to the plasmid origin of replication (orip) and facilitates recognition and replication of the EBV genome by the cellular DNA polymerase [19]. Recombinant DNA vectors that contain orip and express EBNA1 have been developed as molecular biological reagents for maintaining and expressing DNA without integration into the host chromosome [19]. The EBNA2 protein is essential for growth transformation of lymphocytes and is also a transcriptional transactivator [20]. This gene is deleted in viral strains that cannot transform lymphocytes, such as the HR1 strain [21,22]. Transformation-competent recombinants between HR1 and other EBV strains regain the EBNA2 coding sequences and the ability to transform lymphocytes [23]. EBNA2 interacts with several cellular DNA–binding proteins, including PU1 and RBPJk, and regulates the viral promoters for the latent membrane proteins LMP1 and LMP2 [24,25]. EBNA2 also activates expression of cellular genes including cmyc; the B-cell activation marker, CD23; and the EBV receptor, CD21 [26,27]. Two types of EBNA2 have been identiﬁed, encoded by divergent DNA sequences [21]. The type of EBNA2 gene, EBNA2A or EBNA2B, has been used to deﬁne two types of EBV, EBV1 or EBV2. EBV1 is more prevalent than EBV2 in western populations; however, EBV2 infection is prevalent in central Africa, in New Guinea, and among Alaskan Eskimos [28,29]. Coinfection with both EBV types is frequently detected in HIV-infected patients and has also been detected in normal individuals [30]. The EBNA3 genes are also encoded by two types of divergent sequences that usually cosegregate with the EBNA2 type [31,32]. The EBNA3C protein upregulates expression of CD21, the receptor for

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complement, which is also the EBV receptor [1]. All the EBNA3 proteins also interact with RBPJk and modulate transactivation by EBNA2 [33]. Latent membrane protein 1 (LMP1) is located in the cytoplasmic membrane of latently infected cells [34]. LMP1 is essential for EBV transformation of lymphocytes and is the only EBV gene product that has transforming ability in rodent ﬁbroblasts [35,36]. LMP1 has a short cytoplasmic amino terminal domain, six transmembrane domains, and a long cytoplasmic carboxy terminal portion (Fig. 1). Within the carboxy terminal domain, there is an 11 amino acid repeat element and a region of 10 amino acids that is deleted in some strains of EBV [37]. The carboxy

Figure 1 Latent membrane protein 1 (LMP1) structure and signaling. LMP1 has six transmembrane domains and is located within the plasma membrane. Two activating domains (CTAR1 and CTAR2) within the cytoplasmic carboxy terminal domains bind molecules that transduce signals from the tumor necrosis factor receptor family (TRAFs). CTAR1 and 2 both activate the NFkB transcription factor and induce expression of many cellular genes. CTAR1 has the unique ability to induce expression of the epidermal growth factor receptor, and CTAR2 has been shown to activate the c-jun terminal kinase (JNK).

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terminal portion of LMP1 interacts with cellular adapter proteins that transduce signals from the tumor necrosis factor (TNF) family of receptors [38]. These molecules, called TRAFs, for tumor necrosis factor receptor-associated factors, are activated by the receptor clustering that occurs after ligand binding. LMP1 apparently acts as a constitutively activated member of this receptor family. Two domains have been identiﬁed in the carboxy terminus of LMP1 that can both activate NFkB through interactions with TRAFs [39,40]. Activation of NFkB contributes to the activation of expression of most of the cellular genes that are induced by LMP1. LMP1 activates NFkB in both lymphocytes and epithelial cells, and many of the same cellular genes are induced in both cell types [41,42]. LMP1 induces expression of many important cellular genes that would have profound effects on cellular growth, including the epidermal growth factor receptor, antiapoptotic genes such as bcl2 and A20, and B-cell activation markers including MHC class I and adhesion molecules such as ICAM1 [1]. Inhibition of NFkB in EBV-infected lymphocytes using a constitutively active form of the IkB inhibitor of NFkB resulted in rapid death from apoptosis, indicating that NFkB signaling is essential to growth transformation [43]. Latent membrane protein 2 (LMP2) is also an integral membrane protein that has been shown to interfere with signal transduction from the activated immunoglobulin receptor [44]. LMP2 is phosphorylated on tyrosines by the cellular tyrosine kinases, fyn and lyn [45]. LMP2 is thought to sequester these kinases and inhibit translocation of the B-cell receptor into lipid-rich rafts in the plasma membrane [46]. This blocks Bcell activation and thus prevents activation of the viral replicative cycle. In epithelial cells, LMP2 inhibits differentiation and induces cell proliferation through activation of PI3 kinase and the serine/threonine kinase, Akt [47]. The block in epithelial cell differentiation may inhibit activation of EBV replication, which is thought to occur in differentiating epithelial cells. 1.4

Activation of EBV Replication

Epstein-Barr virus must convert to the lytic form of infection in order to be transmitted from cell to cell and from host to host. Lytic EBV infection involves three sequential waves of viral gene expression. The immediateearly (IE) proteins, BZLF1 and BRLF1, are initially expressed [48]. The BZLF1 and BRLF1 proteins, which are transcriptional activators, then activate expression of the viral early genes [48,49]. The viral early genes encode the functions required for the lytic form of viral replication, which

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uses the oriLyt origin of replication and a virally encoded DNA polymerase [50]. Following viral replication, the late viral genes, which generally encode the structural proteins of the virus, are then transcribed. By deﬁnition, host cells containing the latent form of EBV infection do not express the BZLF1 and BRLF1 immediate-early genes, and expression of either EBV IE gene results in the lytic form of infection [51,52]. Thus, host cell transcriptional regulation of the IE genes is the key element determining the status of EBV infection in cells (i.e., latent versus lytic). In the laboratory, the promoters driving the IE genes can be activated by a variety of factors, including phorbol esters, sodium butyrate, TGF-beta, and activation of the B-cell receptor [53]. Although the factors that induce the lytic form of EBV infection in patients remain poorly deﬁned, there is increasing evidence that B-cell stimulation by antigen plays an important role [54]. Each IE protein initially activates expression of the other IE gene, and together the two immediate-early proteins can induce expression of the entire cascade of lytic viral genes [55]. BZLF1 contains a leucine zipper and activates transcription by binding directly to AP1 sites and AP1-like elements known as ZREs in immediate-early and early EBV gene promoters [56]. BRLF1 activates genes through at least two different mechanisms. One mechanism involves direct BRLF1 binding to GC-rich motifs present in some early EBV gene promoters [57]. A second mechanism of transactivation that is required for BRLF1 activation of BZLF1 is mediated through phosphorylation of the cellular c-JUN and ATF-2 transcription factors [58]. The targets for conventional antiviral therapy directed against EBV are expressed during the lytic, rather than latent, form of infection. Like other herpesviruses, EBV encodes kinases, expressed by the early lytic genes BGLF4 and thymidine kinase that phosphorylate the nucleoside analogs ganciclovir and acyclovir into their active antiviral forms [59]. In addition, the EBV-encoded DNA polymerase, the early lytic gene that mediates the lytic form of viral DNA replication, is much more susceptible to the inhibitory effects of the antiviral agents foscarnet and cidofovir than is the cellular DNA polymerase that replicates the EBV genome during latency [60,61]. 2

IDENTIFICATION AND TREATMENT OF EBV-RELATED PATHOLOGY

Most of the diseases associated with EBV represent latent infections with the virus [1]. Primary infection with EBV is known to occasionally result in the syndrome infectious mononucleosis [62]. EBV is also thought to have an etiological role in the development of several malignancies

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including African Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), post-transplant lymphoma, a subset of lymphomas that develop in patients with AIDS, some Hodgkin’s lymphomas, and several unusual carcinomas [63]. 2.1 2.1.1

Primary Infection and Infectious Mononucleosis Clinical Presentation

During primary infection, the virus initially uses the cytolytic form of infection, allowing the virus to be replicated and spread from cell to cell. Infectious mononucleosis is characterized by an extremely vigorous Tcell response, and many of the clinical symptoms of the illness are in fact due to the severity of the immune response. The classical large atypical lymphocytes that are so characteristic of infectious mononucleosis are due to the highly activated cytotoxic T-lymphocyte response to EBVinfected B cells rather than the EBV-positive B cells themselves. In the immunocompetent host, cells infected with the cytolytic form of EBV infection are eventually eliminated by T cells, and only cells containing the most latent type of infection evade the immune response [64]. Primary infection with Epstein-Barr virus is associated with a range of clinical symptoms [65]. Infection in young children appears to cause a milder (usually undiagnosed) illness, whereas infection in adolescents and young adults often results in the classic clinical syndrome, infectious mononucleosis. Primary infection in the elderly often causes an atypical, severe illness characterized by cholestatic liver disease. The classical clinical syndrome of infectious mononucleosis is characterized by fever, pharyngitis, lymphadenopathy, and atypical lymphocytosis. Most patients have an erythematous pharynx (often with an accompanying exudate), bilateral cervical adenopathy, and sometimes splenomegaly [65]. Periorbital edema is often observed early in the disease. Prior treatment with beta-lactam antibiotics may result in the development of a diffuse morbilliform rash. Common laboratory abnormalities include atypical lymphocytosis and mildly elevated liver transaminases (usually less than tenfold normal). However, primary EBV infection in the elderly often has an unusual clinical presentation, without pharyngitis, lymphadenopathy, and atypical lymphocytosis, and more commonly results in a severe, cholestatic liver disease that can progress to fulminant liver failure. 2.1.2

Complications

Almost any organ system can be involved in patients with complicated infectious mononucleosis. Relatively common complications include

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obstruction of the oropharynx due to sever inﬂammation, hepatitis, cholestatic jaundice (particularly in the elderly), myocarditis, thrombocytopenia, hemolytic anemia, and splenic rupture. Less common complications include encephalitis, Guillain-Barre´ syndrome, cranial neuritis, meningitis, transverse myelitis, bone marrow suppression, and hemophagocytic syndrome. Patients who have the rare familiar X-linked lymphoproliferative syndrome are unable to control EBV, and in these patients primary EBV infection results in either immediate, fulminant fatal infectious mononucleosis or, if the patients survive their initial infection, is followed by B-cell lymphoma [66]. This syndrome is caused by a mutation in the SH2D1A gene, which encodes a protein important for regulating B-cell–T-cell interactions [67]. 2.1.3

Chronic Active EBV

Very rarely, patients are unable to control their EBV infection and go on to develop chronic active EBV. This syndrome is characterized by fever, lymphadenopathy, hepatosplenomegaly, and frequently polyclonal gammopathy and bone marrow suppression. Patients have a normal SH2D1A genotype but are nevertheless unable to mount an effective cytotoxic T-cell response against EBV and have extremely high viral loads [68]. EBV-infected T cells appear to play a role in this syndrome as well, and patients not uncommonly eventually succumb to EBV-positive T-cell lymphomas. Chronic active EBV disease should not be confused with chronic fatigue syndrome, in which there is no evidence that EBV plays a role and EBV viral loads are normal [69]. 2.1.4

Diagnosis

Diagnosis of infectious mononucleosis has long been established by the presence of a positive heterophile antibody test (‘‘monospot’’). This test, which detects an antibody that binds to sheep or horse but not guinea pig red blood cells, is positive in over 90% of adolescent patients with classical infectious mononucleosis. In this age group, acute EBV infection is usually associated with polyclonal B-cell activation that results in production of antibodies to heterophile antigens. However, in other age groups this test is much less sensitive, detecting only 33% of primary EBV infections in children. The heterophile antibody test also appears to be less sensitive in patients over 40. False-positive heterophile antibody tests are unusual but can be caused by lymphoma and hepatitis. Speciﬁc serological tests provide a more sensitive and speciﬁc means for diagnosing primary EBV infection in all age groups. Patients with primary EBV infection have a positive IgM antibody to the viral capsid antigen (VCA), which disappears following the acute illness. The

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IgG antibody to VCA, although commonly present in patients with infectious mononucleosis, remains positive for life and cannot distinguish between acute and past infection [70]. Another useful serological marker indicative of primary EBV infection is the absence of antibody to Epstein-Barr virus nuclear antigen 1 (EBNA1) in the presence of any other positive serological marker for EBV. Antibodies to EBNA1 do not develop until several months after infection, and therefore the presence of EBNA1 antibody suggests infection in the past, whereas the absence of EBNA1 antibody in the presence of other EBV-speciﬁc antibodies suggests recent infection [71]. Thus, a deﬁnitive diagnosis of primary EBV can be made by the presence of a positive IgM or IgG VCA antibody titer in the absence of an EBNA1 titer. Positive VCA and EBNA1 titers together suggest past infection. Although antibodies to the viral early antigens are observed in primary, as well as occasionally reactivated, EBV infection, they are not particularly useful for diagnosing primary EBV. Patients with infectious mononucleosis have a much higher EBV load than immunocompetent hosts with past EBV infection. Therefore, polymerase chain reaction (PCR) technology, which can be used to quantify viral load, may help to conﬁrm or reject the diagnosis of acute EBV infection as well as to provide a means for monitoring clinical course. However, because EBV viral load is also sometimes elevated in immunosuppressed patients and patients with EBV-positive tumors, this test is not speciﬁc for primary EBV infection and in most instances should not be required to make the diagnosis of primary EBV infection [72]. 2.1.5

Treatment of Infectious Mononucleosis

Drugs that have been shown to inhibit the lytic form of EBV replication in vitro include acyclovir, ganciclovir, foscarnet, and cidofovir [60,61,73]. Acyclovir and ganciclovir are nucleoside analogs that must be phosphorylated into their active forms in order to be incorporated into DNA, where they act as chain-terminating agents [74]. In contrast to the effects of herpes simplex virus and cytomegalovirus, less is known regarding the precise mechanism(s) by which EBV infection results in acyclovir and ganciclovir phosphorylation. Herpes simplex virus encodes a thymidine kinase that efﬁciently phosphorylates both acyclovir and ganciclovir, whereas cytomegalovirus encodes a kinase (UL97) that preferentially phosphorylates ganciclovir but not acyclovir. EBV encodes homologs to both the HSV thymidine kinase and the CMV UL97 kinase, but it is not clear if one or both of these kinases phosphorylate either drug. Although at least one investigator has

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suggested that EBV infection activates a cellular kinase to phosphorylate these drugs, another has suggested that both the EBV TK and UL97 homolog (BGLF4) can phosphorylate ganciclovir [59]. In any event, cells infected in vitro with lytic EBV infection efﬁciently phosphorylate both acyclovir and ganciclovir, and both drugs inhibit the lytic form of EBV replication at clinically relevant doses [75,76]. Foscarnet, which is an inorganic pyrophosphate analog, directly inhibits the EBV-encoded DNA polymerase as well as all other herpesvirus DNA polymerases, by reversibly binding to the pyrophosphate binding site. Cidofovir is an analog of deoxycytidine monophosphate and is converted into its active diphosphate form by cellular enzymes. Many virally encoded DNA polymerases, including the EBV DNA polymerase, incorporate cidofovir, a chain terminator, more efﬁciently than the host cell DNA polymerase into the growing DNA. Thus, it would be anticipated that all of these antiviral drugs would be effective for inhibiting lytic EBV replication in patients. This has been demonstrated by the ability of acyclovir to treat the lytic EBV infection oral hairy leukoplakia [77]. Given that acyclovir is the least toxic of these agents, it is currently the drug of choice for treating the lytic form of EBV infection in patients. In infectious mononucleosis, host cells are infected with both the latent and lytic forms of EBV, and thus it might be anticipated that drugs that prevent the lytic form of EBV infection would be useful for this syndrome. However, clinical trials to date have not demonstrated a signiﬁcant clinical beneﬁt from using acyclovir to treat infectious mononucleosis, even though the amount of viral shedding in the saliva is decreased [78]. The lack of clinical beneﬁt from acyclovir in infectious mononucleosis may reﬂect the fact that many of the symptoms in this illness are primarily due to the intense host immune response rather than destruction of cells by the virus. In addition, because the clinical trials were performed on otherwise healthy young individuals, who generally recover from primary EBV infection rather quickly in any event, it remains possible that the outcome of primary EBV infection in immunosuppressed or elderly individuals would be improved by acyclovir treatment. Theoretically, at least, the use of acyclovir early in primary EBV infection might reduce the total number of B cells in the host that are latently infected by EBV after recovery from the acute illness. Whether this decrease would correlate with a reduction in risk for EBV-associated B-cell malignancies later in life, perhaps during times of immunosuppression, is an interesting but completely unresolved issue. Many of the symptoms of severe infectious mononucleosis are due to the host immune response, and there is some evidence that a short

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course of high-dose steroids may be useful in speciﬁc complications of the illness, including hemolytic anemia, thrombocytopenia, GuillainBarre´ syndrome or other CNS complications, cardiac involvement, and severe obstructive pharyngitis. However, long-term steroid use is clearly contraindicated following primary EBV infection, because the host immune response is required to prevent the development of lymphoproliferative disease, and short-course steroids should not be routinely administered to all patients with infectious mononucleosis. In patients with uncomplicated infectious mononucleosis, a double-blind, placebocontrolled trial found no difference in the clinical outcome of patients treated with placebo, versus the combination of acyclovir and prednisolone [79]. In the case of the rare patients who develop chronic active EBV infection, there is no evidence to date that acyclovir treatment results in clinical improvement or affects outcome. Whether the infusion of EBVspeciﬁc cytotoxic T cells will be useful for treating such patients is being actively investigated by several groups but is as yet unknown.

2.1.6

Monitoring and Follow-Up

The treatment of infectious mononucleosis is primarily supportive, and most patients can be safely followed as outpatients [65]. Symptoms in the adolescent usually resolve after several weeks. Contact sports should be avoided for several weeks (to prevent splenic rupture) and no beta-lactam drugs given (due to the risk of rash). In general, repeat laboratory tests are not indicated as long as the patient is improving clinically. In rare patients who continue to be ill several months after their initial illness, it may be useful to obtain repeat EBV serology as well as an EBV viral load. There is some evidence that the failure to develop anti-EBNA1 antibody by 6 months after the acute illness is associated with incomplete control of the virus. Likewise, extremely high antibodies to the EBV early antigens may be indicative of poor control. Nevertheless, the absence of EBNA1 antibody, and high antibody titers to early antigen, can also sometimes be seen in apparently healthy individuals. The development of commercially available assays for quantifying EBV viral load promises to be a more accurate way of monitoring patient recovery. Most normal EBV-positive carriers have extremely low or undetectable EBV viral loads, whereas patients who go on to develop chronic active EBV infection have persistently very high viral loads (over 1000 copies of EBV genome per milliliter of blood).

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EBV-Associated Lymphoproliferative Disease in Immunocompromised Patients

Immunoblastic B-cell lymphomas in patients who are immunocompromised by bone marrow or solid-organ transplants or AIDS often contain the EBV genome [80]. This is particularly true in primary CNS lymphomas in AIDS patients, in which virtually 100% of tumors are EBV-positive [81]. Immunoblastic lymphomas in immunocompromised patients likely arise from insufﬁcient T-cell function, allowing EBVpositive B cells expressing immortalizing viral proteins such as LMP1 and EBNA2 to proliferate unchecked. 2.2.1

Diagnosis

Clinically, patients with peripheral EBV-positive lymphomas usually present with lymphadenopathy, commonly with accompanying fever [82]. Speciﬁc organ symptoms reﬂect the particular location of the tumor. The diagnosis of peripheral lymphoproliferative disease is suggested by a high EBV viral load in patients who are at risk for this syndrome [72]. Conﬁrmation of the diagnosis requires biopsy of affected lymph nodes. Commercially available assays that detect expression of the EBV EBER RNAs offer a simple and sensitive method for conﬁrming the presence of EBV in lymphomas. In patients with suspected early EBV-induced lymphoproliferative disease but no obvious lymphadenopathy, liver biopsy with EBER staining may help to conﬁrm the diagnosis [83]. EBVpositive primary CNS lymphomas usually occur in the setting of AIDS and CD4 counts of less than 50, and present with CNS manifestations (the precise symptoms depending upon the location of the lesions) and one or more ring-enhancing lesions in the gray matter of the brain. The appearance of these lesions is most commonly confused with AIDSassociated CNS toxoplasmosis. Although CNS lymphoma and toxoplasmosis can be distinguished by biopsy of the lesions, a positive EBV PCR test in the CSF is highly suggestive for the diagnosis of CNS lymphoma [84]. A positive EBV PCR test in the CSF, with a compatible head CT or MRI scan in a patient with AIDS, may therefore avoid the necessity for brain biopsy. 2.2.2

Treatment

Early on, EBV-associated immunoblastic lymphomas are often polyclonal and may regress following reduction in immunosuppressive regimens or reconstitution of the immune system in AIDS patients with highly active antiretroviral therapy [85]. More advanced disease is usually oligoclonal and has traditionally required chemotherapy and/or

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radiation therapy (in the case of primary CNS disease) for any hope of cure [80]. Because the EBV-positive lymphoma cells primarily contain the latent form of EBV infection and are not sensitive to antiviral drugs directed against the virally encoded DNA polymerase or other lytic gene products, traditional antiviral therapy would not be expected to have a signiﬁcant clinical effect. Nevertheless, there are reports suggesting that prophylactic treatment with ganciclovir reduces the frequency of lymphoproliferative disease in transplant patients in comparison to historical controls and case reports documenting apparent regression of EBV-associated lymphoproliferative disease in immunosuppressed patients following treatment with antiviral agents [86,87]. In addition, interferon-alpha therapy appears to be effective in some patients [88]. In addition to conventional chemotherapy and radiation therapy, there are several recent advancements in the treatment of EBV-positive lymphoproliferative disease. A monoclonal antibody directed against a pan B-cell surface antigen, CD20 (rituximab), has been shown to induce remission in a subset of patients with post-transplantation immunoblastic lymphomas [89]. In addition, infusion of cytotoxic T cells directed against EBV antigens has been shown to be a very effective method if used early for treating EBV-positive lymphomas in transplant recipients [90,91]. At present, the development of EBV-directed cytotoxic T-cell clones is expensive and time-consuming. It is anticipated that in the future EBV-directed cytotoxic T-cell ‘‘banks’’ containing a multitude of HLA types may be available for treatment of patients (such as AIDS patients) for whom it is not practical or possible to derive patient- or donor-derived cytotoxic T cells.

2.3

Oral Hairy Leukoplakia

Hairy leukoplakia (HLP) is an unusual lesion that was ﬁrst described in patients with human immunodeﬁciency virus (HIV) infection and is the only pathological manifestation associated with replication of Epstein-Barr virus (EBV) [92]. HLP has been shown to contain linear virion DNA, viral capsid antigen, and viral particles [18,92]. The antiviral agents cidofovir, desciclovir, and zidovudine, which inhibit EBV DNA replication, induce regression of HLP, conﬁrming that the lesion is caused by EBV replication [77,93]. HLP is also a valuable indicator of the progression of immune impairment in AIDS, although it has been described in some allograft recipients and a few healthy individuals [94].

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Hodgkin’s Disease

Hodgkin’s disease (HD) is an unusual lymphoma where the malignant Reed-Sternberg (RS) or Hodgkin’s cell is a rare cell among a reactive lymphoid inﬁltrate that makes up greater than 98% of the tumor mass. HD is classiﬁed into four histological subtypes: nodular sclerosing, mixed cellularity, lymphocyte-predominant, and lymphocyte-depleted. Early studies had suggested a link between EBV infection and HD, because the same epidemiological factors that distinguished those who developed IM also identiﬁed those at risk for HD [95]. The association of EBV with HD was clearly demonstrated by the identiﬁcation of clonal EBV episomes and the localization of EBV to the malignant ReedSternberg cells [96]. The association of EBV with HD varies between subtypes and geographic regions. Approximately 60–80% of the mixed cellularity and lymphocyte-depleted subtypes are associated, in contrast to 20–40% of the nodular sclerosing subtype [97]. The RS cell is an unusual cell that expresses some lymphocyte activation markers such as CD30 and CD70 and in most cases has rearrangement of the immunoglobulin genes, suggesting that the RS cell is of B-lymphocyte origin [98]. The RS cell represents a latent EBV infection with the type 2 latency pattern including expression of EBNA1, LMP1, LMP2, and the EBER RNAs [99]. It is known that LMP1 activates NFkB; however, both EBV-positive and EBV-negative cases have high levels of activated NFkB, suggesting that activation of this pathway is an important aspect of the altered cell gene expression [100]. In some of the EBV-negative tumors, mutations have been detected in the Ikba gene, suggesting that activation of NFkB can be achieved by LMP1 expression or by genetic change [101]. Importantly, this suggests that treatment of HD with the newly developed inhibitors of NFkB may be particularly effective. 2.5

Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an unusual tumor with intriguing epidemiological and biological characteristics [102]. The tumor occurs worldwide but with exceptionally high incidence in particular populations in speciﬁc geographic regions. The incidence is highest among the Cantonese in southern China and in Hong Kong and Singapore Chinese [103]. It occurs with intermediate incidence in Mediterranean Arabs and in Malays in Singapore. The incidence is quite low in American Caucasians and Europeans. The extraordinarily elevated NPC incidence in distinct populations suggests that environmental and genetic elements and viral infection contribute to the development of this disease.

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Diagnosis

Most NPC tumors develop within the fossa of Rosenmuller, a region of the nasopharynx that is rich in lymphocytes. The primary tumor is frequently not identiﬁed, and more than 50% of patients present with cervical lymph node metastases. NPC is classiﬁed histopathologically with distinguishing differences in the degree of differentiation [104]. In undifferentiated NPC, the cells are almost syncytial in appearance and are arranged in strands or connected with lymphoid stroma. The large nuclei are irregular, with prominent nucleoli [105]. The association of NPC with Epstein-Barr virus was revealed in seroepidemiological studies that showed that patients with nasopharyngeal carcinoma (NPC) had elevated IgG and IgA antibody titers to the EBV viral capsid antigen (VCA) and to an antigen associated with replication, called early antigen (EA) [106]. These titers correlated with tumor burden, remission, and recurrence. EBV serology is an important tool in diagnosis of NPC, because 95% of patients have elevated anti-IgA VCA. These titers are useful in the identiﬁcation of patients with occult NPC. Elevation of IgA antibodies can precede tumor development by 1–2 years, suggesting that EBV infection has reactivated [106]. Other replicative proteins to which antibody titers are elevated in NPC are thymidine kinase, DNAse, and the EBV replication activator protein ZEBRA [107–110]. Overall, the data suggest that reactivation of replication of EBV may precede and indicate the development of NPC and that continued antibody response reﬂects the presence of tumor. Cervical lymphadenopathy is the most common clinical presentation; it is found in 50–70% of patients, and bilateral nodal involvement may occur [111]. Nasal symptoms may affect more than half of patients, with nasal obstruction being a late ﬁnding indicating a large tumor. Hearing loss is due to tumor obstruction of the eustachian tube, and tinnitus occurs in one-third of patients. Blood-stained sputum is common and in the endemic area warrants a thorough head and neck exam. Neurological symptoms may include cranial nerve paralysis, most frequently involving cranial nerves V and VI. Headache is a frequent occurrence and may be unilateral or central or retroorbital. Headache may be due to erosion of the base of the skull or cranial nerve irritation. Clinical diagnosis is conﬁrmed by nasopharyngeal biopsy, which may be performed in the outpatient clinic with local anesthesia. Biopsy is indicated in an endemic area by cervical lymphadenopathy, elevated EBV titers, cranial nerve paralysis involving the V and VI nerves, and radiologically demonstrated lesions. When NPC is strongly suspected

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despite negative biopsy, CT or MRI scans should be performed and can be used to guide an endoscopic biopsy in the suspicious area. If all else fails, the patient should be reexamined under general anesthesia and curettage of the nasopharynx [111]. 2.5.2

EBV Infection

Within the tumor, EBV DNA is clonal, suggesting that the tumor represents a proliferation of a single EBV-infected cell [16]. In the diagnosis of NPC, early stages in malignancy such as dysplasia or carcinoma in situ are extraordinarily rare and in most cases are detected concomitantly with invasive carcinoma [112]. One study identiﬁed rare examples of carcinoma in situ or dysplasia and discovered that all of these samples contained clonal EBV, indicating that they were a clonal proliferation of latently infected cells. The rarity of these premalignant lesions and the usual detection of carcinoma in situ or dysplasia adjacent to invasive cancer suggested that NPC develops from a single EBV-infected cell and that this proliferation rapidly progresses to malignancy. Nasopharyngeal carcinoma represents a latent infection of EBV with consistent expression of speciﬁc viral genes with a latency 2 pattern of expression similar to that found in Hodgkins disease [1]. In addition to the RNAs that are known to encode EBNA1, LMP1, and LMP2, a family of intricately spliced mRNAs transcribed from the BamHI A fragment, were originally identiﬁed in examples of nasopharyngeal carcinoma [113–115]. Sequence analysis of cDNAs has revealed that the RNAs are 30 coterminal but differentially spliced to form novel open reading frames [116]. It is as yet unknown if these open reading frames encode protein; however, interesting molecular properties have been identiﬁed for several of the putative proteins. It is known that in some of the areas with a high incidence of NPC, environmental factors such as phorbol esters may induce EBV reactivation and replication at mucosal surfaces. Infection of epithelial cells would usually result in viral replication as the epithelial cells matured and differentiated. However, the increase in viral replication may also increase the chance of establishing a latent transforming infection in epithelial cells rather than a lytic infection with viral replication. The epithelium may also have sustained previous genetic insults that inhibit cellular differentiation or viral replication and promote expression of the EBV-transforming genes. The expression of the viral genes in combination with any previous cellular genetic changes would result in dysplasia that rapidly progressed to invasive neoplasia without the long

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premalignant phase that is characteristic of the development of most carcinomas.

2.5.3

Genetic Changes in NPC

Infection with EBV is clearly an important factor in the development of NPC, but the extraordinary differences in incidence indicate that other factors inﬂuence the development of this tumor. These factors could include environmental components that affect cellular expression or EBV infection, or the elevated incidence could reﬂect inherent genetic differences in the high-risk populations. The putative genetic changes could work in concert with the viral oncogenic proteins to induce cancer. Genetic changes characteristic of other malignancies such as c-myc rearrangement, p53 mutation, Rb alteration, or ras mutations would not necessarily contribute to this altered growth, and such mutations have not been detected in NPC from Chinese, American, or Arab populations [117–119]. However, the unmutated p53 protein is detected at high levels in NPC, and p53 expression has been shown to be induced by NFkB, a transcription factor that is activated by LMP1 [120]. Several studies have investigated whether an EBV protein possibly interferes with some aspect of p53 function and eliminates a selection for inactivating mutations. In latent infections, EBV does not interfere with the ability of p53 to arrest cells in G1 after DNA damage by inducing expression of the p21 cyclin kinase inhibitor; however, two studies have shown that LMP1 inhibited p53-mediated apoptosis induced by serum withdrawal [121,122]. Speciﬁc protection from p53-mediated apoptosis was conferred by the A20 protein, which is induced by LMP1 expression and protects from apoptosis induced by tumor necrosis factor (TNF) or serum withdrawal [123]. This protection of p53-mediated apoptosis is likely to be responsible for the lack of p53 mutations in EBV-associated cancers. Elegant studies have identiﬁed areas of loss of heterozygosity on several chromosomes including regions on chromosomes 3p24 and 9p21 [124,125]. The p16 gene is a critical regulator of cell cycle progression through G1, and mutations in the retinoblastoma gene, ampliﬁcations of cyclins, or inactivation of cyclin-dependent kinases can all inactivate this component of cellular control. Deletion of p16 and repression of p16 expression in NPC suggest that this pathway is affected in NPC as it is in many other tumors. The identiﬁcation of other speciﬁc genes in the regions displaying chromosomal loss may identify critical cellular genes that contribute to the development of NPC in high-risk populations or a gene that is affected by mutagenic environmental factors.

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OTHER MALIGNANCIES

Although EBV has long been linked to the development of lymphoma and speciﬁc undifferentiated carcinomas, recent studies have linked the virus to a wide spectrum of additional malignancies, including parotid and gastric carcinomas and T-cell and natural killer (NK) cell lymphomas.

3.1

T-Cell Lymphoma

Although long believed to be B-cell trophic, EBV has been detected in an occasional T-cell lymphoma. A particular type of T-cell lymphoma that usually presents in the nasal cavity, also referred to as a midline granuloma, is a common tumor in southeast Asians. The ﬁrst link to EBV was presented in ﬁve Japanese cases, all of which were EBV-positive [126]. The tumors had various T or NK cell markers, suggesting EBV infection of a peculiar undifferentiated cell type. Peripheral T-cell lymphomas that are EBV-positive have also been described in Taiwanese and Japanese populations [127]. In some cases, EBV was detected in only some cells, suggesting that the virus infected the tumor secondarily. However, the proportion of EBV-infected cells seemed to increase over time with emergence of a clonal EBV-infected population, indicating that the virally infected cells have some growth advantage and that the fastest growing clone will eventually predominate [128].

3.2

Parotid and Gastric Carcinomas

Other undifferentiated carcinomas that develop in other tissues are also associated with EBV. Clonal EBV episomes have also been detected in all examined samples of undifferentiated carcinoma of the parotid gland [129]. Undifferentiated carcinoma of the parotid gland is an extremely rare cancer that has been most often detected in Eskimo populations who also have a high incidence of NPC. Infection with EBV has also been detected in approximately 10% of undifferentiated gastric carcinoma, a tumor that occurs in both Oriental and Caucasian populations [130]. These ﬁndings indicate that EBV may gain access to epithelial cells outside the naso/oropharynx and that in some instances when this occurs it leads to the development of carcinoma.

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EBV-SPECIFIC THERAPY

The consistent association of EBV with speciﬁc cancers suggests that viral infection and expression are essential factors in tumor development and growth. This provides unique opportunities to prevent or possibly treat the tumor by enhancing the immune response or developing pharmacological agents that speciﬁcally target the function of viral proteins or essential cellular pathways that are activated by viral proteins. 4.1

Vaccine Development

The ability to develop vaccines against herpesviruses is impaired by the possibility that herpesviruses can establish latent, persistent infections without any viral replication. Thus the virus may not initially replicate in the cells infected at mucosal surfaces but may directly infect cells where the viral genome persists in a state invisible to immune detection. Because most of the diseases linked to EBV develop many years postinfection, a successful vaccine would have to completely prevent EBV infection by total neutralization of EBV at mucosal surfaces, a process that requires efﬁcient induction of IgA antibodies. This could theoretically be accomplished with a transformation-defective EBV that would replicate at mucosal surfaces. Several studies indicate that there are naturally occurring EBV strains that lack the EBNA2 gene and are transformation-negative [131]. A genetically engineered strain of EBV that lacked essential transforming proteins yet replicated efﬁciently at mucosal surfaces might be useful as an attenuated vaccine. An alternative approach is the development of a subunit vaccine. The viral glycoprotein gp350 is the most abundant viral glycoprotein and is essential for viral binding and infection [132]. The protective ability of antibodies to gp350 has been tested in cottontop marmosets parenterally challenged with a lymphoma-inducing dose of EBV [133,134]. Some protection was provided that seemed to be cell-mediated rather than antibody-mediated. It is possible that gp350 vaccination could produce sufﬁcient antibody and cell-mediated response to prohibit infection of lymphocytes at mucosal epithelial/lymphoid sites. A second approach tested a gp350/vaccinia recombinant in nine seronegative children in China. After one year, all 10 of the control group had seroconverted and had antibodies to VCA, indicating wild-type EBV infection, in comparison with the test group, in which only three vaccinated participants had seroconverted [135]. Even in the absence of complete protective immunity, vaccination that produced serum-neutralizing antibodies would possibly be protective in the development of lymphoma in solid-organ transplant

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seronegative recipients, who frequently acquire the virus from the donor organ. Thus the serum-neutralizing antibodies produced to recombinant gp350 may be effective protection for this high-risk group. An alternative approach would induce EBV-speciﬁc cytotoxic lymphocyte (CTL) recognition using synthetic peptides representing the predominant CTL epitopes presented by prevalent class I molecules. Expression of a cocktail of EBV epitopes in vaccinia virus indicated that all of the epitopes were processed correctly for the individual HLA classes and that the virus could induce the correct EBV epitope-speciﬁc CTLs in vitro [136]. Many EBV CTL epitopes have been identiﬁed, and it is possible that an appropriate cocktail could be selected to protect the majority of individuals. It may also be possible to enhance a speciﬁc CTL response. In NPC and Hodgkins’s disease, the viral proteins that induce the immunodominant CTL response are not expressed. It is possible that the viral expression could be manipulated, perhaps through the use of demethylating agents such as azacitidine, to induce expression of the viral proteins that would make the cells susceptible to CTL killing [137]. Alternatively, the CTL response to weaker immunogens such as LMP1 or LMP2 could theoretically be enhanced, which may enable immune recognition and control of the tumor [138].

4.2

Immunotherapy

It is known that the immunoblastic lymphomas that develop in transplant recipients express the EBNA2 and EBNA3 proteins, which are the major CTL targets [13,139]. These lymphomas remain susceptible to T-cell control, and in some cases the lymphomas may regress with reduced immunosuppression [85]. It has also been possible to prophylactically suppress the development of these lymphomas by treating with EBV-speciﬁc CTLs expanded in vitro [90]. In most EBV-associated cancers, the EBNA2 and EBNA3 proteins are not expressed; however, LMP1 and LMP2 are frequently expressed. Although CTLs have been identiﬁed that are speciﬁc to these proteins, they are a minor component in the total cytotoxic T-cell response. In vitro expansion of CTLs directed against LMP1 and LMP2 for infusion into patients who have failed conventional therapy is currently being tested for Hodgkin’s lymphoma [140]. It may also be possible to develop therapeutic vaccination to induce a vigorous CTL response to these proteins in vivo.

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643

Treatment of Latent Transforming Infection

Many of the malignancies associated with EBV, including NPC, HD, and PTLD, express viral proteins that are essential to maintain the latent infection and also affect cell growth. Thus, novel treatments might prevent their expression or inhibit their function. Because EBNA1 is essential for maintaining viral infection, initial attempts used antisense RNAs or oligonucleotides to inhibit EBNA1 expression [141,142]. More recent attempts have targeted expression of the EBV oncogene LMP1 [143]. As with all antisense approaches, their utility will be limited by lack of efﬁcient delivery to the tumor site and by cellular uptake. The viral proteins LMP1 and LMP2 signiﬁcantly affect cellular gene expression, usurp cellular signaling pathways, and are expressed in most of the EBV-associated cancers. LMP1, in particular, activates the transcription factor NFkB and induces expression of many cellular genes (Fig. 1). A recent study used a dominant inhibitor of NFkB and induced apoptosis in EBV-infected lymphocytes in vitro [43]. Novel inhibitors that speciﬁcally target the kinases that lead to activation of NFkB are being developed, and some of them are currently being tested in phase 1 trials. These compounds may be particularly effective against cancers associated with EBV and LMP1 expression. Similarly, the induction of high levels of expression of the epidermal growth factor receptor (EGFR) by LMP1 in NPC is likely important for the continual proliferation of the tumor. Thus, NPC may be treatable by combinations of radiation and EGFR inhibitors.

4.4

Future Development of Novel EBV-Based Therapies for Cancer

Additional EBV-targeted therapies, which take advantage of the consistent presence of the EBV genome in certain tumors, are currently being developed and hopefully will be available for clinical use in the future. For example, there is increasing interest in using EBV itself to kill tumor cells. Several groups are investigating methods for converting the viral infection from a latent transforming infection to a lytic, cytotoxic form of infection in tumor cells (Fig. 2). The alteration in the state of infection could directly result in cell killing. In addition, the expression of additional viral proteins would render the cell recognizable to prevalent cytotoxic T cells. Furthermore, the expression of virally encoded kinases would activate cytotoxic nucleoside analogs that would be incorporated into cell DNA and induce cell death.

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Figure 2 Therapeutic consequences of activation of EBV replication. Expression of the viral immediate-early genes, Z and R, in tumor cells would reactivate viral replication. This would induce cell death and lysis. Expression of viral kinases could phosphorylate cytotoxic nucleoside analogs that would be incorporated into the cellular genome and induce cell death. In addition, expression of Z and R would increase recognition of the tumor cells by cytotoxic T cells that speciﬁcally recognize these proteins.

Theoretically, activation of EBV replication could be accomplished either by inducing transcription of either of the two EBV immediate-early genes in tumor cells or by directly introducing either gene into tumor cells using gene delivery technology (Fig. 3). As discussed previously, the two EBV immediate-early proteins are transcriptional activators, and expression of either protein is sufﬁcient to reactivate the lytic form of EBV infection. In a mouse model, adenovirus vectors expressing either EBV immediate-early protein (BZLF1 or BRLF1) can induce the lytic form of EBV infection when directly inoculated into EBV-positive tumors [144]. Injection of either the BZLF1 or BRLF1 adenovirus vector dramatically inhibits the growth of these tumors, curing a portion of them. Thus,

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Figure 3 Gene therapy of EBV-associated tumors. It is possible to treat EBV tumors using expression vectors such as adenovirus to directly deliver the immediate-early genes Z or R into the tumor cells. This would directly induce cell death and induce expression of the viral kinases to potentially activate nucleoside analogs such as ganciclovir. The phosphorylated ganciclovir could potentially enter adjacent cells where EBV replication had not been activated and induce a ‘‘bystander’’ cytotoxic effect.

delivery of either EBV IE gene into EBV-positive tumors may prove to be an effective method for treating small, well-localized tumors that are accessible to direct inoculation. There is also great interest in developing pharmaceutical agents that speciﬁcally activate the lytic form of EBV infection in tumor cells. Recent studies have shown that many agents that are stressful to the host cell and are used to treat cancer, such as chemotherapy agents and gamma irradiation, also reactivate the lytic form of EBV infection in a portion of tumor cells [145]. The induction of lytic EBV infection by these agents involves activation of several different cellular signaling pathways, including protein kinase C, P13 kinase, and the p38 stress map kinase. Thus induction of lytic EBV infection could contribute to the effectiveness of chemotherapy and radiation for EBV-positive tumors. This effect can be ampliﬁed by combining the antiviral drug ganciclovir with either chemotherapy or radiation. In this situation, the EBVencoded kinases BGLF4 and thymidine kinase that phosphorylate the prodrug ganciclovir into its active form are expressed. Phosphorylated

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ganciclovir inhibits not only the virally encoded DNA polymerase but also the host cell DNA polymerase and is thus cytotoxic [146]. This phosphorylated ganciclovir can also be transferred into nearby cells that are unable to phosphorylate ganciclovir, thus inducing ‘‘bystander’’ killing [147]. The combination of chemotherapy or radiation and ganciclovir might be more effective than either agent alone for treatment of EBV-positive tumors, because chemotherapy or radiation treatment would induce expression of the EBV-encoded kinases that phosphorylate ganciclovir in a portion of tumor cells, and the phosphorylated ganciclovir could then induce bystander killing in tumor cells where the lytic form of EBV infection was not induced by chemotherapy. This, in fact, appears to be the case; it was recently shown that ganciclovir dramatically enhances the efﬁcacy of both radiation and chemotherapy for the treatment of EBV-positive tumor models in mice [144,145]. 5

SUMMARY

Epstein-Barr virus is a fascinating virus that is linked to a myriad of pathologies that can develop in various cell types. Although there are likely other genetic and environmental factors that also contribute to the development of the disease, the presence of EBV and expression of viral proteins are consistent features. As our understanding of the function of EBV proteins in latent infection, the factors that regulate EBV replication, and the immune response to EBV continues to increase, it is increasingly likely that this information can be used to develop effective therapies that speciﬁcally target the viral infection. ACKNOWLEDGMENTS We gratefully acknowledge the contribution of artwork by Natalie Thornburg, Tem Morrison, and Bruce Israel. REFERENCES 1.

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19 The Human Herpesviruses HHV-6, HHV-7, and HHV-8 Dharam V. Ablashi Georgetown University Medical School, Washington, D.C., and Advanced Biotechnologies Inc., Columbia, Maryland, U.S.A.

Gerhard R. F. Krueger University of Texas Medical School at Houston, Houston, Texas, U.S.A.

1

HISTORICAL ASPECTS OF HHV-6, HHV-7, AND HHV-8

Human herpes viruses HHV-6 and HHV-7 belong to the Roseolaviridae genus of the beta herpesvirus subfamily and were discovered in 1986 [1] and 1990 [2], respectively. Prior to the isolation of HHV-6, the last human herpesvirus reported was EBV in 1966. However, after the documentation of HHV-6, within four years HHV-7 [2] and HHV-8 [3] were discovered. Initially, HHV-6 was designated ‘‘HBLV,’’ because it was isolated from peripheral blood mononuclear cells (PBMCS) from patients with AIDS and other lymphoproliferative disorders. Later on, the designation was changed to HHV-6 [4] to conform to the rules of the International Committee on Taxonomy of Viruses. After the initial isolation of HHV-6 at the National Institutes of Health (NIH), two independent isolates were reported—one known as U1102 from Uganda [5], and the other, called Z-29 by the CDC, from Zambia [6]. HHV-7 was initially isolated from CD4þ T cells of a healthy donor at NIH [2], the second independent isolate of HHV-7 was identiﬁed at NIH in the PBMC 659

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of a chronic fatigue syndrome patient [7]. HHV-8, also known as Kaposi’s sarcoma–associated herpesvirus (KSHV), was discovered by Chang et al. [3] using representational difference analysis. By using this unique molecular approach, herpes viral DNA sequences were detected in lesions from AIDS patients with Kaposi’s sarcoma. KSHV (or HHV-8) is a member of the genus Rhadinoviridae within the human gamma herpesvirus subfamily. HHV-8 is also called a gamma-2 human herpesvirus [8].

2

STRAIN CLASSIFICATION OF HHV-6, HHV-7, AND HHV-8

The GS strain of HHV-6 was the ﬁrst reported [1] and was later designated as the prototype of HHV-6A variants [9]; U1102 was also classiﬁed as variant A. The HHV-6 Z-29 strain was classiﬁed as the prototype of HHV-6B variants [9]. Unlike HHV-6A and 6B, HHV-7 isolates did not show variability; however, a more recent report by Franti et al. [10] showed that on the basis of HHV-7 phosphoprotein P100, glycoprotein B, and major capsid protein, the isolates can be grouped into two major groups (i.e., CO1 and CO2). CO1 isolates are found in Africa and Asia. The CO2 group isolates are found in Europe, North America, and Mongolia. More recently, another group of researchers [11] classiﬁed HHV-7 isolates as variants 1 and 2, based on molecular analysis of gB and gH and R-2 repeat regions. Only the JI strain [7] was representative of variant 1. The rest of the known isolates from America, Europe, Japan, and elsewhere were classiﬁed as variant 2. Regarding HHV-8 strain variants, extensive work has been done by Hayward and colleagues [12]. Based on the analysis of the ORF-K1 and ORF-15 genes of HHV-8, variants were divided into groups A–E. Group B is dominant in Africa, and groups D and E are conﬁned to the populations of the Paciﬁc islands and Amerindians. In North America and Europe, the A and C groups are predominant [8,13]. Whether a particular group or variant of HHV-7 or HHV-8 would be associated with a certain disease is yet to be investigated. However, at least with HHV-6, it is evident that variant B is the causative agent of most of the exanthema subitum cases and also prevalent in transplant recipients. Variant A is consistently found in the cerebral spinal ﬂuids and plasma of multiple sclerosis (MS) and chronic fatigue syndrome patients [14,15]. HHV-6A strains were also predominantly found in AIDS patients with Kaposi’s sarcoma. Moreover, HHV-6A downregulates MHC class 1, but HHV-6B does not [16].

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GENERAL MORPHOLOGY AND MOLECULAR BIOLOGY OF HHV-6, HHV-7, AND HHV-8

The virions of HHV-6, HHV-7, and HHV-8 are 160–200 nm in diameter and have typical morphological features of herpesvirus virions, consisting of a central core, a capsid, and a tegument layer surrounded by a membrane structure. The HHV-6A genome (Fig. 1) has been fully sequenced (strain U1102), and its variant size is 159–170 kilobase pairs (kbp); the HHV-6B genome (strain Z-29) has also been sequenced (Fig. 1). The genes in HHV-6A and B belonging to conserved gene blocks have greater than 94% amino acid identity. There are seven gene blocks in the central region designated as core genes common to all herpesviruses. The genomic architecture of HHV-6 and HHV-7 is unique among human herpesviruses and resembles that of channel catﬁsh virus. HHV-6 genomes contain reiterations of the hexanucleotide (GGTTA) near the end of the DRs and are present in both A and B strains. The function of GGTTA is not known. Both HHV-6A and HHV-6B have been characterized by restriction enzyme analysis as well [17–20]. In addition to showing variability in in vitro propagation in T-cell lines, they also show different reactivities with monoclonal antibodies [21]. HHV-6A and B and HHV-7 are closely related to HCMV, the prototype virus of the human b-herpesvirus subfamily. However, HHV-6 and HHV-7 are more closely related to each other than to HCMV, as shown by their limited serological cross-reactivity, DNA hybridization, and nucleotide sequence similarity [17–19]. The major capsid protein (MCP) of HHV-6A shares 61.3% amino acid identity with MCP of HHV-7 and 43.3% amino acid identity with HCMV but less than 30% identity with EBV (gamma herpesvirus) and HSV-1 (alpha herpesvirus). More extensive details concerning the molecular aspects of HHV-6 and HHV-7 have been reviewed [17–20]. The genome of HHV-7 is 145 kbp (Fig. 1) with a G þ C content of 55%. The sizes of different HHV-7 strains have been estimated to be 140–150 kbp, which is probably due to the variation of the heterogeneous sequences located at the genome termini. Restriction enzyme pattern analysis of saliva isolates of HHV-7 indicate that HHV-7 strains are well conserved with the exception of the ‘‘het region’’ [22,23]. However, as described above, they have recently been reclassiﬁed into two groups [10,11]. Several lines of evidence suggest that HHV-7 DNA is very stable both in vitro and in vivo. This observation was based on the het sequences of speciﬁc strains [22]. The genome obtained from puriﬁed HHV-8 virions has a size of 165–170 kb (Fig. 1) [24]. Like EBV, the latent HHV-8 genome has a circular conformation, but the active DNA is linear

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Figure 1 (Top) Structures of the HHV-6 (A and B variants) genomes. The genomic size is 159–170 kbp. There is 94% amino acid identity between A and B strains. The ﬁgure shows the genomic variation of HHV-6A (strain u1102) and HHV6B (strain z-29). (Middle) The genomic structure of HHV-7 (strain J1). Its genomic size is 145 kbp. HHV-7 strains have been estimated to be 140–150 kbp, perhaps due to the variation of the heterogeneous sequences located at the genome termini. (Bottom) The genomic structure of HHV-8 (KSHV), which is 165–170 kbp. The HHV-8 genome has a circular conformation, but the active DNA is linear during replication. The HHV-8 genome contains close to 100 open reading frames (ORFs), some of which are unique to HHV-8.

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during replication [25]. It has been found that a number of viral genes are responsible for the pathogenesis of Kaposi’s sarcoma (KS): K1, K2, VMIPS, K4, K4.1, K5, K9, K12, ORF-6, ORF-71, ORF-74, and K15 [26]. 4

BIOLOGY OF HHV-6

Human herpesvirus-6 (HHV-6) readily infects CD4þ cells and leads to degeneration of these cells. The infection and replication of HHV-6A and B were also shown in a variety of human T-cell lines (i.e., HSB2, MoLT-3, Jihan, SupT1, and PHA-stimulated PBMC) [17–19,21]. HHV-6 has also been shown to be present in monocytes/macrophages [17,18]. In addition, HHV-6 can also infect B cells, neural cells, and, to some degree, human ﬁbroblasts. An HHV-6A and B cellular receptor (CD46) was recently identiﬁed [27], and its ubiquity is in keeping with the apparently broad cellular tropism of HHV-6 [18]. The propagation of HHV-6A and B in T-cell lines varies considerably [21]. HHV-6 can be propagated in the immature T-cell line HSB2, and HHV-6B grows to a high titer in MoLT-3 cells (fully matured T cells). Human umbilical cord mononuclear cells (HCBMCs) grow both strains to a higher titer than established cell lines. However, these cells have to be PHA-stimulated prior to infection. For viral isolation, HCBMCs have generally been used instead of established cell lines. Typically, infected cells show multinucleated giant cells that are enlarged by two-to ﬁvefold and generally have a ballooning appearance [1]. When such cells are tested with HHV6 speciﬁc monoclonal antibody, they show localization of antigens in the nucleus as well as in the cytoplasm. The infected lymphocytes tend to aggregate in small-to-medium clusters. Even though morphological changes indicate a high degree of infection, the yield of infectious virus is lower than expected, suggesting that the majority of the virus particles are noninfectious. The virus has also been reported to induce apoptosis in uninfected bystander cells—CD8þ and T lymphocytes as well as natural killer (NK) cells [18]. Moreover, HHV-6, particularly variant A, has been shown to induce CD4þ expression in infected cells such as HSB2, NK, and CD8þ T cells [17,19]. The genes responsible for transactivation of the CD4þ promoter include U86 and U89 [28]. HHV6 is a potent inducer of IL-1b, TNF-alpha, and IL-6 [29] as well as IL-10 and IL-12 expression [30] in monocytes/macrophages. Virus infection downregulates CD3 in infected T cells, which probably alters immunemediated transmembrane signaling [18]. Furthermore, infection of PBMCs has been found to suppress T-cell function, including reducing IL-2 synthesis and cell proliferation [31]. Downregulation of CD46 has also been observed following HHV-6 infection [27].

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CLINICAL PATHOLOGY OF HHV-6

A variety of clinical symptoms and diseases are presumed to be associated with HHV-6 infection [18,32–34], although a direct causal relationship between infection and disease is frequently hard to prove. Considering viral effects, primary infections need to be distinguished from reactivated ones. HHV-6 type A must be separated from HHV-6 type B. And ﬁnally—similarly to EBV—eventual pathological sequelae of latent (i.e., nonreplicative) HHV-6 should occasionally be entertained (e.g., via NF-kB activation or defective viral antigen expression and molecular mimicry [35,36]). Considering the host’s reaction in HHV-6 infection, a direct lytic effect of the virus itself for infected target cells needs to be distinguished from virus-induced inﬂammatory and immune (or autoimmune) reactions. These can be mediated by a large variety of HHV-6-induced or altered cytokine and chemokine patterns as well as by modulation of cell membrane receptors [16,29–31,37–46]. Target cells for HHV-6 infection possess CD46 as part of the viral receptor [27,47]. CD46 is strongly expressed on epithelial cells of salivary gland ducts and on kidney tubular cells, moderately well on lymphocytes and vascular endothelial cells, and weakly on cells of the interstitial space and on muscle cells. It may also be present on various tumor cells [48]. CD46 participates in downregulating complement activity (binding of C’3b and C’4b); thus the interaction of CD46 with HHV-6 may interfere with this function and cause inﬂammation via the alternative complement pathway. The tissue distribution of CD46 suggests what may be targets for HHV-6 infection and immediate virus-induced pathology: salivary glands, lymphoid tissues, the vascular system, kidneys, and apparently also neuroglial cells in the central nervous system. Damage to primarily noninfectable cells and tissues must therefore originate from indirect effects of viral infection such as complement activation and immune and/or autoimmune reactions. Although salivary glands appear to be primary targets of HHV-6 infection and sites of latent persistence of the virus [49,50], they rarely show signs of acute disease (sialoadenitis or tumors). Similarly, HHV-6 antigen can be shown in renal tubular cells, yet their infection results in practically no kind of nephritis. Lymphoid tissues instead may reveal a variety of inﬂammatory and proliferative diseases following HHV-6 infection or reactivation. There are also increasing numbers of publications relating vascular diseases and necrotizing or demyelinating brain diseases to HHV-6 infections. These examples clearly show that the presence of virus or its antigens in tissues does not automatically imply a

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virus-induced disease. A critical evaluation of all clinical, immunovirological, and pathological parameters is necessary, therefore, before assuming that a certain disease is induced by HHV-6 (the same pertains to HHV-7 infections to be discussed later). There are only a few diseases in which primary HHV-6 infection plays a causative role: certain infantile febrile diseases with or without convulsions, exanthem subitum, heterophile antigen negative infectious mononucleosis, and possibly Kikuchi-Fujimoto’s disease. There are larger numbers of disorders to which persistent or reactivated HHV-6 infections may be contributory, yet a direct pathogenetic connection remains frequently suggestive rather than proven. Such diseases include chronic fatigue syndrome (CFS), certain autoimmune and lymphoproliferative diseases, and demyelinating diseases of the central nervous system [34,51]. Finally, there are sporadic reports of other supposedly HHV-6-related disorders. All are brieﬂy discussed according to the organ systems affected. 5.1

Systemic Appearances

General clinical symptoms in HHV-6 infection are similar to an inﬂuenza-like disease with fever, sweats and chills, fatigue, malaise, occasional convulsions, and exanthema (the latter two preferentially in children [52]). There may be arthritic signs and iridocyclitis [53]. Postinfectious chronic fatigue syndrome (CFS) occurs in a certain percentage of adult persons with reactivated and/or persistent HHV-6 infection [54,55]. Although this has been repeatedly challenged by some authors [56,57], HHV-6 has been isolated from patients with CFS as deﬁned by CDC criteria [15,55]. HHV-6 infection only rarely causes acutely fatal systemic disease [58]. 5.2

Skin and Appendages

The most frequent manifestation of acute primary HHV-6 infection is exanthem subitum (ES; also known as roseola infantum) in babies and young children [59,60]. Children suffer from ES, usually during their ﬁrst year of life, with up to 4 days of fever, a macular/papular rash on face and/or trunk, pharyngitis and cough, lymphadenopathy, occasional convulsions, and mild diarrhea. Antibody titers against HHV-6 (IFA) rise after about 1 week with IgM up to 1:40 followed by IgG peaking at up to 1:2560 a month later. Virus, preferentially yet not exclusively HHV-6B, can be isolated from peripheral blood mononuclear cells (PBLs) during the ﬁrst week of illness. Patients usually recover rapidly and completely.

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In addition to ES, nonspeciﬁc exanthems were observed repeatedly in HHV-6 infection associated with infectious mononucleosis or CFS and in bone marrow allograft recipients (graft-versus-host disease excluded [61]). 5.3

Lymphatic and Hematopoietic System

Next to ES, the disease most closely related to acute primary HHV-6 infection is Kikuchi-Fujimoto’s disease (KFD) or histiocytic necrotizing lymphadenitis [62,63]. KFD is a self-limited subacute lymphoproliferative disease that usually affects young adults. Its onset bears certain similarities to that of Hodgkin’s disease, yet proliferating lymphoid cells ﬁnally undergo massive apoptosis with histiocytic reaction leading to recovery. Most frequently affected are neck lymph nodes, with a distribution similar to that of infectious mononucleosis. Virus antigen and DNA (preferentially HHV-6A) can be easily shown with various methods in these lymph nodes. About 6% of heterophile-negative infectious mononucleosis (IM) cases are due to HHV-6 infection [33,64,65]. Both subtypes of the virus, HHV-6A and HHV-6B, can be found in such patients. More frequent, however, is the reactivation of latent HHV-6 in patients with classical EBV-induced IM, which may result in a somewhat more protracted course of the disease with elevated liver enzymes [66]. IM represents another example of a benign lymphoproliferative disorder with spontaneous recovery that affects predominantly young adults. Among other benign diseases of the lymphatic and hematopoietic tissues, which may be occasionally (and not exclusively) caused by HHV-6, preferentially variant B, are angioimmunoblastic lymphadenopathy (AIL) [67,68] and hemophagocytic syndromes [69,70]. In addition, there is an interesting publication relating HHV-6 to Langerhans cell histiocytosis [71], which deserves further attention. Because HHV-6 was initially isolated from patients with malignant lymphomas [1], and all of the above diseases include a lymphoproliferative response, the question arises as to whether HHV-6 may be causing or somehow assisting in the development of malignant lymphomas. Unfortunately, this topic has been discussed quite controversially without a search having been made for a comprehensive approach across specialties. Reactivated HHV-6 (preferentially subtype A) and/or increased viral DNA loads were found in certain malignant lymphomas including subtypes of Hodgkin’s disease [72,73] and a few non-Hodgkin’s lymphomas (i.e., large-cell T- or B-cell type, one Burkitt’s lymphoma [74–78]) as well as in a number of prelymphomatous

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‘‘atypical polyclonal lymphoproliferations’’ (APLs) that progressed to malignant lymphoma [74]. There are many more publications available than the few cited here. Some authors, however, have refuted their own previous diagnosis of an eventual causal relationship to HHV-6 infection [79,80]. Because immediate oncogenic effects of the HHV-6 genome, although once suggested [81,82], have not been proven so far, there may well exist other mechanisms through which the virus may contribute to atypical lymphoproliferation [63,83]. Besides its potential relation to lymphoproliferative disorders, HHV-6 apparently has other effects on the lymphohematopoietic tissues that are of more immediate importance: Both subtypes can infect hematopoietic progenitor cells and exert suppressive effects on the engraftment of these cells in transplant recipients [84–87]. In addition to failure of engraftment, lymphocytopenia, suppression of myelopoiesis, or erythrocytopenia may follow HHV-6 reactivation [88–90], and the virus can even be transmitted by the graft itself [91]. HHV-6 reactivation also occurs frequently in patients with allotransplants other than bone marrow or stem cells, yet pathological sequelae appear to threaten only when they coincide with active cytomegalovirus infection [92]. Besides these structural changes in the lymphoid tissues following HHV-6 infection, the virus, when reactivated and/or persistent, may cause obvious functional disturbances of the immune system. Antibody titers against HHV-6 rise signiﬁcantly in allergies, drug-induced hypersensitivity reactions, and certain autoimmune disorders such as systemic lupus erythematosus, Sjogren’s syndrome, and progressive systemic sclerosis [33,93–96], and the virus may constitute a risk factor for additional immune dysregulation and for increasing the severity of the adverse reaction [97,98]. 5.4

Central Nervous System

Human herpesvirus-6 can replicate with low efﬁciency in certain neuroglial cells [99]. Viral DNA and antigen have been successfully demonstrated in human brain tissue, in healthy organs as well as in diseased tissues, with subtype A being about three times as frequent as subtype B [100]. The central nervous system was suggested to be another site for persistence of HHV-6 after primary infection [101]. There is suggestive but not yet conclusive evidence for a pathogenetic role of HHV-6 in necrotizing and demyelinating brain diseases in immunodeﬁcient patients and in persons suffering from multiple sclerosis [15,102– 108].

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Cardiovascular System

Because vascular endothelial cells and muscle cells express to a certain extent the HHV-6 receptor molecule CD46, such cells may become infected and theoretically undergo virus-induced pathology. HHV-6 infection and replication were indeed shown in endothelium of the aorta and of the umbilical vein [100,109,110]. In striking contrast to the widespread distribution of such cells, however, is the rarity of suggested associations of HHV-6 with cardiovascular diseases. There are single reports relating HHV-6 infection to leukocytoclastic vasculitis and to Kawasaki’s disease [111,112]. HHV-6 genomic material was found in coronary arteries of heart allografts [113], suggesting viral reactivation at this site. Finally, a case of fulminant myocarditis was described in a patient receiving steroid therapy for hepatitis. HHV-6 antibodies showed a fourfold increase in this patient, and HHV-6 DNA was demonstrated by PCR in liver and heart tissue [114]. 5.6

Other Organs and Tissues

Among other diseases described in association with active HHV-6 infection were interstitial pneumonitis [115,116] and fulminant hepatic failure with HHV-6 present in liver tissue and in portal vein endothelium [117]. Finally it should be mentioned that HHV-6 can apparently activate other viral infections such as Epstein-Barr virus, HIV-1, measles, and papillomavirus and parvovirus infections and may thus contribute indirectly to the pathological effects of these viruses [37,41,118–125]. Dual active infections appear especially frequently with other herpesviruses (CMV, EBV, HHV-7) as well as with HIV-1. 6

BIOLOGY OF HHV-7

The RK strain of HHV-7 was initially isolated from CD4þ T-cell-enriched PBMCs [2], and its replication in these cells suggests that it has CD4þ tropism. These data are further supported by the fact that HHV-7 (JI strain)-infected PBMCs are largely CD4þ T cells (42%) with approximately 4% CD8þ T cells. More recent evidence also suggests that CD4 is a critical component of the cellular receptor for HHV-7. In addition, these studies show that there is selective CD4 downregulation during HHV-7 infection [22,23]. Furukawa et al. [126] showed that during HHV-7 infection there is less surface CD4 expression on the infected cell. Speciﬁcally, it was shown that preabsorption of uninfected CD4þ T cells with HHV-7 resulted in inhibition of HIV-1 replication, a ﬁnding that

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might have clinical relevance. Over the years, many attempts have been made to infect T-cell lines with HHV-7 strains, but initially without success. The exception was SupT1 cells (an immature T-cell line). SupT1 cells were able to replicate HHV-7 regardless of the strain used [127]. Typically, infected SupT1 cells become extremely enlarged, often vacuolated, and are multinucleated. HHV-7 antigens can be detected in these cells by the use of HHV-7-speciﬁc monoclonal antibody [127]. Not only is HHV-7 more cell-associated than HHV-6, it also seems that HHV7 cell tropism is more restricted than HHV-6. 7

CLINICAL PATHOLOGY OF HHV-7

The clinical pathology of HHV-7 infections most closely resembles that of HHV-6 infection, although the course of the diseases is usually milder [128–131]. Similar to HHV-6, it is frequently difﬁcult to establish a direct causal relationship between HHV-7 infection and disease, and in many cases the virus is probably a cofactor rather than an etiological agent; it functions through interference with the cytokine/chemokine network and cellular receptor expression [45,126,132–138]. In addition, HHV-7 may activate other latent viruses, preferentially other herpesviruses, and enhance the pathogenicity of these viruses [139–142]: this was demonstrated especially in renal transplant recipients, where active HHV-7 infection constitutes a risk factor for more severe CMV-induced disease. Target cells for HHV-7 infection possess the CD4 cell membrane receptor, although CD4 itself appears not be the only molecule for virus binding and internalization of viral particles [143–145]. HHV-7 replicates in activated CD4þ T lymphocytes and has also been shown to be present in many other normal cells and tissues without signs of disease including hematopoietic progenitor cells, macrophages, salivary gland cells, cells in the gastric mucosa, and normal brain tissue [146–150]. This tissue distribution identiﬁes potential targets for HHV-7-induced disease, although, as in HHV-6 infection, other sites may become involved secondary to virus-mediated effects by the action of cytokines, chemokines, molecular mimicry, and autoimmune reactions. Diagnostic criteria for relating HHV-7 infection to diseases must be applied with care, as described for HHV-6 infection. 7.1

Systemic Appearances

General clinical symptoms in HHV-7 infection are those of a nonspeciﬁc inﬂuenza-like or mononucleosis-like febrile illness with occasional skin rash, anorexia, irritability, pharyngitis, cervical lymphadenopathy, and

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mild diarrhea [128–131]. Other patients may suffer from chronic fatigue syndrome (CFS)–like syndromes associated with HHV-7 reactivation [151].

7.2

Skin and Appendages

Human herpesvirus-7 (HHV-7) was identiﬁed as the likely causal agent in a few cases of HHV-6-negative exanthem subitum (roseola infantum) [152–155]. In other dermatological diseases such as atopical dermatitis, psoriasis, and pityriasis rosea, HHV-7 infection was occasionally suggested, yet its etiological importance remains questionable [61,156– 158]. There is one report identifying HHV-7 infection in an adult patient with Sweet’s syndrome and necrotizing lymphadenitis [159]. Sweet’s syndrome is a rather rare acute febrile neutrophilic dermatosis with sometimes painful papulonodules or pustules of unknown cause. Its possible relation to HHV-7 infection needs further conﬁrmation. Finally, HHV-7 has been suggested but not conﬁrmed as the etiological agent in lymphomatoid papulosis, a T-cell lymphoproliferative disorder of the skin [160].

7.3

Lymphatic and Hematopoietic System

Frequently present in rather low DNA copy numbers in normal lymph node and bone marrow from healthy individuals, HHV-7 shows increased expression in immuno deﬁcient AIDS patients, for example [161–163]. Although HHV-6 exerts suppressive effects on bone marrow stem cell proliferation, HHV-7 does not. Occasional febrile lymphadenopathy in primary or reactivated infection shows histologically a nonspeciﬁc hyperplasia of T and B zones (follicular and paracortical hyperplasia) with prominent postcapillary venules, focal edema, and focal areas of lymphocyte apoptosis. A very few pronounced cases may resemble infectious mononucleosis-like clinical and pathological changes [131,164]. HHV-7 has been frequently demonstrated in various lymphoproliferative diseases such as Hodgkin’s lymphoma, cutaneous T- and Bcell lymphomas, and primary ocular lymphoma, yet an etiological role of the virus in the development of such lymphomas is rather doubtful [165– 169]. It does not exclude, however, that these lymphotropic viruses (HHV-6 and HHV-7) may inﬂuence the course of such diseases by interfering with the normal immune response, cell proliferation, and cell differentiation [63,83]. Such aspects deserve more intense investigation.

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Central Nervous System

Children with acute primary HHV-7 infection may suffer from febrile seizures, yet the virus is not necessarily isolated from their spinal ﬂuid [170–172]. Severe neurological symptoms occasionally may accompany HHV-7-associated exanthem subitum and are suggested to represent viral encephalopathy due to viral invasion of the brain [173]. In children and young adults with positive PCR tests for HHV-7 in their cerebrospinal ﬂuid, there are also isolated cases of meningoencephalitis as well as occasional facial palsy and vestibular neuritis [174]. Such complications appear to occur more frequently in company with other viral infections including HIV-1 infection and associated immunodeﬁciency [175,176]. Like HHV-6, HHV-7 is frequently reactivated in patients with demyelinating diseases such as multiple sclerosis (MS) without conclusive evidence so far of its etiological role in these diseases [177,178]. There is also no indication for a causal role of HHV-7 in primary brain tumors [179]. 7.5

Other Organs and Tissues

Similar to situations of virus-induced immunosuppression, HHV-7 reactivation is frequently observed in recipients of organ transplants including allografts of the kidney, liver, and lungs as well as allogeneic and autologous stem cell transplants [180–182]. Although itself not pathogenic, HHV-7 obviously increases the risk for CMV-induced pathology in transplanted organs [183,184]. In lung allografts HHV-7 may cause bronchiolitis obliterans and pneumonitis [185]. HHV-7 is frequently isolated from saliva, and the salivary gland is thought to be a major site for viral persistence in latency [186–188]. HHV-7 antigens are frequently found by immunohistochemical techniques in salivary gland epithelia [189]. HHV-7 may be reactivated in immunocompromised patients, but pathological changes in salivary glands directly associated with HHV-7 are usually not identiﬁed. Finally, there is some indication that HHV-7 may cause liver dysfunction and hepatitis, but more conclusive studies are needed [190,191]. Like HHV-6, active HHV-7 was described in cases of Kawasaki’s disease (leukocytoclastic vasculitis [192]), which may be a virus-induced, immunologically mediated disorder, not speciﬁc for one individual virus. 8

BIOLOGY OF HHV-8

Human herpesvirus-8 (HHV-8) has been detected in tissues from KS and multicentric Castleman’s disease (MCD) by PCR and from primary

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effusion lymphoma (PEL). Thus far it has been difﬁcult to obtain infectious HHV-8 from MCD or KS. HHV-8 can, however, be obtained from PEL. Various PEL lines have been established that carry HHV-8 virions that, when treated with chemicals such as PMA or butyrate produce virus particles in large numbers. The inducibility of different PEL cell lines varies considerably (from 20% to more than 70% induction). The KS-1 cell line is known to express abundant virions after 72 hr (> 70%), and most of the major lytic proteins are expressed after induction [8]. Despite the high expression of viral proteins in KS-1 cells, the yield of virus is between 109 and 1010 per mL, and a considerable number of the virions are defective. Some of the better known PEL lines are BCBL, BC-1, BC-3, BCP-1, JSC-1, and KS-1. PEL cell lines express CD45 but no other B- or T-cell markers. HHV-8 from PEL cell lines has been transmitted to primary and secondary cells. HHV-8 from KS lesions has also been propagated in embryonic kidney 293 cells [193], but there was limited transmission and only viral DNA was detected by PCR. HHV-8 is a transforming virus [194]. Microvascular endothelial cells transformed with papilloma virus type 16 E6 and E7 genes were found to be permissive for HHV-8 derived from BCBL-1 cells. Primary cultures of monocytes/macrophages from patients with AIDS were also permissive for HHV-8 infection. Nontransformed primary fetal dermal microvascular endothelial cells derived from large blood vessels or capillaries have also been infected with HHV-8 obtained from JSC-1, BC3, and BCP-1 cell lines [195,196]. Infection of primary bone marrow cells with HHV-8 obtained from BC-3 cells led not only to increased long-term proliferation and survival but also to the acquisition of telomerase activity and anchorage-dependent growth [194]. Approximately 3 months later, <5% of the cells expressed LNA, but the phenotype of these cells was distinguishable from that of the primary uninfected cells. HHV-8-associated malignancies (KS, MCD, PEL) express a unique protein called kaposin, which is encoded by an abundant latent-cycle transcript of 0.7 kb, T0.7 or HHV-8 ORF-K12 [197]. The kaposin gene induced tumorigenic transformation in transfected Rat-3 cells that contained kaposin mRNA LNA-1 (ORF-73), the protein most frequently identiﬁed in KS tumor cells [8]. It has also been found in HHV-8 transformed primary rat embryo cells. Heparin sulfate has been found to be a receptor that interacts with the K8.1 envelope protein [198]. In some ways HHV-8 is unique in this group of human herpesviruses, because some genes are expressed only by HHV-8 [26]. HHV-8 also encodes homologous cytokines and cytokine response genes, e.g., KILG. HHV-8 also contains ORFs homologous to

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cellular oncogenes involved in lymphogenesis [24]. These are viral cyclin D (ORF 72), which is homologous to the BCL-1 gene and the viral BCL-2 (ORF 16). The cellular and molecular biology of HHV-8 has been reviewed in more detail by many authors [8,13,199–201]. 9

CLINICAL PATHOLOGY OF HHV-8

Since the detection of HHV-8 in Kaposi’s sarcoma tissues from AIDS patients [3,200], the search for the pathogenesis of HHV-8 in other diseases has continued. Some of the evidence of HHV-8 in malignancies such as KS (all forms), PEL, and MCD has been consistent. The evidence has been supported by molecular biology, i.e., the presence of viral DNA detected by PCR (RT-PCR or in situ hybridization, or real-time PCR), and by immunological methods such as serology. On the other hand, there have been some reports of involvement of HHV-8 in diseases such as multiple myeloma, Kikuchi disease, Bowen’s disease, and salivary gland tumor [8]. Involvement in HIV-infected patients having interstitial pneumonitis or pemphigus vulgaris has been disputed because either there was only a single report or solid evidence for the presence of HHV8 was lacking. Infectious mononucleosis–like illness has been reported with an HHV-8 [5] primary infection. This ﬁnding is similar to those of infections caused by EBV, CMV, HHV-6, and HHV-7. HHV-8 DNA was reported in non-KS lesions in transplant recipients, but this was in only a single report [8,13,199–201]. 9.1

Kaposi’s Sarcoma

Moriz Kaposi reported in 1872 a condition consisting of multifocal pigmented sarcoma occurring in elderly Viennese men [202]. These patients exhibited cutaneous lesions, typically on the lower extremities. All of these patients eventually died of the disease, which is now known as classical Kaposi Sarcoma (KS) and is predominantly observed in older men of Mediterranean and Eastern European origin. Later, three other forms of KS were recognized, i.e., endemic, iatrogenic, and a very aggressive type identiﬁed in otherwise healthy homosexual men [8]. The endemic KS, slightly more aggressive than classical KS and involving lymph nodes in addition to skin lesions, is common in Africa, often affecting HIV-negative adults and children. The iatrogenic form of KS occurs after solid organ transplantation in patients on immunosuppressive medication. The most aggressive type of KS, occurring in homosexual AIDS patients, involves the skin as well as lymph nodes, often spreading to the lung, gastrointestinal tract, liver, and spleen. The

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skin lesions are purple and widespread over the body [8]. All four clinical/epidemiological forms of KS are indistinguishable histologically. KS is composed of a mixture of ectatic irregularly shaped round capillary and slitlike endothelium-lined vascular spaces and spindle-shaped cells. Red blood cells and hemosiderin pigment are frequently observed. The spindle cells become the predominant cell population, forming fascicles that compress the vascular slits, and the lesions become progressively nodular [8,13,199–201]. The early KS lesions contain few spindle cells compared to the surrounding inﬂammatory cells. Cultured KS cells are dependent upon exogenous growth factors. When implanted in nude mice, they can stimulate the production of inﬂammatory and angiogenic molecules but do not induce tumors one would expect of fully transformed cells. More recent studies revealed that KS can occur with varying monoclonality, oligoclonality, and polyclonality in different patients [203]. Neoplastic cell lines are often established from KS lesions. The likely possibility is that KS starts as a hyperplastic polyclonal lesion that later gives rise to clonal cells only under speciﬁc circumstances, such as immunosuppression. KS may be similar to post-transplantation lymphoproliferative disorders, which are driven to progress by polyclonal hyperplasia. All four forms of KS showed the presence of HHV-8 or KSHV by PCR in PBMCs. KS lesions and plasma from patients contain HHV-8 primer sequences. Furthermore, serological evidence indicated that KS patients contain antibody to HHV-8 [8,13,199–201]. Although the indication of an involvement of HHV-8 in the pathogenesis of KS is quite strong, other cofactors such as cytokines are necessary for the lesions to develop. 9.2

Primary Effusion Lymphoma

The ﬁrst reported cases of malignant lymphoma appearing as body cavity lymphoma were described as AIDS-associated lymphohematopoietic neoplasm exhibiting an indeterminate immunophenotype [204]. Of three early cases described, two showed lymphomatous effusions, a B-cell lineage, and the presence of EBV footprints. Subsequent studies recognized that these lymphomatous effusions occur relatively frequently in HIV-infected individuals. These were, however, unusual AIDS-related cases, because a quite distinct clinicopathological entity exhibits the presence of HHV-8 or KSHV. PEL are even rarer lymphomas, and some PELs had both EBV and HHV-8 DNA. HHV-8 is not found in other AIDS-related non-Hodgkin’s lymphomas. Primary effusion lymphoma contained very large amounts of viral DNA, ranging

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between 40 and 80 copies per cell, so the virus was easily identiﬁed by Southern blot analysis. This is in contrast to KS tissues, some of which contain one copy per cell or less. Primary effusion lymphoma occurring in non-HIV-infected men and women has also been reported to contain HHV-8 [205–207]. In most cases, PEL histopathological criteria include the presence of effusion in the pleural cavity of origin in 80% of cases and a morphology bridging large-cell immunoblastic lymphoma and neoplastic cell lymphoma (100%). The histology was recently described in detail by Ablashi et al. [8]. These tumors express CD45 and one or more activation-associated antigens (95%) and immunoglobulin expression (79%). B-cell origin is demonstrated by the presence of clonal immunoglobulin gene rearrangements (97%). There is no CMYC gene rearrangement as is observed in Burkitt’s lymphoma, and PEL also lacks bcl-2, ras, and P53 gene alteration (87%). An additional complication of the PEL is that lymphomas containing HHV-8 can present as a solid tissue mass, usually extranodally, similar to that in AIDS-related non-Hodgkin’s lymphoma. These are often diagnosed as diffuse large-cell lymphomas in which the presence of HHV-8 could be detected in practically all the cells by in situ hybridization. These lymphomas lack expression of B-cell antigen and immunoglobulin and have an immunoblastic or anaplastic morphology. These are usually infected with EBV. Epidemiological evidence showed a clear association between the presence of KS and immunoblastic lymphomas in patients with AIDS [208]. PEL containing HHV-8 comprise approximately 5% or less of all AIDS-related lymphomas. 9.3

Non-PEL Cases

Additional cases of lymphomas, which do not have the features of PEL containing HHV-8, have been identiﬁed [209]. Also, a case of Burkitt’s lymphoma having no EBV was found by PCR to contain HHV-8 in a child who had IgG antibody to HHV-8 lytic antigens at 1:640 titer. The spectrum of HHV-8-associated lymphomas will therefore expand and will need conﬁrmation by molecular as well as serological means. PEL cell lines have been established and contain HHV-8 genome (LNA-1), as detected by PCR and antibody to ORF-73 protein (LNA-1 latent protein). The cell lines are inducible to express lytic proteins and are a source of infectious virus. PEL cell lines have numerous abnormalities, e.g., trisomy7m, trisomy12, and aberrations of chromosome bands 1q21– q25. Translocations speciﬁc to other lymphoma types have not been identiﬁed [8].

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Multicentric Castleman’s Disease

Human herpesvirus-8 (HHV-8) has been detected in multicentric Castleman’s disease (MCD) [25]. MCD is a poorly understood, atypical lymphoproliferative disorder thought to be related to immune dysregulation. MCD has been described as a polyclonal, neoplastic disorder. It is found in two distinct histological types with different clinical manifestations. The more common type is the hyaline vascular form, which presents a solitary mass that is usually curable surgically. The other type is the plasma cell form, which is associated with more generalized lymphadenopathy and immunological abnormality. This disorder occurs more commonly in older men. The patients usually present multiple lymphadenopathy, thus giving rise to the name multicentric Castleman’s disease, and a variety of constitutional symptoms. They may develop autoimmune phenomena, cytopenias, skin rashes, and intercurrent infection. Patients with MCD usually develop malignancies, most commonly KS and non-Hodgkin’s lymphoma [210]. Human herpesvirus-8 is present in most cases of MCD that occur in AIDS patients. The detection of HHV-8 in AIDS and non-AIDS MCD patients supports a closer relationship between MCD and KS than was previously thought. HHV-8 may, in fact, play a crucial role, although other lymphotropic viruses such as EBV and HHV-6 have also been identiﬁed in non-AIDS-related MCD [74,211,212]. It has been found that HIV-positive MCD patients develop KS more frequently than others. MCD has also been called multicentric angiofollicular hyperplasia, because it is characterized by vascular proliferation in the germinal centers, reminiscent of KS. Interestingly, the germinal centers of hyperplastic lymph nodes produce large quantities of IL-6. As mentioned before, HHV-8 encodes a viral IL-6 homolog, which is also expressed in MCD in scattered cells surrounding the lymphoid follicles [213]. HHV-8 has, moreover, been found in mantle zone large immunoblastic B cells by using anti-IRF-73 antibody. These cells are monotypical for the lambda chain and give rise to monoclonal expressions and frank lymphomas called plasmablastic lymphomas, which are distinct from PEL. 9.5

Is HHV-8 Involved in the Pathogenesis of Other Diseases?

The evidence for the involvement of HHV-8 in the pathogenesis of other diseases is weak [8]. Further studies are needed to assess the role of HHV-8 in such diseases. Pemphigus vulgaris and pemphigus foliaceus are autoimmune diseases of the skin characterized by separation of the

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dermis and epidermis. KS is a frequently reported lesion in pemphigus vulgaris patients. HHV-8 DNA has been localized in skin lesions, but some reports failed to show association of HHV-8 infection in pemphigus [214]. The coexistence of KS and sarcoidosis in a lesion from an HIV-negative patient revealed HHV-8 DNA; however, the occurrence of a high frequency of HHV-8 has been attributed to PCR cross-contamination. The most controversial involvement of HHV-8 in multiple myeloma (MM) generated several reports, some in favor of HHV-8 being involved in the pathogenesis of MM. The evidence was based on molecular ﬁndings (PCR). On the other hand, none of the reports found HHV-8 antibodies in MM [8]. Because monoclonal gammopathy of undetermined signiﬁcance (MGUS) usually progresses to MM, Ablashi et al. tested sera from MGUS patients, some of whom progressed to MM. There was no serological evidence that MGUS serum supports HHV-8 antibodies. It is possible that a distinct HHV-8-like virus, yet to be conﬁrmed in MM, has homology to HHV-8 molecularly but is serologically distinct from HHV-8 [8]. 9.6

HHV-8 Association in Transplant Patients

Infection with HHV-8 has been detected in transplant recipients of bone marrow and kidney [215]. These patients developed KS and/or PEL. The risk of developing HHV-8-associated malignancy in transplant recipients is high because these patients are immunosuppressed, regardless of whether HHV-8 came from the donor or the patient had latent HHV-8 that became reactivated. HHV-8 DNA was found in kidney allografts after transplantation, and the presence of HHV-8 antibody indicated that these patients would develop KS. Monitoring of these patients for HHV8 after transplantation would therefore certainly aid in antiviral therapy and may prevent the development of KS or PEL [8].

10 10.1

EPIDEMIOLOGY OF HHV-6, HHV-7, AND HHV-8 HHV-6 Prevalence

Based on serological studies, HHV-6 is ubiquitous in general populations, with at least 90% of adults being seropositive [17–19,51]. It is difﬁcult to assess the infection rate of HHV-6A and B because the existing serological tests are unable to distinguish between the antibodies to HHV-6A and those to HHV-6B. A serological evaluation of 234 sera from Malaysians of diverse ethnic origins revealed 4.7% differences in IgG antibody titers to HHV-6A and B [216]. It has also been reported that

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HHV-6B infection is predominant in children; in Zambia 44% of the children are infected with HHV-6A [217]. Seroepidemiological studies of HHV-6 in children show that HHV6 antibody can be detected in 67% of the children aged 0–5 months, compared to 83% in children aged 6–11 months and 92% in those aged 2–8 years. Thus there is an increase of HHV-6 antibody with age [218]. Infection with HHV-6 usually occurs within the ﬁrst two years of childhood. Maternal antibodies are present in the neonates and decline to undetectable levels within 6 months; from the age of 6 months onward, infants are susceptible to HHV-6 primary infection. HHV-6 DNA was detected in 87% of children aged 1 year or older, and it increased with age. In children with exanthema subitum, HHV-6B DNA continued to increase. A high DNA copy number was prevalent not only in the acute phase but also in the convalescent phases of the exanthema subitum. Although the seroprevalence of HHV-6 is approximately 60–90% in the adult population from Tanzania, Malaysia, Thailand, and Brazil, no signiﬁcant ethnic differences have been found between natives and Brazilian and Japanese immigrants. One study reported a modest increase in the seroprevalence of HHV-6 in females compared to males, but another study did not ﬁnd any gender differences. Reports from European countries and the United States showed that the seroprevalence of HHV-6 ranged between 72% and 95% in adults and children. In addition to serology, molecular techniques have been employed to assess the HHV-6 infection rate, e.g., restriction fragment analysis, PCR, PCR-RFLP, PCR in situ hybridization, and reactivity of HHV-6speciﬁc monoclonal antibodies [17]. Examination of different anatomical sites by PCR showed that HHV-6B was more prevalent in the saliva. Therefore, salivary contact may be the major source for the spread of HHV-6B. Furthermore, HHV-6B is more frequently detected in the PBMC of healthy donors. On the other hand, true prevalence of HHV6A is underestimated. Coinfection with both HHV-6A and B was detected in 22–34% of lung specimens examined [219]. HHV-6A was also detected at a high frequency in healthy skin biopsies and cerebral spinal ﬂuid from MS and CFS patients [220]. The prevalence of HHV6A and B was also assessed in PBMC and saliva from healthy donors from Austria. Among 44 healthy adult donors, HHV-6B DNA was detected by nested PCR in 98% in the PBMC, and 95% secreted HHV-6B in the saliva. These studies emphasize that saliva is the route of transmission of HHV-6B, but the route of transmission of HHV-6A remains unknown [18,19].

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HHV-7 Prevalence

In comparison with HHV-6, children are infected with HHV-7 later in childhood (i.e., mostly after HHV-6 primary infection) [23,221]. The virus can be isolated from the saliva of normal healthy donors in addition to exanthema subitum patients and subjects with immundeﬁciency. Like HHV-6, HHV-7 is widely distributed in the general population (83–93% by adulthood). However, one study suggested that HHV-7 infection was less prevalent in Japan than in the United States [22,23,221]. HHV-7 infection can occur in patients who already have antibodies to HHV-6, and the apparent antibody titer to HHV-6 then increases. This ﬁnding is supported by the fact that HHV-7 induces HHV-6 reactivation [22,23]. A study by Tanaka-Taya et al. [222] showed that HHV-7 infection increased with age and reached a maximum in adulthood. Another study of blood donors, using PCR, showed that 97% were positive for HHV-7. Furthermore, HHV-7 DNA was detected in the oral secretions (95%) and buffy coats (66%) of 112 donors, indicating transmission by the oral route as well as during blood transfusion [223]. In a study of 100 umbilical samples, 96% of the plasma contained IgG antibody to HHV-7. Because there may be overlapping or cross-reacting antibody to HHV-6, a subset of the plasma samples, after adsorption with HHV-6-infected cells, revealed that 89% remained positive for HHV-7. A comparative study of antibody to HHV-6 and HHV-7 in children showed that HHV-7 seroprevalence was 42% in those who were 1–2 years of age. Some reports on the prevalence of HHV-6 in saliva have turned out to be misleading [22,23,221,224], indicating that more speciﬁc PCR probes or monoclonal antibodies should be used in the identiﬁcation of HHV-6 and HHV-7 from clinical specimens [23]. 10.3

HHV-8 Prevalence

Many studies have been conducted with immunological assays to assess the prevalence of HHV-8 in various populations [225]. Despite all these results, there are discrepancies in the sensitivity and speciﬁcity of the assays used. There is no gold standard against which to measure the efﬁcacy of these immunological techniques, so some may under- or overestimate the number of HHV-8 antibody positives. Despite all these differences, it is clear that, unlike other human herpesviruses, HHV-8 is not widely distributed in healthy populations [226]. The highest seroprevalence in healthy populations was found in sub-Saharan Africa (40%), compared to 2–4% positive in northern Europe, Asia, and the Caribbean [226]. The prevalence of HHV-8 IgG antibody in HIV-positive patients ranged much higher [225,226]. In Japan fewer than 0.2% of

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healthy adults had HHV-8 antibody, in contrast with 11% in HIV-1positive patients. HIV-1-positive individuals have a higher risk of developing Kaposi’s sarcoma. In Italy and other parts of the world there are areas with a high seroprevalence of HHV-8, and incidence of KS is also high in these areas [8,226]. In the United States the prevalence of HHV-8 ranged between 5% and 20%, depending on the assay used to measure the antibody. Sera from KS patients indicated that more than 95% are antibody-positive regardless of country of origin. Although children outside Africa are rarely positive for HHV-8 antibody, in Cameroon, French Guyana, and Uganda the seroprevalence of HHV-8 increased with age, usually reaching the adult prevalence rate before puberty. To support this, in Cameroon the overall prevalence in children and young adolescents was found to be 27.5% and rose to 48% with age. Studies in Central Africa, moreover, revealed a measurable rate of mother-to-child infection. In Italy, two out of 57 infants carried HHV-8 antibody, and in older children the rate rose to 4.4%. This clustering and the Cameroon study therefore support the existence of nonsexual transmission of HHV-8 [8,13,199–201].

10.4

Transmission of HHV-8

Some reports indicate that HHV-8 can be transmitted sexually [8]. HHV8 has been detected in PBMCs obtained from AIDS patients. For example, HHV-8 DNA was found in the saliva from KS-positive patients. In another study HHV-8 DNA was detected in the saliva of 75% of HIVpositive patients. Mucosal shedding of HHV-8 was also reported, suggesting oral transmission. The sexual transmission of HHV-8 was indicated by ﬁnding HHV-8 DNA in the semen rather than in spermatocytes, suggesting secretion into seminal ﬂuid. On the assumption that HHV-8 is transmitted sexually, the virus has to be at a higher titer than in PBMCs. The presence of HHV-8 in HIV-1-positive KS patients is most likely due to sexual transmission of infection. The presence of HHV-8 DNA in semen of healthy donors is controversial. HHV-8 has also been detected in the genital tract of women who were HHV-8 seropositive [227]. HHV-8 was also found in young men who had had sex with other men. The study showed that high-risk sexual behavior and drug use acted as cofactors for the presence of HHV-8 in a group of 1295 women from the United States [228]. HHV-8 has also been transmitted via blood through needle sharing. The fact that most studies about sexual transmission found that the semen from a very low number of KS patients contains HHV-8 DNA suggests that it is rare to transmit

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HHV-8 by the genital route in HIV-1 and HHV-8 antibody–positive patients [8,13,199–201].

11

DIAGNOSING HHV-6, HHV-7 AND HHV-8 INFECTIONS

Various molecular and immunological assays are currently used to assess the infection of human herpesvirus-6, -7, and -8. At least two independent test systems should be used simultaneously for the identiﬁcation of HHV-6-associated diseases: serology in combination with virus isolation or with antigen-capture ELISA for p41, ideally with follow-up testing. In situ hybridization or polymerase chain ampliﬁcation reaction (PCR) for viral DNA may show increased viral load but not necessarily viral activity. In addition to these tests, eventual tissue biopsies should be positive for antigen and/or DNA deposits in diseased tissues but negative in adjacent healthy tissue of the same individual (internal control). 1.

Acute (primary) infection (a)

Indirect immunoﬂuorescence assays (IFA) for IgM and IgG using HHV-6A-infected HSB2 cells or HHV-6Binfected MOLT3 cells with respective follow-ups (b) Virus isolation 2. Chronic persistent or reactivated infections (a)

IFA for HHV-6 IgG (occasionally IgA; IgM is not necessarily positive: ‘‘anamnestic response’’) with followups (b) p41 antigen-capture ELISA or quantitative PCR or virus isolation (c) If tissue is available, antigen (p41) and DNA in situ demonstration by immunohistology and in situ hybridization If one separates PBMCs from the peripheral blood and propagates them in vitro with indicator cells [i.e., PHA-stimulated HCBMCs or ﬁbroblasts (MRC-5)], there may be a spread of HHV-6/HHV-7 from latency in the cells to a reactivation state. The cocultured cells are then tested by IFA with HHV-6- or HHV-7-speciﬁc monoclonal antibodies [15]. A number of studies have employed PCR to detect HHV-6 in examining serum/plasma, PBMCs, saliva, and/or cerebral spinal ﬂuids from MS, CFS, and AIDS patients and those with Rosai-Dorfman disease [229]. However, to detect active infection, it is necessary to employ

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quantitative assays such as nested PCR and real-time PCR. An assessment of the viral load is important in patients who are undergoing treatment. PCR results have been conﬁrmed with Southern blot hybridization to detect HHV-6 DNA [230–232]. Molecular and serological techniques have been employed to detect HHV-8 infection in cells, tissues, and body ﬂuids, but none of the existing assays is 100% accurate. It is also necessary to employ more than one assay to make certain that one does not miss the presence of HHV-8. Some studies showed that serological assays were more sensitive than PCR, because PCR failed to detect the presence of HHV-8 DNA, but serology was positive in such individuals [8]. In the majority of studies on KS tissues, PEL lymphoma cells, and MCD, clinical materials were tested by PCR. It was found that clinical materials from classical KS, endemic KS, and KS in HIV-1-positive patients contained HHV-8 DNA (95%). HHV-8 DNA can also be detected in frozen or fresh tissues by PCR. Ablashi et al. [8] showed that KS patients’ PBMCs, biopsy materials, and plasma were PCR-positive. By employing either RT-PCR or real-time PCR, the number of HHV-8 genome copies can be calculated. Parafﬁn-embedded tissues from KS and MCD and lymphoid tissue from PEL were found to be HHV-8positive by PCR. HHV-8 DNA was also detected by nested PCR from KS patients’ clinical samples. In situ hybridization can be used to localize the speciﬁc cell that harbors HHV-8 [8,13,199–201]. Recently the in situ method has been replaced by immunocytochemistry, using commercially available HHV-8-speciﬁc monoclonal antibody to ORF-73, ORF K8.1, and ORF-59. Immunocytochemistry was found useful in identifying cells from KS, MCD, and PEL. Serology has played a major role in assessing HHV-8 infection as well as in screening large numbers of samples for epidemiological purposes [225]. IFA, ELISA, and immunoblots detected antibody to lytic and latent proteins. Latent IgG antibody can be detected by IFA with LANA and an induced PEL cell line. Here a positive serum stains the nuclei of the cell [8]. To detect antibody by IFA, one must use a PEL cell line after induction. The differences between LANA-1 and lytic cell positivity is that almost 100% of the cells are LANA-1-positive, whereas for lytic infection the number of positive cells may vary from 5% to 60%, depending on the rate of lytic antigen activation. If a serum is latent antibody–positive, it may or may not contain lytic antigen antibody. The same may be true for serum that is lytic antigen–positive. It is therefore necessary that both antibodies be measured, so one does not miss the HHV-8 infection. Various investigators have used different sources of antigen for the ELISA to measure HHV-8 antibody. For example, the

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antigens used to coat ELISA in different studies included puriﬁed virions, ORF-65, ORF-26, ORF-K8-1, and ORF-73 proteins. The sensitivity and speciﬁcity of ELISA may vary, but ELISA is more sensitive than IFA. Further details about these assays can be found in review articles [8,13,199–201]. Some investigators found that serology was more sensitive that PCR. It is therefore suggested that PCR data should be supported by serology. In one study investigators used nine different assays for comparison of speciﬁcity and sensitivity. Statistical analysis revealed that concordance with AIDS-KS sera. The use of healthy donor sera, however, showed a great variability in detecting HHV-8 antibody. In ELISA, some native antigens have been replaced with antigen expressed in a vector [8]. It is important that the vector-expressed protein and native protein should be compared before deciding to use only vector-expressed antigen in the assay. ELISA kits for latent (ORF-73) and lytic antigens are now commercially available. It is anticipated that in the near future the serological assays will be researched further, so it will no longer be necessary to use more than one assay to detect HHV-8 infection.

12 12.1

SUSCEPTIBILITY OF HHV-6, HHV-7 AND HHV-8 TO ANTIVIRAL TREATMENT Low Molecular Weight Compounds

Some studies using aciclovir (ACV) showed an inhibitory effect on HHV6, but other studies reveal a lack of inhibitory activity [18]. In some studies, ganciclovir (GCV), which is a potent inhibitor of HCMV, was similarly found to inhibit HHV-6 infection, whereas other reports found either that GCV was partially effective against HHV-6 infection or that it did not inhibit HHV-6 replication in vitro. In a trial, four CFS patients showing active HHV-6A infection were treated with GCV. None of the four patients improved, and HHV-6 could be isolated after GCV treatment (Ablashi and Peterson, unpublished data). In another study of MS patients, valaciclovir was used to inhibit active HHV-6A infection. These patients exhibited HHV-6-speciﬁc IgM antibody and showed no signiﬁcant differences between treated and placebo groups except that there were fewer recurrences of MS attacks in the untreated patients (Friedman et al., unpublished data). Although aciclovir has little or no effect on HHV-6 in vitro, Wang et al. [85] found fewer HHV-6 PCRpositive blood samples in bone marrow transplant patients who received a high dose of aciclovir than in those without the drug. Therefore, aciclovir may be effective against HHV-6 in vivo. On the other hand,

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GCV successfully blocked HHV-6 infection in bone marrow transplant patients with HHV-6 encephalitis [233]. Two CFS patients treated with foscarnet showed considerable improvement. HHV-6 active infection disappeared, one patient fully recovered, and the other is steadily recovering (Ablashi and Peterson, unpublished data). Foscarnet and nucleoside analogs are limited in their action because they have no effect on latent HHV-6 genome, which may become active later on when the drug is discontinued. Foscarnet and phosphonoacetic acid (PAA) inhibit viral DNA polymerase and reverse transcriptases. Streicher et al. [234] and Williams [235] investigated other antiviral compounds (e.g., dUrd, 5mercuri-20 -deoxyuridine (HgdUrd), dithiothreitol-HgdUrd, 2-thiouracilHgdUrd, 6-mercaptoguanosine-HgdUrd) but did not detect any inhibition of HHV-6 replication. Antiviral effects of these compounds and other nucleoside analogs were reviewed extensively by Williams [235]. Newer antiviral compounds effective against HHV-6 and HHV-7 have been extensively reviewed by De Clercq et al. [236]. One of the major problems in assessing the inhibitory effect of test compounds on HHV-8 replication is that, at present, there is no suitable in vitro test system available. Inhibitors of viral DNA polymerase are, moreover, effective in controlling lytic, but not latent, DNA infection. Although ganciclovir, which is a potent inhibitor of CMV, showed partial or no inhibition of HHV-6, it was reported to cause regression of KS lesions in a small trial in HIV-1-infected patients, and in three follow-up studies [237] HHV-8 was found to be sensitive to cidofovir when tested in vitro. In another study, foscarnet, a potent inhibitor of all the herpesviruses, was only moderately effective against HHV-8 [238], and ganciclovir showed similar efﬁcacy. These antiviral drugs do not inhibit episomal HHV-8 DNA polymerase, suggesting that host DNA polymerase replicates the latent form of viral DNA. Aciclovir, which inhibits EBV replication in hairy leukoplakia in HIV-infected patients, did not inhibit HHV-8 infections, although both of these are gamma herpesviruses. If an antiviral drug blocks virus reactivation it could still keep the virus in check; however, the ideal antiviral agent would be one that inhibits the latent form of HHV-8. Antiretroviral therapy is useful in combating KS in HIV-infected patients, most likely by improving immune surveillance [239]. Highly active antiretroviral therapy, despite having no direct antiviral effect, was found to reduce the HHV-8 load and suggests a relationship between tumor burden and HHV-8 activity. To treat HHV-8 infection, therefore, more new antiviral drugs are needed as well as a more sensitive in vitro test system to aid in screening these compounds.

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Biologicals

Ampligen, a mismatched double-stranded RNA, also known as poly(I): poly C12 (U), is an interferon inducer having broad-spectrum antiviral activity. Ampligen blocked the HHV-6 DNA polymerase in vitro [240] and also showed dose-dependent inhibition of HHV-6A (GS strain) in an immature T-cell line (HSB2). In one study, Ampligen was given to 15 HHV-6-positive CFS patients for 12–48 weeks. Of these, 13 patients improved, and no HHV-6 could be isolated from the PBMC during the period these patients were on Ampligen [241]. Kutapressin (KU) is effective against HHV-6 in vitro [240,242] and has been used for over 50 years by physicians for the prevention of neurasthenia and for dermatological conditions. Immunomodulators such as transfer factor (TF) have been used in an effort to inhibit HHV-6. Even though some CFS patients receiving HHV-6-speciﬁc TF showed improvement, no systematic baseline data were generated to show in vivo inhibition of HHV-6 infection [243]. Ampligen and TF have yet to be tested in HHV-8 infections, but newer antiviral compounds are being examined for efﬁcacy against the virus, so the future is promising [236]. The variability of antiviral drugs to inhibit HHV-6 could be due to HHV-6 strains/variants or to assay conditions. Current knowledge indicates that with the exception of foscarnet, ACV, GCV, valciclovir, KU, and Ampligen are unlikely to effectively suppress HHV-6 or HHV-7 infection [244,245]. Cidofovir has been shown to have an inhibitory effect on HHV-6 replication, however. Both HHV-6 and HHV-7 have been shown to enhance CMV disease in transplant recipients, and in HIV-infected people there may be a faster progression to HIV disease, especially in children with vertical HIV-1 infection [18]. Thus, new anti-herpesvirus compounds that can be administered orally without toxicity are urgently needed to treat HHV6 and HHV-7 infection. A compound that would inhibit HHV-8 infections and also reduce the size of HHV-8-related tumors would be ideal.

13

CONCLUSION

In essence, both HHV-6 and HHV-7 show a widespread distribution with lifelong persistence. They are frequently reactivated and remain clinically inapparent unless the patient is immunodeﬁcient in some way. Even then, HHV-6 and HHV-7 reactivation may simply enhance the pathogenicity of other viruses or of certain autoimmune processes rather

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than becoming pathogens themselves. Future studies need to focus on such indirect viral inﬂuences mediated through molecular mimicry and interference with cell receptor expression and cytokine and chemokine network regulation. Such disturbances, nevertheless, may afford therapeutic intervention to disrupt herpesvirus interference with certain disease processes. There are only a few diseases for which an immediate causal relationship to HHV-6 or HHV-7 infection has been suggested. HHV-8, on the other hand, is endemic in particular geographical areas and is clearly associated with KS, which itself has a limited and uneven distribution. In a way, HHV-8 behaves much the same as EBV in that both appear to have tumorigenic properties. As diagnostic tools improve, it is likely that the true role of HHV-8 in diseases such as PEL and MCD will be elucidated.

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Index

Abacavir (ABC; Ziagen2; Trizivir2), 401, 409, 485–488, 515 ABC (see Abacavir) Abortion, 6 ABT-378 (see also Lopinavir, LPV), 535 ABT-538 (see also Ritonavir, RTV), 535 3R,4R, 5S-4-Acetamido-5-amino-3-(1ethylpropoxyl)-1-cyclohexene1-carboxylic acid, 58–64 Aciclovir (ACV), 175–176, 210, 600– 603, 628–633, 684 Acquired immunodeﬁciency syndrome (AIDS; see also HIV/ AIDS), 3, 8, 369–380, 399–413, 457, 523–532, 547–548, 587, 589, 597, 607, 634–635, 660, 674–675 Acute hepatitis C, 299, 324, 342 ACV (see Aciclovir) Acyclic nucleoside phosphonate analogs (ANP), 491–493, 498– 499

ADA (see Azodicarbonamide) Adefovir (PMEA)/Adefovir dipivoxil, 491–492 Adefovir dipivoxil (ADV), 287, 288 Adenovirus, 644 adenovirus vectors, 644 Adult T-cell leukemia, 3 ADV (adefovir dipivoxil), 287, 288 Adverse drug effects aciclovir, 175, 179–180, 210 adefovir, 287 cardiovascular disease, 409, 545 central nervous system, 409, 508 cidofovir, 182 drug interactions, 404–406, 412, 468, 534, 542, 545 famciclovir, 179 foscarnet, 601 ganciclovir, 601 gastrointestinal, 409, 487, 545 hepatic steatosis, 409 707

708 [Adverse drug effects] hepato- (liver-) toxicity, 409, 545– 547 insulin resistance, 409 interferon-a 286, 325–329, 331 alopecia, 326 arthralgia, 326 depression, 326 discontinuation, 326 fatigue, 326 fever, 326 headache, 326 leukopenia, 326 loss of appetite, 326 loss of vision, 326 lumbalgia, 326 myalgia, 326 nausea, 326 retinopathy, 326 thrombocytopenia, 326 tinnitus, 326 weight loss, 326 kidney stones, 545 lactic acidosis, 409 lipid metabolism, 409, 545–546 lipodystrophy syndrome, 409, 410, 546 mitochondrial toxicity, 409, 587 neutropenia, 601 non-nucleoside reverse transcriptase inhibitors (NNRTIs), 409, 487, 508 nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), 409–410, 487, 587 pancreatitis, 409, 487 pleconaril, 151 polyneuropathy, 409, 487 protease inhibitors, 404–406, 409– 410, 412, 468, 531–537, 542, 544–548 ribavirin, 325 cough, 325 dyspnea, 325 exanthema, 325 hemoglobin decline, 325

Index [Adverse drug effects] pharyngitis, 325 pruritus, 325 skin rash, 409, 487, 508 Stevens-Johnson syndrome, 508 AG 7088 (ruprintivir), 147, 154–155, 265 AG1004, 529 AG1549 (capravirine), 508 Agenerase2 (amprenavir, AMP), 401, 540–541, 543–547 Aichi virus, 3, 16–17 AIDS (see Acquired immunodeﬁciency syndrome) Alaninyl AZTMP, 497 Alaninyl d4TMP, 497 Aldara2 (imiquimod), 238 ALX40, 557, 568 ALX40–4C, 568 Amantadine (1-amino adamantane hydrochloride), 57–60, 338– 339 AMD 3100, 557, 568 AMD 3451, 569 AMD 7049, 568–569 Amdoxovir [(-)-b-D-2,6diaminopurine dioxolane, DAPD], 490, 494, 491 1-Amino adamantane hydrochloride (amantadine), 57–60 Aminooxypentane-RANTES (AOPRANTES), 557, 566 Aminothiazolylphenyl derivatives (HSV helicase-primase inhibitors), 177 AMP (see Amprenavir) Ampligen [poly(I):poly C12 (U)], 685 Amprenavir (AMP, Agenerase2), 401, 540–541, 543, 545, 547 Anal cancer, 233 Anemia, 630 Anogenital warts, 232 ANP (acyclic nucleoside phosphonate analogs), 498– 499, 492

Index Antiretroviral therapy/treatment (ART), 373–375, 398–412, 433, 446, 457–458, 460–461, 468, 514–516, 541–542 Antisense oligonucleotide, 113, 347– 348, 569–570, 643 Antiviral agents abacavir (ABC; Ziagen2; Trizivir2), 401, 409, 485–488, 515 ABC (see Abacavir) ABT-378 (see also Lopinavir, LPV), 535 ABT-538 (see also Ritonavir, RTV), 535 3R,4R, 5S-4-Acetamido-5-amino-3(1-ethylpropoxyl)-1cyclohexene-1-carboxylic acid, 58–64 ACV (see Aciclovir) acyclic nucleoside phosphonate analogs (ANP), 491–493, 498–499 ADA (see Azodicarbonamide) adefovir (PMEA)/adefovir dipivoxil, 287, 288, 491–492 adenovirus vectors, 644 ADV (adefovir dipivoxil), 287, 288 AG 7088 (ruprintivir), 147, 154–155, 265 AG1004, 529 AG1549 (capravirine), 508 Agenerase2 (amprenavir, AMP), 401, 540–541, 543–547 alaninyl AZTMP, 497 alaninyl d4TMP, 497 Aldara2 (imiquimod), 238 ALX40, 557, 568 ALX40–4C, 568 amantadine (1-amino adamantane hydrochloride), 57–60, 338– 339 AMD 3100, 557, 568 AMD 3451, 569 AMD 7049, 568–569

709 [Antiviral agents] amdoxovir [(-)-b-D-2,6diaminopurine dioxolane, DAPD], 490, 491, 494 1-amino adamantane hydrochloride (amantadine), 57–60 aminooxypentane-RANTES (AOPRANTES), 557, 566 N-[5-(aminosulfonyl)-4-methyl-1,3thiazole-2-yl]-N-methyl-2[4-(2pyridinyl)phenyl]acetamide (BAY 57–1293, HSV helicase-primase inhibitor), 177 aminothiazolylphenyl derivatives (HSV helicase-primase Inhibitors), 177 AMP (see Amprenavir) ampligen [poly(I):poly C12 (U)], 685 amprenavir (AMP, Agenerase2), 401, 540–541, 543, 545, 547 ANP (acyclic nucleoside phosphonate analogs), 498– 499, 492 antisense oligonucleotide, 113, 347– 348, 569–570, 643 AOP-RANTES (aminooxypentaneRANTES), 557, 566 AR177 (Zintevir), 557–558 atazanavir (BMS-232632), 546–547 azidothymidine (AZT, zidovudine, ZDV; Retrovir2, Combivir2, Trizivir2), 399, 401, 409, 485–494, 512–515, 533–534, 537, 540 azodicarbonamide (ADA), 557, 564 AZT (see Azidothymidine) BAY 38-4766, 592 BAY 41-4109, 284 BAY 57-1293, 183 BIRR 4 (soluble ICAM-1), 152 bis(SATE)ddAMP, 497

710 [Antiviral agents] BMS 806, 557, 559 BMS-232632 (see Atazanavir) BRI2923 (phenyldicarboxylic acid), 557–558 calanolide A, 508 capravirine (AG1549), 508 4-carbamoyl-1-b-D-ribofuranosylimidazolium-5-olate, 111 carbocyclic 3-deaza adenosine, 112 CCR5 inhibitors, 566–567 CCR5 peptides, 557–558 CD4 fusion proteins, 556–558 CD4-IgG2, 556–558 cetirizine (2-[2-[4-(4-chlorophenyl)phenylmethyl]-1piperazinyl]ethoxy-acidic acid), 116 chalcone, 265 2-[2-[4-(4-Chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy-acidic acid, 16 cholestyramine, 269 CI-1012, 557, 564 cidofovir, 176, 592, 606–607, 628– 635, 686 clevudine (L-FMAU), 287, 288 Combivir2 (azidothymidine + lamivudine; AZT + 3TC), 401, 409, 487 consensus interferon, 326 coreceptor inhibitors, 557, 565–569 Crixivan2 (Indinavir, IDV), 401, 536–537 CXCR4 inhibitors, 568–569 cyanovirin N, 557–558 cyclopentenylcytosine, 112 cyclosporin A, 561 Cytovene2 (see Ganciclovir) d4A, 497 d4T (20 ,30 -dideoxy-20 ,30 didehydrothymidine, Stavudine, Zerit2), 401, 409, 485–490, 496–499, 540 d4TMP, 496–497

Index [Antiviral agents] d4TTP, 494 DAPD [(-)-b-D-2,6-diaminopurine dioxolane, amdoxovir], 287, 490–494 ddA, 496, 497 ddAMP, 496 ddC (20 ,30 -dideoxycytidine, zalcitabine, HIVID2), 401, 409, 485–489, 498–499, 534 ddI (20 ,30 -dideoxyinosine, Didanosine, Videx2), 401, 409, 485–489, 497–499, 512, 515 ddN (20 ,30 -dideoxynucleoside analogs), 485, 488–489, 494 ddNMPs (20 ,30 -dideoxynucleoside 50 -monophosphates), 494– 497, 499 3-deazaguanine, 111 3-deaza-50 -noraristeromycin, 113 7-deaza-50 -noraristeromycin, 113 1,4-dehydro-2,3-bonzodithiin, 116 delavirdine (DLV, Rescriptor2), 401, 506, 508, 513–514 20 -deoxy-20 -ﬂuororibonucleoside, 113 20 -deoxy-30 -oxa-40 -thiocytidine (dOTC), 489, 490, 491 40 -E-20 -deoxyribofuranosyl-2,6diaminopurine, 491 desciclovir, 635 dextran sulfate, 558 (-)-b-D-2,6-diaminopurine dioxolane (DAPD, amdoxovir), 490, 491, 494 DIBA-1 (dithiobenzamide), 557, 564 didanosine (ddI, Videx2), 401, 487, 485–489, 497–499, 512, 515 20 ,30 -dideoxycytidine (see also ddC, Zalcitabine), 487 20 ,30 -dideoxy-20 ,30 didehydrothymidine ( see also d4T, Stavudine), 487 20 ,30 -dideoxyinosine (see also ddI, Didanosine), 487

Index [Antiviral agents] 20 ,30 -dideoxynucleoside (ddN) analogs, 485, 488–489, 494 20 ,30 -dideoxynucleoside 50 monophosphates (ddNMPs), 494–497, 499 diketo acid compounds, 557, 562 3-[3,5-dimethyl-4-[[3-(3-methyl-5isoxazolyl)-propyl]oxy]phenyl]-5-(triﬂuoromethyl)1,2,4-oxadiazole (VP63843), 149 dioxolane guanine (DXG ), 490, 494 distamycin analogs, 569 dithiobenzamide (DIBA-1), 557, 564 DLV (see Delavirdine) dOTC (20 -deoxy-30 -oxa-40 thiocytidine), 489, 490, 491 DPC083, 508 DXG (dioxolane guanine), 490, 494 Efavirenz (EFV, Sustiva2), 401, 409, 506–508, 512–515, 544, 547 EICAR (5-ethynyl-1-b-Dribofuranosylimidazole-4carboxamide), 111 emivirine, 508 emtricitabine (FTC), 288, 287 enfuvirtide ( see T20), 401 entecavir (ETV), 287, 288 enviroxime, 148, 149 Epivir2 (see also Lamivudine, 3TC), 401, 487 40 -ethynyl nucleoside analogs, 491 5-ethynyl-1-b-Dribofuranosylimidazole-4carboxamide, 111 ETV (entecavir), 287, 288 famciclovir, 175–176, 210–211 5’-ﬂuorouracil, 238 ﬂuticasone (Flonase2), 146 fortovase2 (see also Saquinavir, SQV; Invirase2), 401, 409, 532–534 foscarnet (phosphonoformic acid, PFA), 176, 506, 592, 601, 606–607, 628, 631–632, 685

711 [Antiviral agents] fozivudine tidoxil (FZD), 489 FP-21399, 557–558 FTC (emtricitabine), 287–288, 489– 491 Fuzeon2 (enfuvirtide; see also T20), 401 ganciclovir (GCV, Cymevene2, Cytovene2), 592, 600, 605– 607, 628–635, 684–685 GCV (see Ganciclovir) gene therapy, 44–645 gp120 inhibitors, 556–559 gp41 inhibitors, 557, 560–562 HBY 097, 513–515 5-helix, 557, 560 HIV entry inhibitors, 557, 560–562, 565–569 HIVID2 (see also Zalcitabine, ddC), 401, 487 HSV helicase-primase inhibitors (aminothiazolylphenyl derivatives), 177, 183 hyperimmune globulin, 603 ICAM-1, soluble, BIRR 4, 147, 152– 154 IDV (see Indinavir) IL-12 (interleukin 12), 350–351, 557, 571–572 IL-2 (interleukin 2), 350, 557, 571 imiquimod (1-(2-methylpropyl)1H-imidazol[4,5-c]quinolin4-amine; Aldara2, Zartra2), 238 immune globulin (Ig), 107–109, 207–209, 271 immune response modiﬁer, 351– 352 immunotherapy, 642 IMPDH Inhibitors, 109–112 indinavir (IDV, Crixivan2), 401, 536–538, 540–541, 545 integrase inhibitors, 557, 562–564 interferon, 11, 117–118, 145–148, 155–157, 269, 285–289, 317– 318, 557, 570–572, 635, 686

712 [Antiviral agents] a, 11, 155–157, 286, 318, 322–327, 337 a-2a, 328–329 a-2b, 324, 329–331 pegylated/peginterferon, 327– 340 intravenous immunoglobulin (IVIG), 599, 602 invirase2 (Saquinavir, SQV; see also Fortovase2), 401, 532– 534 ipratropium, 148 IVIG (intravenous immunoglobulin), 599, 602 Kaletra2 (lopinavir/ ritonavir, LPV/r), 401, 409, 412, 536– 538, 541 kutapressin (KU), 686 lamivudine (3TC; Epivir2, Zefﬁx2; Combivir2, Trizivir2), 284– 289, 401, 409, 485–499, 514– 515, 537–540 L-dT (telbivudine), 288 L-Fd4C, 287, 288 L-FMAU (clevudine), 287, 288 lopinavir (LPV, ABT-378), 536–538, 540–541, 544–545, 548 lopinavir/ritonavir (LPV/r, Kaletra2), 401, 406, 412, 536–538, 540–541, 544– 548 lovastatin, 116 LPV/r (see Lopinavir/ ritonavir, Kaletra2) LY253963, 112 a-methyl-1-adamantane methylamine hydrochloride (rimantadine), 57–60 methylenecyclopropane nucleoside analogs, 491 60 -(R-60 -C-methyleneplanocin) A, 112 metoclopramide, 269 microbicides, 374, 516, 558–559 MIV-150, 508

Index [Antiviral agents] mizoribine (4-carbamoyl-1-b-Dribofuranosylimidazolium5-olate), 111 naphtalene sulfonate polymer (see PRO 2000) naproxene, 148 nelﬁnavir (NFV, Viracept2), 401, 537–541, 543–545, 547–548 neplanocin, 113 nevirapine (NVP, Viramune2), 401, 506–509, 512–516 NFV (see Nelﬁnavir) NMSO3, 116 NNRTIs (non-nucleoside reverse transcriptase inhibitors), 401, 497–499, 505–521 NNY-RANTES, 567 non-nucleoside reverse transcriptase inhibitors (NNRTIs), 401, 497–499, 505–521 non-steroidal antiinﬂammatory drugs (NSAID), 148 50 -noraristeromycin, 113 Norvir2 (see also Ritonavir, RTV), 401, 536 NRTI phosphorylation, 493–495 NRTIs (nucleoside reverse transcriptase inhibitors), 401, 485–504 NtRTIs (nucleotide reverse transcriptase inhibitors), 401, 485–504 nucleoside/nucleotide analogs, 109–113, 238, 284–289, 485– 504, 628, 643 nucleoside reverse transcriptase inhibitors (NRTIs), 401, 485– 504 nucleotide reverse transcriptase inhibitors (NtRTIs), 401 NVP (see also Nevirapine), 401 oseltamivir [3R,4R, 5S-4acetamido-5-amino-3-(1ethylpropoxyl)-1-

Index [Antiviral agents] cyclohexene-1-carboxylic acid; Tamiﬂu2], 58–64 PALA [N-(phosphonoacetyl)-Laspartate], 112 palivizumab, 107 penciclovir, 175–176, 211 peptide T, 557 PFA (see Phosphonoformic acid, foscarnet) phenyldicarboxylic acid (see BRI2923) phosphonoformic acid (foscarnet, PFA), 506 PI (see Protease inhibitors, PRI) pirodavir, 145 pleconaril (VP63843), (3-[3,5dimethyl-4-[[3-(3-methyl-5isoxazolyl)propyl]oxy]phenyl]-5-(triﬂuoromethyl)1,2,4-oxadiazole), 149–152, 265 PMEA (adefovir), 491, 492 PMPA (tenofovir), 485, 491, 492 PNU-140690 (see Tipranavir) podophyllin, 238 polyanionic compounds, 558 POM (polyoxometalates), 114 PRI (see Protease inhibitors, PI) PRO 140, 557, 567 PRO 2000, 557, 559 PRO 542, 556–558 prodrugs, 491, 494–497, 547 protease inhibitors (PI, PRI), 401, 04-406, 409–412, 469, 523– 554 pyrazofurin [3-(b-Dribofuranosyl)-4hydroxypyrazole-5carboxamide], 112, 113 R61837, 265 RD3–0028 [1,4-dehydro-2,3bonzodithiin], 116 Relenza2 (zanamivir), 60–61 Rescriptor2 (see also Delavirdine, DLV), 401, 598

713 [Antiviral agents] resiquimod, 1 76 RespiGam2, 107 Retrovir2 (zidovudine, ZDV, azidothymidine, AZT; see Combivir2, Trizivir2), 401, 409, 487 RFI 641, 115 ribavirin (1-b-D-ribofuranosyl1,2,4-triazole-3carboxamide; trade names: Cotronak2, Rebetol2, Virazole2, Vilona2, Viramid2, Virazide2), 11, 110–111, 325, 331–340 1-b-D-ribofuranosyl-1,2,4-triazole3-carboxamide, 110, 111 ribozymes, 348, 557, 570–571 rimantadine (a-methyl-1adamantane methylamine hydrochloride; Flumadine2), 57–60 ritonavir (ABT-538, RTV, Norvir2), 401, 412, 534–538, 541, 544– 548 rituximab, 635 Ro 31–8959 (see also Saquinavir, SQV), 532, 539 RTV (see Ritonavir, Norvir2) ruprintrivir (AG 7088), 147–149, 154–155, 265 RWJ-270201, 63 S-1360, 557, 562 saquinavir (Ro 31–8959, SQV, Fortovase2, Invirase2), 401, 529–539, 541 SCH38057, 265 SCH-C, 557, 567 SDS NIM811, 557, 564 single chain antibodies (SFv), 570 soluble CD4 (sCD4), 556–558 SQV (see Saquinavir) stavudine (d4T, Zerit2), 401, 485– 487, 485–490, 496–499, 540 steroids, 633

714 [Antiviral agents] Sustiva2 (see also Efavirenz, EFV), 401, 598 Synergis2, 107 T1249, 557, 560–561 T-134, 568 T-140, 568 T-20 (enfuvirtide, Fuzeon2), 557, 560–562 T-22, 557, 568 TAK-779, 557, 567 Tamiﬂu2 (oseltamivir), 60–61 3TC (20 ,30 -dideoxy-30 -thiacytidine, Lamivudine, Epivir2; Combivir2, Trizivir2, Zefﬁx2), 287, 401, 409, 485– 499, 514–515, 537–540 TDF (tenofovir disoproxil fumarate), 401, 406, 409, 485–487, 491–492 tenofovir (PMPA), 485, 491, 492 tenofovir disoproxil fumarate (TDF, Viread2), 401, 406, 409, 485–487, 491–492 TIBO (tetrahydroimidazobenzodiazepinone), 512 tipranavir (U-140690, PNU140690), 547 TMC120, 508 TMC125, 508 topical microbicides, 558–559 topoisomerase I inhibitors, 557 topotecan, 557 transfer factor, 686 Tremacamra2 (soluble ICAM-1), 147, 152–154, 264 Trizivir2 (azidothymidine + lamivudine + abacavir, AZT + 3TC + ABC), 401, 409, 487 TSAO (tertbutyldimethylsilylspiroa-minooxathioledioxid-ethymines), 512 U-140690 (see Tipranavir) UC781, 513–514, 516 valaciclovir, 175–176, 210–211, 686

Index [Antiviral agents] valganciclovir, 592 Videx2, Videx-EC (see also Didanosine, ddI), 401, 487 Viracept2 (nelﬁnavir, NFV), 401, 537–540 Viramune2 (nevirapine, NVP), 401, 598 Virazole2 (ribavirin), 110–111 Viread2 (see also Tenofovir disoproxil fumarate, TDF), 401, 487 VP14637, 115 VP63843 (pleconaril), 149–152, 265 VX497, 112 WIN52035, 265 9238X, 113 zalcitabine (ddC, HIVID2), 401, 409, 485–489, 498–499, 534 zanamivir (see Relenza2), 58 Zartra2 (see Imiquimod) ZDV (see Zidovudine) Zefﬁx2 (see Lamivudine, 3TC; Epivir2), 284, 287–289 2 Zerit (see also Stavudine, d4T), 401, 487 Ziagen2 (Abacavir, ABC; see Trizivir2), 401, 409, 487 zidovudine (ZDV; Azidothymidine, AZT; Retrovir2, Combivir2, Trizivir2), 401, 409, 485–499, 512–515, 533–534, 537, 540, 635, 685 zintevir (see AR177) AOP-RANTES (aminooxypentaneRANTES), 557, 566 Aplastic anemia, 3 AR177 (zintevir), 557–558 Arenaviridae, 7 Asthma, 92, 99, 140 Astroviridae, 3 5 Atazanavir (BMS-232632), 546–547 Autoimmune hepatitis, 322 Autoimmune phenomena, 279

Index Azidothymidine (AZT, zidovudine, ZDV; Retrovir2, Combivir2, Trizivir2), 399, 401, 409, 485– 494, 512–515, 533–534, 537, 540 Azodicarbonamide (ADA), 557, 564 AZT (see Azidothymidine) BAY 38-4766, 592 BAY 41-4109, 284 BAY 57-1293, 183 B-cell lymphoma (see also Lymphoma), 630 BDV (Borna disease virus), 18 Bell’s palsy, 212 BIRR 4 (soluble ICAM-1), 152 bis(SATE)ddAMP, 497 BMS 806, 557, 559 BMS-232632 (see Atazanavir) Borna disease virus (BDV), 4, 18, 20 Bornaviridae, 18 Bovine spongiform encephalopathy (BSE), 18, 19 BRI2923 (phenyldicarboxylic acid), 557–558 Bronchiolitis, 99 BSE (bovine spongiform encephalitis/encephalopathy), 4, 18–20 Bunyaviruses, 7 Calanolide A, 508 Caliciviridae, 2, 20 Cantalago virus, 4, 17, 20 Capravirine (AG1549), 508 4-Carbamoyl-1-b-Dribofuranosylimidazolium-5olate, 111 Carbocyclic 3-deaza adenosine, 112 Carcinoma (see also Tumor), 232, 234 Carcinoma in situ, 234 Cardiovascular disease, 669 CastlemanC ¸ s disease, 4, 10, 672, 677 CCR5 inhibitors, 566–567 CCR5 peptides, 557–558 CD4 fusion proteins, 556–558 CD4-IgG2, 556–558

715 Central nervous system disease, 196, 672, 668–669 Cervical cancer, 228, 233, 235, 245 Cervical intraepithelial neoplasia (CIN), 233, 236 Cetirizine (2-[2-[4-(4chlorophenyl)phenyl-methyl]1-piperazinyl]ethoxy-acidic acid), 116 Chalcone, 265 CHC (chronic hepatitis C), 295, 323, 342 2-[2-[4-(4-chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxyacidic acid, 116 Cholestatic liver disease, 629 Cholestyramine, 269 Chronic active EBV infection, 630 Chronic active hepatitis (CAH), 319 Chronic fatigue syndrome, 660 Chronic hepatitis C (CHC), 295, 299– 300, 304, 318–319, 325, 342 Chronic obstructive pulmonary disease (COPD), 92, 140 Chronic persistent hepatitis (CPH), 319 CI-1012, 557, 564 CID (cytomegalic inclusion disease), 598 Cidofovir, 176, 592, 606–607, 628–635 CIN (cervical intraepithelial neoplasia), 233, 236 Circoviridae, 12 Cirrhosis, 278, 285–287, 295–297, 296, 318, 322–325 Clevudine (L-FMAU), 287, 288 CMV (cytomegalovirus), 587–622 CMV disease, 596 Combivir2 (azidothymidine + lamivudine; AZT + 3TC), 401, 409, 487 Common cold, 139 Common warts, 231, 228 Condyloma, 228, 232–236 Congenital CMV infection, 589, 598 Congenital heart malformation, 109

716 Conjunctivitis, 4 Consensus interferon, 326 COPD (chronic obstructive pulmonary disease), 92, 140 Coreceptor inhibitors, 557, 565–569 Coxsackie A21, 152 Creutzfeld-Jakob disease, 4 Crixivan2 (indinavir, IDV), 401, 536– 537 Croup, 56 Cryoglobulinemia, 279 CXCR4 inhibitors, 568–569 Cyanovirin N, 557–558 Cyclopentenylcytosine, 112 Cyclosporin A, 561 Cystic ﬁbrosis, 109, 140 Cytomegalic inclusion disease (CID), 598 Cytomegalovirus (CMV), 587–622 ACIF (anticomplement immunoﬂuorescence test), 589, 590 acquired immunodeﬁcency syndrome (AIDS), 587 acyclovir, 600, 603 AD169, 587 adoptive immunotherapy, 608 adults, 587 AIDS (acquired immunodeﬁcency syndrome), 587, 589, 597, 607 allogeneic stem cell transplant, 596, 604 allograft recipient, 596, 601, 602 anticomplement Immunoﬂuorescence Test (ACIF), 589 antigenemia, 590, 592, 595, 599–607 antivirals, 592–608 aciclovir, 600, 603 cidofovir, 592, 606, 607 foscarnet, 592, 601, 606, 607 ganciclovir, 592, 600, 605–607 resistance, 593, 594, 607, 608 valganciclovir, 592 asymptotic infection, 595

Index [Cytomegalovirus (CMV)] autologous stem cell transplant, 603 blood, 591 breast milk, 598 bronchoalveolar lavage (BAL), 600 CF (complement ﬁxation antigen), 590 children, 588 CID (cytomegalic inclusion disease), 598 cidofovir, 592, 606, 607 clinical management, 590 clinical resistance, 594 CMV disease, 596 Cobas Amplicor CMV Monitor2 assay2, 590 complement ﬁxation antigen (CFA), 589 congenital infection, 589, 598 cross-resistance, 594 CTL (cytotoxic T-lymphocyte), 596 cytomegalic inclusion disease (CID), 598 cytopathology, 589 cytotoxic T-lymphocyte (CTL), 596 deafness, 599 diagnosis, 588 digene hybrid capture2, 592 DNA, 605 DNA polymerase (UL54), 92–593 early treatment, 600 ELISA (enzyme-linked immunosorbent assay), 590 endogenous virus, 595 enzyme-linked immunosorbent assay (ELISA), 590 established CMV disease, 607 exogenous virus, 595 foscarnet, 592, 601, 606, 607 ganciclovir, 592, 600, 605–607 genotypic assay, 593, 595 graft rejection, 597 graft versus host disease (GvHD), 595, 602, 605 GvHD (graft versus host disease), 595, 602, 603

Index [Cytomegalovirus (CMV)] HAART (highly active antiretroviral therapy), 597 hemagglutination test, 590 hepatosplenomegaly, 598 herpesvirus, 588 highly active antiretroviral therapy (HAART), 597 high-risk patient, 602, 603 HIV (human immunodeﬁciency virus), 589 HIV infection, 597 HLA-peptide tetramers, 608 human cytomegalovirus (HCMV), 587–622 human immunodeﬁciency virus (HIV), 589 hyperimmune globulin, 603 IE antigen (immediate early antigen), 588 IFA (indirect ﬂuorescent antibody), 589 immediate early antigen (IE antigen), 588 immune response, 606 indirect ﬂuorescent antibody (IFA), 589 intrauterine infection, 598 intravenous immunoglobulin (IVIG), 599, 602 IVIG (intravenous immunoglobulin), 599, 602 jaundice, 598 latency, 595 late-onset CMV disease, 601 latex agglutination assay, 590 Light Cycler2, 594 maintenance dose, 601 microcephaly, 598 monoclonal antibody, 588 mononucleosis, 587 mortality, 603 mutation, 594 neonate, 587 neutropenia, 601

717 [Cytomegalovirus (CMV)] nucleic acid hybridization, 592 NucliSens2, 591 pathogenesis, 595 PCR (polymerase chain reaction), 588, 594 perinatal infection, 589, 598 phenotypic assay, 593 phenotypic resistance, 595 plaque reduction assay, 593 pneumonia, 604, 607 polymerase chain reaction (PCR), 588, 590, 604 postnatal infection, 598, 590 post-transplant CMV infection, 590 pp65, 588, 605 preemptive therapy, 600, 603, 604 pregnancy, 597, 598 prophylaxis, 591, 599, 601, 603, 605 prophylaxis strategy, 602 radioimmunoassay, 590 renal toxicity, 601 resistance, 593, 607, 608 retinitis, 598 RNA, 605 seronegative patient, 599 seropositive patient, 595, 599 shell vials, 588, 604 solid organ transplant, 607 stem cell transplant, 595, 607 surrogate marker, 595 thymidine kinase (UL97), 592 transmission, 598 transplantation, 594 UL54 (DNA polymerase), 593, 595 UL97 (thymidine kinase), 592, 594, 595, 607 urine, 598 valganciclovir, 592 viral load, 591, 595–597, 605 viremia, 604 Cytovene2 (see Ganciclovir)

718 d4A, 497 d4T (20 ,30 -dideoxy-20 ,30 didehydrothymidine, Stavudine, Zerit2), 401, 409, 485–490, 496–499, 540 d4TMP, 496–497 d4TTP, 494 Dane particle, 277 DAPD [(-)-b-D-2,6-diaminopurine dioxolane, amdoxovir], 287, 490–494 ddA, 496, 497 ddAMP, 496 ddC (20 ,30 -dideoxycytidine, Zalcitabine, HIVID2), 401, 409, 485–489, 498–499, 534 ddI (20 ,30 -dideoxyinosine, Didanosine, Videx2), 401, 409, 485–489, 497–499, 512, 515 ddN (20 ,30 -dideoxynucleoside analogs), 485, 488–489, 494 ddNMPs (20 ,30 -dideoxynucleoside 50 monophosphates), 494–497, 499 Deafness, 599 3-Deazaguanine, 111 3-Deaza-50 -noraristeromycin, 113 7-Deaza-50 -noraristeromycin, 113 1,4-Dehydro-2,3-bonzodithiin, 116 Delavirdine (DLV, Rescriptor2), 401, 506, 508, 513–514 20 -Deoxy-20 -ﬂuororibonucleoside, 113 20 -Deoxy-30 -oxa-40 -thiocytidine (dOTC), 489, 490, 491 40 -E-20 -Deoxyribofuranosyl-2,6diaminopurine, 491 Dermatologic disorders, 671 Desciclovir, 635 Dextran sulfate, 558 (-)-b-D-2,6-Diaminopurine dioxolane (DAPD, amdoxovir), 490, 491, 494 Diarrhea, 3, 6, 16–17, 388 DIBA-1 (dithiobenzamide), 557, 564 Didanosine (ddI, Videx2), 401, 487, 485–489, 497–499, 512, 515

Index 20 ,30 -Dideoxy-20 ,30 didehydrothymidine (see also d4T, stavudine), 487 20 ,30 -Dideoxycytidine (see also ddC, zalcitabine), 487 20 ,30 -Dideoxyinosine (see also ddI, didanosine), 487 20 ,30 -Dideoxynucleoside (ddN) analogs, 485, 488–489, 494 20 ,30 -Dideoxynucleoside 50 monophosphates (ddNMPs), 494–497, 499 Diketo acid compounds, 557, 562 3-[3,5-Dimethyl-4-[[3-(3-methyl-5isoxazolyl)propyl]oxy]phenyl]-5(triﬂuoromethyl)-1,2,4oxadiazole (VP63843), 149 Dioxolane guanine (DXG ), 490, 494 Distamycin analogs, 569 Dithiobenzamide (DIBA-1), 557, 564 DLV (see Delavirdine) Dobrava virus, 3, 7 dOTC (20 -deoxy-30 -oxa-40 thiocytidine), 489, 490, 491 DPC083, 508 Drug resistance cytomegalovirus (CMV), 593–595 607, 608 hepatitis B virus (HBV), 287 hepatitis C virus (HCV), 318 herpes simplex virus, 176–177, 181–182 HIV/AIDS, 399, 410–411, 433–434, 457, 461, 464, 497–499, 508– 516, 534–544, 559, 567–570 inﬂuenza virus, 59–60, 64–65 rhino virus, 151–152 DXG (dioxolane guanine), 490, 494 Ebola virus, 3, 6, 20 EBV (see also Epstein-Barr virus), 623– 658 Efavirenz (EFV, Sustiva2), 401, 409, 506–508, 512–515, 544, 547

Index EICAR (5-ethynyl-1-b-Dribofuranosylimidazole-4carboxamide), 111 Emerging viruses, 1–38 Emivirine, 508 Emtricitabine (FTC), 288, 287 EMV (equine morbilli virus), 15, 16 Encephalitis, 3, 4, 9, 15, 56, 166, 196 Encephalopathy, 297 Enfuvirtide (see also T20), 401 Entecavir (ETV), 287, 288 Enterovirus 71 (EV71), 4, 16 Enviroxime, 148, 149 Epidemiology animal reservoirs, 8, 13–20 cytomegalovirus (CMV), 598 Epstein-Barr virus (EBV), 623 hepatitis A virus (HAV), 259, 266, 269, 270 hepatitis B virus (HBV), 281 hepatitis C virus (HCV), 296 human herpes virus 6 (HHV-6), 678–682 human herpes virus 7 (HHV-7), 678–682 human herpes virus 8 (HHV-8), 678–682 human Immunodeﬁciency virus type 1 (HIV-1), 373–380, 433–434, 443, 457, 464, 471, 516, 542, 559 papillomavirus, 228 zoonosis, 20 Epidermodysplasia verruciformis (EV), 228–239 Epivir2 (see also Lamivudine, 3TC), 401, 487 Epstein-Barr virus (EBV), 623–658 acquired immunodeﬁciency syndrome (AIDS), 634, 635 aciclovir, 628, 631–633 adenovirus vectors, 644 adolescents, 629 adults, 629 allograft recipient, 624 anemia, 630

719 [Epstein-Barr virus (EBV)] antibody, 630 antisense oligonucleotide, 643 B-cell lymphoma, 630 BRLF1, 627–628, 644–645 BZLF1, 627–628, 644–645 CD20, 635 children, 629 cholestatic liver disease, 629 chronic active EBV infection, 630 cidofovir, 628, 631, 632, 635 clinical presentation/ manifestation, 628–633 complications, 629–630 cytotoxic T cells/lymphocytes (CTL), 635, 642 desciclovir, 635 diagnosis, 630–631 DNA polymerase, 624, 628, 632 early genes, 627, 628 EBNA (EBV nuclear antigen), 625, 626, 631–635, 640, 642 EBV nuclear antigen 1 (EBNA-1), 631, 633 elderly, 629, 632 episome, 624 fever, 629 foscarnet, 628, 631, 632 ganciclovir, 628, 631, 632, 635, 646 gastric carcinoma, 640 gene expression, 625 gene therapy, 644–645 HAART (highly active antiretroviral therapy), 634 HD (Hodgkin’s disease), 635, 642 hepatitis, 630 herpesvirus, 623, 628 heterophile antibody test, 630 highly active antiretroviral therapy (HAART), 634 Hodgkin’s disease (HD), 635, 642 ICAM-1 (intercellular adhesion molecule 1), 627 immediate early genes, 646 immediate early proteins, 627

720 [Epstein-Barr virus (EBV)] immunosuppressed/ immunocompromised patients, 631, 632, 634–635 immunotherapy, 642 infectious mononucleosis, 628–635 interferon, 635 jaundice, 630 late membrane protein (LMP), 625, 626–627 latency, 625 latent infection, 623, 643 LMP (late membrane protein), 625, 626–627, 634, 635, 640, 642 lymphadenopathy, 629, 634, 637 lymphocytes, 623, 624, 637 lymphocytosis, 629 lymphoma, 624, 634, 635, 642 lymphoproliferative disease, 634– 635 lytic infection, 627, 643–645 memory B Cell, 624 MHC-class I (major histocompatibility complex class I), 627 myocarditis, 630 nasopharyngeal carcinoma (NPC), 636–639 nasopharynx, 623, 637 NFkB (nuclear factor kB), 627, 626, 635 NPC (nasopharyngeal carcinoma), 636–639 nuclear antigen (EBNA), 625, 626 nucleoside analogs, 628, 643 nuclear factor kB (NFkB), 626, 627 oral hairy leukoplakia, 632 origin of replication, 625, 628 oropharynx, 623 p53, 639 parotid carcinoma, 640 parotid gland, 623 PCR (polymerase chain reaction), 631, 634 permissive infection, 624 pharyngitis, 629

Index [Epstein-Barr virus (EBV)] phosphorylation, 628, 631 polymerase chain reaction (PCR), 631, 634 primary infection, 629, 630 rash, 629 Reed-Sternberg cell, 636 repeat elements, 624 replication, 627, 628, 644 rituximab, 635 saliva, 623 splenomegaly, 629 steroids, 633 T-cell lymphoma, 640 thrombocytopenia, 630 thymidine kinase, 628, 631, 637, 645 TNF (tumor necrosis factor), 627 tonsil, 623 TRAF (tumor necrosis factor receptor family), 626 transcription factor AP-1, 628 ATF-2, 628 c-JUN, 628 transformation, 624 transmission, 623 transplant patients, 634 transplantion, 641–642 treatment/therapy, 631–637, 641– 646 tumor, 631, 636–640, 644 tumor necrosis factor (TNF), 627 tumor necrosis factor receptor family (TRAF), 626 vaccine, 641–642 VCA (viral capsid antigen), 641 viral capsid antigen (VCA), 630, 637, 641 viral glycoprotein (gp350), 641 viral load, 631, 633 zidovudine, 635 Equine morbilli virus (EMV), 15–16 Erythema infectiosum, 6 Erythema multiforme, 166 Established CMV disease, 607 40 -Ethynyl nucleoside analogs, 491

Index 5-Ethynyl-1-b-D-ribofuranosylimidazole-4-carboxamide, 111 ETV (entecavir), 287, 288 EV (epidermodysplasia verruciformis), 228–239 EV 71 (enterovirus 71), 16 Exanthema subitum, 3, 9, 660, 666, 671 Experimental drugs/compounds (see antiviral agents) Famciclovir, 175–176, 210–211 Fever, 666 Fifth disease, 3 Filoviridae, 6 Flaviviridae, 10, 12, 17 50 -Fluorouracil, 238 Fluticasone (Flonase2), 146 Fortovase2 (see also Saquinavir, SQV; Invirase2), 401, 409, 532–534 Foscarnet (phosphonoformic acid, PFA), 176, 506, 592, 601, 606– 607, 685, 628, 631–632 Four corners disease, 3, 7 Fozivudine tidoxil (FZD), 489 FP-21399, 557–558 FTC (emtricitabine), 287–288, 489–491 Fulminant hepatitis, 278 Fuzeon2 (enfuvirtide; see also T20), 401 Ganciclovir (GCV, Cymevene2, Cytovene2), 592, 600, 605–607, 628–635, 684–685 Gastric carcinoma, 640 Gastroenteritis, 4, 5, 17 GCV (see Ganciclovir) Gene therapy, 644–645 Genital warts, 232–233, 245 gp120 inhibitors, 556–559 gp41 inhibitors, 557, 560–562 graft versus host disease (GvHD), 9, 595, 602, 603, 605 HAART (highly active antiretroviral therapy; see HIV/AIDS— antiretroviral therapy/ treatment)

721 HAM (HTLV-1 associated myelopathy), 3 Hand, foot and mouth disease (HFMD), 16 Hanta virus pulmonary syndrome (HPS), 3, 7 Hantaan virus, 3, 7 Hantavirus, 7 HAV (see also Hepatitis A virus), 11, 259–276 HBV (see also Hepatitis B virus), 277– 294 HBY 097, 513–515 HCC (hepatocellular carcinoma), 10, 281, 285 HCMV (human cytomegalovirus; see Cytomegalovirus) HCV (see also Hepatitis C virus), 3, 10–11, 295–368, 545 HD (Hodgkin’s disease), 635, 642 HDV (hepatitis D virus), 285 5-Helix, 557, 560 Hemorrhagic fever, 3, 6, 7 Hemorrhagic fever with renal syndrome (HFRS), 3 Hendra virus, 4, 15–16, 20 Hepatic failure, 295 Hepatitis, 3, 10–12, 259, 266–269, 277– 368, 630 Hepatitis A virus (HAV), 259–276 active immunization, 269–271 adolescents, 269 adults, 269 AG7088, 265 alanine aminotransferase (ALT), 267 alkaline phosphatase (AP), 267 bilirubin, 267 canyon, 264 chalcone, 265 children, 269, 270 cholestyramin, 269 clinical manifestation, 267–268 clotting factor disorders, 269 diagnosis, 267 drug users, 269

722 [Hepatitis A virus (HAV)] epidemiology, 266 g-glutamyl transpeptidase (g-GT), 267 HBV (hepatitis B virus), 259 hepatitis, 259, 266–269 hepatitis B virus (HBV), 259 hydrophobic pocket, 264 immune globulin (Ig), 271 interferon, 269 jaundice, 259, 267, 268 life cycle, 263 men, 269 metoclopramide, 269 molecular biology, 260–263 pathogenesis, 266–267 picornaviridae, 260 pleconaril, 265 polymerase, 261 postexposure prophylaxis, 271 prevention, 269–271 proteinase 3C, 261–262, 265 R61837, 265 receptor, 262, 264 replication, 260–263 rhinovirus, 260, 264, 265 SCH38057, 265 structure, 260 therapy/treatment, 268–269 transmission, 266 treatment/therapy, 268–269 tremacamra2, 264 vaccine, 269–271 adverse effects, 270 formalin-inactivated, 269 HAVRIX2, 269–270 Vaqta2, 269–270 WIN52035, 265 Hepatitis B virus (HBV), 277–294 3TC (lamivudine, Zefﬁx2), 287 adefovir dipivoxil (ADV), 287, 288 adults, 278 ADV (adefovir dipivoxil), 287, 288 alanine aminotransferase (ALT), 278, 282

Index [Hepatitis B virus (HBV)] anti-HBc, 280–282 anti-HBe, 280–282 anti-HBs, 280–283 bilirubin, 278 cccDNA (covalently closed circular DNA), 283–284 children, 281, 278 cirrhosis, 278, 285, 287 clevudine (L-FMAU), 287, 288 clinical manifestation, 278 covalently closed circular DNA (cccDNA), 283–284 cross-resistance, 287 cytokines, 285 Dane particle, 277 DAPD, 287 diagnosis, 277, 280 emtricitabine (FTC), 288, 287 entecavir (ETV), 287, 288 ETV (entecavir), 287, 288 FTC (emtricitabine), 287, 288 fulminant hepatitis, 278 genotypes, 288 granulocyte-macrophage colony stimulating factor (GMCSF), 287 HB core antigen (HBcAg), 277 HBcAg (HB core antigen), 280 HBe antigen (HBeAg), 277, 282, 289 HBeAg (HBe antigen), 280, 282, 286, 289 HBs antigen (HBsAg), 277, 282, 289 HBsAg (HB surface antigen), 280, 282, 289 HCC (hepatocellular carcinoma), 281, 285 HDV, 285 health care workers, 283 hemodialysis, 283 hepatitis, 277 hepatitis D virus, 285 hepatocellular carcinoma (HCC), 281, 285 HIV, 287 immune globulin (Ig), 280

Index [Hepatitis B virus (HBV)] inﬂammation, 277 interferon, 285, 289 a-interferon, 286 jaundice, 277, 278 lamivudine (3TC, ZefﬁxTM), 284, 287, 289 L-dT (Telbivudine), 288 L-Fd4C, 287, 288 L-FMAU (clevudine), 287, 288 liver failure, 278 liver transplantation, 284 nephrotoxicity, 287 newborns, 278, 281, 283 nucleoside analogs, 284–289 3TC (lamivudine2), 284, 287, 289 adefovir dipivoxil, 287, 288 ADV, 287, 288 clevudine, 287, 288 emtricitabine, 287, 288 entecavir, 287, 288 ETV, 287, 288 FTC, 287, 288 lamivudine (3TC), 284, 287, 289 L-dT, 288 L-Fd4C, 287, 288 L-FMAU, 287, 288 resistance, 287 telbivudine, 288 polymerase, 277, 279, 284, 287 replication, 283–284 resistance, 287 reverse transcriptase, 285, 287 seroconversion, 280, 289 structure, 278 telbivudine (L-dT), 288 therapy/treatment, 282 thymosin, 286 transaminase, 289 transfusion, 283 treatment/therapy), 282 vaccine, 280–283 variants, 288 YMDD motif, 287 mutation, 289

723 [Hepatitis B virus (HBV)] Zefﬁx2 (lamivudine), 284, 287, 289 Hepatitis C virus (HCV), 3, 10–11, 295–368, 545 acute hepatitis C, 299 therapy/treatment, 324, 342 RT-PCR, 302, 304 alanine aminotransferase (ALT), 301 alcohol, 297 amantadine, antiviral efﬁcacy, 338 amantadine, combination, 338–339 Amplicor HCV2, 302, 309, 322 antibody, detection, 296, 299, 300 antibody, testing, 322 antisense, 347–348 antiviral therapy (see therapy/ treatment), 323–352 ascites, 297 autoimmune hepatitis, 322 bDNA (branched DNA), 310, 322 bilirubin, 325 biochemical parameters, 322 blood-screening, 296 branched DNA (bDNA), 310, 322 C100, 299–301 C-100 (see C100), 299–301 C22, 299–301 C33, 299–301 carcinogenesis, 297 CHC (see Chronic hepatitis C) chronic hepatitis C (CHC), 295, 299–304, 318, 319, 325 chronic active hepatitis (CAH), 319 chronic lobular hepatitis (CLH), 319 chronic persistent hepatitis (CPH), 319 grading/staging, 319 histology, 319 treatment, 342 cirrhosis, 295–297, 318, 322, 323, 325 clearance, 323 clinical manifestation, 296–299

724 [Hepatitis C virus (HCV)] CLIP2, 314 combination therapy non-responder, 338 safety proﬁle, 341 virological response, 335, 337 conﬁrmatory assay, 301 consensus interferon, 326–327 contraindications, 326 core antigen assay, 301 core protein, 297, 298 death, 296 detection limit, 304 diagnosis, 299–322 clinical, 319 differential, 299, 322 diagnostics, 299–323 algorithm, 322, 323 enzyme Immunoassays (EIA), 299–301 false positive results, 302, 304 genotyping, 322 histological, 318–322 molecular, 302–318 serological, 299–302 drug users, 313 enzyme immunoassays (EIA), 299– 301 encephalopathy, 297 epidemiology, 296 eradication, 299, 323 false positive results, 302, 304 ﬁbrosis (see Liver ﬁbrosis), 295, 318 GEN-ETI-K2 DEIA, 315, 316 genome, 300, 317 genotype/subtype, 296–297, 304– 309, 313–318, 325, 337–338 genotyping, 322 HAART (highly active antiretroviral therapy), 341 HBV (hepatitis B virus), 296, 297 HCC (hepatocellular carcinoma), 296, 322 HCV-superquant2, 309 helicase inhibitors, 345–347 hepatic failure, 295

Index [Hepatitis C virus (HCV)] hepatitis B virus (HBV), 296, 297 hepatocellular carcinoma (HCC), 296, 297, 322, 323 histology, 297, 298, 318, 319, 322, 324 human immunodeﬁciency virus type 1 (HIV-1), 297 IgG antibody, 299, 300 IgM antibody, 299, 322 immune globulin (Ig), 299 immune response modiﬁer, 351– 352 immunoblotting Matrix HCV2, 300 RIBA2, 300 immunocompromised patient, 304, 340 immunotherapy, 348–351 infection, 295, 298 acute, 304 biochemical course, 300 chronic, 304 cured, 304 resolved, 304 risk, 296 serological course, 300 symptoms, 298 inﬂammation, 319, 322, 323 inﬂammatory hepatitis, 297 Inno-LiPA2, 314–316 interferon-a, 317, 318, 322 pegylated, 327 resistance, 318 side effects, 325–331 alopecia, 326 arthralgia, 326 depression, 326 discontinuation, 326 fatigue, 326 fever, 326 headache, 326 leukopenia, 326 loss of appetite, 326 loss of vision, 326 lumbalgia, 326

Index [Hepatitis C virus (HCV)] myalgia, 326 nausea, 326 retinopathy, 326 thrombocytopenia, 326 tinnitus, 326 weight loss, 326 interferon a-2a, 328–335 interferon a-2b, 324–335 interferon-a monotherapy, 324– 327, 377 interferon sensitivity determining region (ISDR), 317 IRES inhibitors, 345–347 ISDR (interferon sensitivity determining region), 317 Ishak score, 320, 321 jaundice, 295, 297 Knodell score, 319, 320 ligase chain reaction, 304–305 Light Cycler2, 310 liver biopsy, 298, 319 liver damage, 299 histology, 299 molecular classiﬁcation, 299 serological classiﬁcation, 299 ultrasound Imaging, 299 liver enzymes, 322, 324 liver failure, 296, 297 liver ﬁbrosis, 297, 318, 319, 323 Matrix2 HCV, 300 matrix metalloproteinase, 318 METAVIR score, 320, 321 molecular diagnostics, 302–318 qualitative, 302–308 quantitative, 308–313 monoclonal antibodies, 301 mortality, 297 mothers, 304 MUREX HCV2 serotyping 1-6, 314 NAT (nucleic acid testing), 296 nested PCR, 302, 315, 316 newborns, 304 NS3, 299, 300 NS4, 299, 313, 315 NS5A, 317, 318

725 [Hepatitis C virus (HCV)] NS5B, 314, 315 nucleic acid sequence-based ampliﬁcation (NASBA), 307 nucleic acid testing (NAT), 296 organ transplantation, 304 pathogenesis, 297, 298 PCR (see Polymerase chain reaction) peginterferon, 328–340 polymerase chain reaction (PCR), 302, 308, 323 quantitative, 309, 322 polymerase inhibitors, 345–347 portal hypertension, 297 prevalence, 296 primary hepatocellular carcinoma, 295 PRK (RNA-dependent protein kinase), 318 progressive ﬁbrotic disease, 297 protease inhibitors, 345–347 proteomic map, 300 Quantiplex2 HCV RNA, 312 quasi-species, 316 relapse, 324, 337 response rate, 308 reverse transcriptase polymerase chain reaction (RT-PCR), 301–304, 322 RIBA2, 300, 315 ribavirin, 325–332 combination, 332–340 side effects, 325 therapy, 331–332 ribozymes, 348 RNA, 297, 322–328 quantiﬁcation, 308 seropositivity, 300 test, 323, 332 RNA-dependent protein kinase (PRK), 318 RT-PCR (reverse transcriptase polymerase chain reaction), 301, 302, 304, 315, 322

726 [Hepatitis C virus (HCV)] contamination, 302, 315 scoring systems, 319 second-generation enzyme immunoassays (EIA-2), 299 sequencing, 314 serological diagnostics, 299–302, 304 seropositivity, 300 serovonversion, 299 side effects, 328–329 subtypes (see also Genotypes), 313, 316 symptoms of infection, 299 TaqMan2 assay, 310 TGF-beta 1 (transforming growth factor-b1), 318 therapy/treatment, 322–352 acute hepatitis C, 324 chronic hepatitis C, 324–342 combination, 332–340 guidelines, 342–345 monitoring, 308 non-responder, 337 perspectives, 345–352 relapse, 336 third-generation antibody testing, 300 TMA (see Transcription-mediated ampliﬁcation), 305–307, 322 toxic liver damage, 322 transaminase, 324, 333 transcription-mediated ampliﬁcation (TMA), 305– 307, 322, 323 genotypes, 307 sensitivity, 307 transforming growth factor-b1 (TGF-beta 1), 318 transmission, 295, 296, 298 blood products, 296 blood transfusions, 296 colonoscopy, 296 hemodialysis, 296 intravenous drug use, 296 nosocomial, 296

Index [Hepatitis C virus (HCV)] risk, 296 surgery, 296 treatment (see Therapy) triple antiviral therapy, 339–340 TruGene2 HCV, 314, 315 typing, 313–318 genotyping, 314, 315 sequencing, 314 ultrasound imaging, 322 United States, 296 untranslated region (UTR), 300, 302, 314 vaccine, 352 variceal hemorrhage, 297 Versant2 HCV RNA assay, 322, 313 viral load, 308 viremia, 297, 300, 325, 332 virological response, 324, 325, 326, 328–332, 333 serotyping, 313 Hepatitis D virus (HDV), 285 Hepatitis E virus (HEV), 3, 11, 20 Hepatitis F virus (HFV), 3, 12 Hepatitis G virus (HGV), 3, 12 Hepatocellular carcinoma (HCC), 10, 281, 285, 296, 297, 322, 323 Hepatosplenomegaly, 598 Herpes genitalis, 167, 178–180 Herpes gladiatorum, 165 Herpes labialis, 165–166, 178 Herpes simplex virus, 165–192, 371, 441, 559 antivirals, 175–182 aciclovir, 176–177 aminothiazolylphenyl derivatives, 177 BAY 57–1293, 183 foscarnet, 176 immune response modiﬁer, 176 nucleoside analogs, 175–180 helicase-primase inhibitors, 177 diagnosis, 167–172 drug resistance, 176–177, 181– 182

Index [Herpes simplex virus] acyclovir, 176–177, 183 foscarnet, 182 epidemiology, 165–167 exogenous re-infection, 167 fetus, 180 HIV infection, 172, 181 latency, 167 neonate, 171–172, 180–181 pathogenesis, 165–167 pregnancy, 171–172, 180 primary infection, 165, 167 recurrent infection, 167 replication, 175 thymidine kinase, 176 transmission, 165, 170 treatment, 177–180 drug resistance, 181–182 during pregnancy, 180 immunocompromised, 181 neonate, 180–181 trigeminal ganglia, 175 vaccine, 173–174 Herpes zoster, 198–206, 371 Herpesvirus, 3–4, 9, 165–226, 371, 441, 559, 588, 623, 628, 659–705 Herpetic sycosis (folliculitis), 165 HEV (hepatitis E virus), 3, 11, 20 HFMD (hand, foot and mouth disease), 16, 17 HFRS (hemorrhagic fever with renal syndrome), 3, 7 HFV (hepatitis F virus), 3, 12 HGV (hepatitis G virus), 3, 12 HHV-6 (see Human herpes virus 6), 3, 9, 659–705 HHV-7 (see Human herpes virus 7), 3, 9, 659–705 HHV-8 (see Human herpes virus 8), 4, 9, 659–705 High grade squamous intraepithelial lesions (HSIL), 235, 236, 239, 247 Highly active antiretroviral therapy (HAART; see also HIV/AIDS, antiretroviral therapy/

727 [Highly active antiretroviral therapy (HAART; see also HIV/AIDS, antiretroviral therapy/treatment)] treatment), 400, 407, 410, 458, 537, 541, 548, 555, 562, 571–573 HIV/AIDS, 369–585 abacavir (ABC, abacavir, Ziagen2; Trizivir2), 401, 409, 485–488, 515 accessory proteins, 383–386 acquired immunodeﬁciency syndrome (AIDS), 369–380, 399–413, 457, 523–524, 529– 532, 547–548 acute HIV infection, 371, 387–388, 405, 434 acute retroviral syndrome, 387– 388, 405 adherence (compliance), 402, 406, 410, 472 adolescents, HIV infection of, 370, 371, 404 adults, HIV infection of, 370, 371, 375–377, 404 adverse drug effects/events, 405– 412, 487, 508, 524, 531–537, 544–548, 555–559, 567–575 cardiovascular disease, 409, 545 central nervous system, 409, 508 drug interactions, 404–406, 412, 468, 534, 542, 545 gastrointestinal, 409, 487, 545 hepatic steatosis, 409 hepato- (liver-) toxicity, 409, 545– 547 insulin resistance, 409 kidney stones, 545 lactic acidosis, 409 lipid metabolism, 409, 545–546 lipodystrophy syndrome, 409, 410, 546 mitochondrial toxicity, 409, 487

728 [HIV/AIDS] NNRTIs, 409, 508 NRTIs, 409, 487 pancreatitis, 409, 487 polyneuropathy, 409, 487 protease inhibitors, 409, 545– 547 skin rash, 409, 508, 487 Stevens-Johnson syndrome, 508 Agenerase2 (amprenavir, AMP), 401, 540–541, 543–54 AIDS (acquired immunodeﬁciency syndrome), 369–380, 399– 413, 457, 523–524, 529–532, 547–548 algorithm for serological HIV diagnostics, 435, 441, 443 AMPLICOR HIV-1 MONITOR2 test, 450–451, 460, 462, 463 amprenavir (AMP, Agenerase2), 401, 540–541, 543–547 antibodies to HIV (see also HIV/ AIDS-immune response to HIV and HIV/AIDSimmunoassays), 395–396, 433–445 antiretroviral drugs/agents (see also antiviral agents), 399, 400–401, 408, 409–412, 433– 586 antiretroviral therapy/treatment (ART), 373–375, 398–412, 433, 446, 457–461, 468, 514– 516, 541–542 cellular drug targets, 557, 565– 571 combination therapy, 405–407, 411, 458, 506, 512–515, 534–537, 540–543 drug level monitoring, 468–469, 472, 542 drug resistance/susceptibility testing, 464–472, 497–499, 512–515 emerging therapies, 555–586

Index [HIV/AIDS] failure, 406, 410, 458–460, 472, 544 gene therapy, 569–571 guidelines, 373, 392, 399, 402– 405, 441, 457, 460, 472 immunomodulators, 571–572 interruption/discontinuation, 400, 544, 572–574 quality of life, 544 ritonavir-boosted regimen, 534, 541, 545–547 side effects (see HIV/AIDS, adverse drug effects/ events) structures treatment Interruption (STI), 572–574 viral load assays, 445–463 aspartic acid proteases (aspartyl proteases), 524–532 assembly of HIV virions, 383, 386, 526, 564 asymptomatic HIV infection, 371 attachment, 382–386, 566 azidothymidine (AZT, Zidovudine, ZDV; Retrovir2, Combivir2, Trizivir2), 399, 401, 409, 485–494, 512–515, 533–534, 537, 540 AZT (see Azidothymidine, zidovudine, ZDV) branched chain DNA assay (bDNA assay; see also HIV/ AIDS-testing), 399, 403, 446– 448, 457, 461 capsid (CA; see also HIV/AIDSp24), 382–386, 557, 564 CCR5 (see also HIV/AIDS, coreceptors), 383–386, 565– 657 delta-32 allele, 565–566 inhibitors (see also Antiviral agents), 566–567 natural ligands, 565 physiological function, 565

Index [HIV/AIDS] role in viral entry, 566 CD4, 383, 386, 457, 461, 555–560, 565, 569 CD4+ T-lymphocyte count, 370, 387, 391, 399, 402–408, 555, 567, 571–573 CD4+ T-lymphocytes, 370, 389–395, 397 CD8+ T-cells, 394, 396, 572 CDC (U.S. Centers for Disease Control and Prevention), 369–370, 443 chemokines and chemokine receptors (see also HIV/ AIDS-CCR5 and CXCR4), 557, 565–569 chemotherapy (see HIV/AIDSantiretroviral therapy/ treatment) children, HIV infection of, 375–378, 402 chronic HIV infection, 387, 389– 392 clades (see also HIV/AIDS, genotypes, subtypes, and diversity), 380–382, 460– 462, 468–472, 526–527, 542, 559 classiﬁcation system of disease, 370–371 clinical categories, 371 clinical latency, 387, 389 clinical manifestation, 370–371, 382, 387–392, 402–405 combination therapy (see Antiretroviral therapy/ treatment) Combivir2 (azidothymidine + lamivudine; AZT + 3TC), 401, 409, 487 competitive reverse transcription PCR assay for HIV RNA, 451–453 compliance (adherence), 402, 406, 410, 472

729 [HIV/AIDS] conﬁrmatory assays/tests for HIV infection, 434–435, 438, 440– 445 coreceptors (see also HIV/AIDSCCR5 and CXCR4), 383, 386, 470, 557, 565–569 inhibitors (see also Antiviral agents), 565–569 Crixivan2 (indinavir, IDV), 401, 536–537 cross-resistance (see Drug resistance) CTL (see Cytotoxic T-lymphocytes) CXCR4 (see also HIV/AIDS, coreceptors), 383, 565–566, 568–569 inhibitors (see also Antiviral agents), 568–569 natural ligands, 568 physiological function, 565, 568 cyclophilin A interaction with HIV capsid, 557, 564 cyclosporin A, 557, 564 cytochrome P450 (see also HIV/ AIDS, drug interactions, adverse drug effects/ events), 409, 412, 534, 542, 545 cytokines, 571–572 cytomegalovirus infection, 371, 441 cytotoxic T-lymphocyte (CTL), 396–398, 572–573, 576 d4T (stavudine, Zerit2), 401, 409, 485–490, 496–499, 540 DC-SIGN, 393 ddC (Zalcitabine, HIVID2), 401, 409, 485–489, 498–499, 534 ddI (didanosine, Videx2), 401, 409, 485–489, 497–499, 512, 515 delavirdine (DLV, Rescriptor2), 401, 506, 508, 513–514 diagnosis, 382, 398–399, 433–484 diagnostic window in HIV infection, 439, 445 diagnostics, 373, 433–484

730 [HIV/AIDS] didanosine (ddI, Videx2), 401, 487, 485–489, 497–499, 512, 515 disease progression, 399, 405, 434, 457, 524, 536, 565 diversity/ heterogeneity/ variability (see also HIV/ AIDS, genotypes, subtypes and diversity), 380–382, 458, 462, 526–527, 541–542, 559 DLV (see Delavirdine) drugs (see also Antiviral agents), 399, 400–401, 408, 409–412, 433–586 drug interactions (see also HIV/ AIDS, adverse drug effects/ events), 404–406, 412, 468, 534, 542, 545 drug level monitoring, 468–469, 472, 542 drug resistance, 399, 410–411, 433– 434, 457, 461, 464, 497–499, 508–516, 534–544, 559, 567– 570 cross-resistance, 471, 497–499, 512–516, 537, 540, 543–546 multidrug resistance, 411, 497– 499, 537, 540, 515–516, 574 NNRTIs, 508–516 NRTIs, 497–499 protease inhibitors, 534–544 drug resistance/susceptibility testing, 405, 410, 464–472, 497–499, 512–515, 542–544 advantages and disadvantages of assays, 471–472 Antivirogram2, 465, 468–469 clinical beneﬁt, 472, 464, 499, 548 GENChec2, 465–466 GeneSeq2, 465, 466 genotypic assays, 464–468, 471 home brew assays, 466 homologous recombination, 468, 470 Phenoscript2, 465, 470

Index [HIV/AIDS] PhenoSense2, 465, 469–470 phenotypic assays, 468–472 pseudotyped virus, 469 RC assay, 469 recombinant virus, 462, 464, 468– 470 TRUGENE2, 466–466 VERSANT2 HIV-1 protease resistance assay, 465, 467 VERSANT2 HIV-1 RT resistance assay, 465, 467 viral quasi species, 471 ViroSeq2, 465–466 VirtualPhenotype2, 465, 467– 468 drug toxicity (see Adverse drug effects/events) drug users (see Injection drug users) efavirenz (EFV, Sustiva2), 401, 409, 506–508, 512–515, 544, 547 electron microscopy, 526, 533 ELISA (enzyme-linked immunosorbent assay), 398, 435–441 emerging therapies, 555–586 enfuvirtide (T20, Fuzeon2; see also Antiviral agents), 401 entry, 383–386, 556–557, 560–562, 566 inhibitors (see also Antiviral agents), 557, 560–562, 565– 569 envelope glycoprotein (Env; see also HIV/AIDS, gp120; gp41), 383–385, 435, 444, 469–470, 556, 550–561 epidemiology, 373–380, 443, 471 Epivir2 (see also Lamivudine, 3TC), 401, 487 eradication, 400, 573 evolution, 380–382, 567 experimental drugs (see also Antiviral agents), 433–586

Index [HIV/AIDS] ﬁtness/replicative capacity, 411, 469–470, 542–544 Fortovase2 (see also Saquinavir, SQV; Invirase2), 401, 409, 532–534 fusion, 383, 386, 556–557, 560–562, 566 inhibitors (see also Antiviral agents), 406, 557–562 Fuzeon2 (enfuvirtide, T20; see also Antiviral agents), 401 gag (group-speciﬁc antigen)/gag proteins, 383–385, 396, 435, 449–450, 462, 466, 525–526, 529, 564 gene therapy of HIV infection, 569– 571 genotypes (see also HIV/AIDS, clades, diversity, subtypes), 467, 542 genotypic assays for drug resistance/susceptibility testing, 464–468, 471 Gen-Probe HIV-12 viral load assay, 453–454, 462 gp120 (surface glycoprotein, SU), 383–385, 435, 444, 469–470, 556–559 CD4 binding pocket, 559 inhibitors (see also Antiviral agents), 556–559 structure, 559 gp41 (transmembrane glycoprotein, TM), 384, 386, 435, 441, 557, 560–562 fusion peptide, 560 helical region (HR), 560–561 inhibitors (see also Antiviral agents), 557, 560–562 structure, 560, 568 guidelines (see also HIV/AIDS, antiretroviral therapy/ treatment), 373, 399, 392, 402–405, 441, 457, 460, 472

731 [HIV/AIDS] HAART (see Highly active antiretroviral therapy; see also HIV/AIDS, antiretroviral therapy/ treatment) helper T-cells (see CD4+ Tlymphocytes) hepatitis C virus (HCV) infection (see also HIV/AIDS, adverse drug effects/events), 545 herpes simplex virus (HSV), 371, 441, 559 herpes zoster, 371 highly active antiretroviral therapy (HAART; see also HIV/ AIDS, antiretroviral therapy/treatment), 400, 407, 410, 458, 537, 541, 548, 555, 562, 571–573, 573 HIV-1 (see: Human immunodeﬁciency virus type 1) HIV-2 (see Human immunodeﬁciency virus type 2) HIVID2 (see also Zalcitabine, ddC), 401, 487 home HIV antibody test, 434, 440 human immunodeﬁciency virus type 1 (HIV-1), 380–381, 398, 434, 438–440, 443–472 genotypes/groups/subtypes, 380–381, 438–439, 460– 462, 468–472, 526–527, 542, 559 human immunodeﬁciency virus type 2 ( HIV-2), 380–381, 398, 434, 438–444, 455, 511 IDU (see Injection drug users) IDV (see Indinavir) immature particles, 526, 533 immune escape, 396–398 immune response, 395–398, 405, 435, 571–573

732 [HIV/AIDS] immunoassays (see also HIV/AIDS, testing), 433–445, 453, 476 algorithm for serological testing, 435, 441, 443 conﬁrmatory assays/tests, 434– 435, 438, 440–445 cross-reactive antibodies, 439, 443 diagnostic window, 439, 445 home HIV antibody test, 434, 440 immunoﬂuorescence assay (IFA), 444, 437 INNO-LIA, 445 LIA (line immunoassay), 444, 445 licensed/approved assays, 435– 437, 440–445 radioimmunoprecipitation assay/test (RIPA), 444– 445 rapid tests, 434, 440 screening assays/tests, 434–440 western blot, 434–435, 440–444 indinavir (IDV, Crixivan2), 401, 536–538, 540–541, 545 infants, infection of, 374, 457 injection drug users (IDU), 374, 377 innate antiviral pathways, 557, 571 integrase (IN), 383–386, 562–564 enzymatic activities, 562–563 inhibitors (see also Antiviral agents), 562–564 interferon, 557, 570–572 interleukin, 557, 571–572 Invirase2 (saquinavir, SQV; see also Fortovase2), 401, 532–534 Joint United Nations Program on HIV/AIDS (UNAIDS), 376, 379 Kaletra2 (lopinavir/ritonavir, LPV/r), 401, 409, 412, 536– 538, 541 Kaposi’s sarcoma, 369, 372, 390 lamivudine (3TC, Epivir2; Combivir2, Trizivir2,

Index [HIV/AIDS] Zefﬁx2), 401, 409, 485–499, 514–515, 537–540 Latency, 398, 407, 555, 573 LCx HIV RNA assay2, 451–453 lentivirus, 381 licensed/approved immunoassays for HIV diagnostics, 437 lopinavir (LPV, ABT-378), 536–538, 540–541, 544–545, 548 lopinavir/ritonavir (LPV/r, Kaletra2), 401, 406, 412, 536–538, 540–541, 544–548 LPV/r (see Lopinavir/Ritonavir, Kaletra2) lymph nodes, 392–395 lymphadenopathy, 388, 392 lymphoid organs/tissues, 387, 390, 392–393 macrophages in HIV-infection, 389–390, 394–395, 556, 565 maturation of virion, 383, 386, 526 meningitis/meningoencephalitis, 388 microbicides, 374, 516, 558–559 morbidity and mortality, 408, 529, 546–547 mother-to-child transmission, 374, 457, 516 multidrug resistance (see Drug resistance) mutations (see also HIV/AIDS, drug resistance), 405, 410–411, 433, 464–475, 534–538, 540– 544, 564 myalgia, 388 nanoxyl N-9, 374 natural history of infection, 387– 392 nelﬁnavir (NFV, Viracept2), 401, 537–541, 543–545, 547– 548 neutralization of HIV-1 (see also HIV/AIDS, immune response), 558

Index [HIV/AIDS] nevirapine (NVP, Viramune2), 401, 506–509, 512–516 new therapeutic targets, 555–568 NFV (see Nelﬁnavir) NNRTIs (see Non-nucleoside reverse transcriptase inhibitors; see also Antiviral agents) non-nucleoside inhibitor binding pocket (NNIBT), 509 non-nucleoside reverse transcriptase inhibitors (NNRTI; see also Antiviral agents), 401, 405–406, 409, 466–467, 497–499, 505–521 combination therapy (see also HIV/AIDS, antiretroviral therapy/treatment) 514– 516 mechanism of action, 506–612 resistance (see also HIV/AIDS, drug resistance), 508–516 structures, 507 Norvir2 (see also Ritonavir, RTV), 401, 536 NRTI (see Nucleoside reverse transcriptase inhibitor) NtRTI (see Nucleotide reverse transcriptase inhibitor), 401, 485–504 nucleocapsid protein (p6, p9), 383– 386, 557, 564 nucleoside/nucleotide analogs (see also Antiviral agents), 485– 504 nucleoside reverse transcriptase inhibitors (NRTIs; see also Antiviral agents), 401, 405– 406, 409–410, 467, 485–504 clinical use (see also HIV/AIDS, antiretroviral therapy/ treatment), 399–411, 487– 490 mechanism of action, 486 phosphorylation, 493–495

733 [HIV/AIDS] resistance (see also HIV/AIDS, drug resistance), 497–499 nucleotide reverse transcriptase inhibitors (NtRTIs; (see also Antiviral agents), 401, 485– 504 NucliSens2 HIV-1 QT Test, 449, 463, 447–450 number of deaths, 408 NVP (see Nevirapine) opportunistic disease/infections, 382, 395, 404, 412 oral contraceptives, 374 p24 (see also HIV/AIDS, capsid), 385, 396, 435, 439, 441, 462, 564 pandemic, 375–378 pathogenesis, 382–395 PCR (see Polymerase chain reaction) peripheral neuropathy or radiculopathy, 388 PGL (progressive generalized lymphadenopathy ), 370, 390 phenotypic assays in drug resistance/susceptibility testing, 468–470, 471–472 PI (see Protease inhibitors, PRI) pill burden, 409 PMPA (tenofovir), 485, 491, 492 pneumocystis carinii, 369, 372, 390 pneumonia, 369, 371, 372, 404 polymerase (Pol; see also HIV/ AIDS, reverse transcriptase), 383–385, 435, 446–451, 462, 466, 468 polymerase chain reaction (PCR) in HIV diagnostics, 398, 450– 467 post-exposure prophylaxis, 375 PR (see Protease) pregnancy, 374, 402, 439 prevalence, 377

734 [HIV/AIDS] PRI (see Protease inhibitors, PI) primary HIV infection, 371, 387, 397, 402, 405 primate immunodeﬁciency viruses, 380 progressive generalized lymphadenopathy (PGL), 370, 390 protease (PR; see also HIV/AIDS, aspartic acid proteases), 384–385, 399, 401, 405–406, 411, 464–470, 523–532 active site, 524–528, 538, 540, 542–543 structure, 526–527 substrate, 524, 527, 529–531 protease inhibitors (PI, PRI; see also Antiviral agents), 401, 404– 406, 409–412, 466, 469, 523– 554 bioavailability, 533, 537–538, 541 clinical use (see also HIV/AIDS, antiretroviral therapy/ treatment), 399–406, 541– 542 discovery and development, 532–541 dosing, 534, 536–537, 540–541, 546–547 drug level monitoring, 542 drug-interactions (see also HIV/ AIDS, adverse drug effects/events), 412, 541– 542, 546 effects on lipid metabolism, 409– 410, 545–546 ﬁtness of mutants (see also HIV/ AIDS, drug resistance), 544 pharmacokinetic interactions (see also HIV/AIDS, adverse drug effects/events), 412, 534, 541–542, -545–547 protein crystallography, 527, 530–531, 534, 536–538

Index [HIV/AIDS] resistance (see also HIV/AIDS, drug resistance), 534–538, 540, 542–544, 546 sequential use, 548 side effects (see also HIV/AIDS, adverse drug effects/ events), 409–410, 534, 541, 544–545 structure-based drug design, 529–531, 538, 540 trough levels, 536, 547 quantiﬁcation (see also HIV/AIDS, HIV viral load assays), 445– 446, 450, 454–455, 458, 460– 462 quasi-species (see Clades; diversity; subtypes) rapid tests for HIV infection, 434, 440 rash, 388 real-time PCR assay, 454–457 receptor (see also HIV/AIDS, CD4), 383, 470, 565–569 replication, 383, 386, 400, 405, 408, 410–411, 464, 468–470, 526, 533, 542, 548 rescriptor2 (see also Delavirdine, DLV), 401, 598 reservoirs (see also HIV/AIDS, latency), 389, 407, 573 resistance (see Drug resistance) retroviridae, 383 Retrovir2 (Zidovudine, ZDV, Azidothymidine, ZDV, Combivir2, Trizivir2), 401, 409, 487 reverse transcriptase (RT), 384–385, 399, 401, 405, 464–468, 470, 486–489, 505–521 active site, 505 DNA polymerisation reaction, 505–506 enymatic activities, 486–489, 505–506 hydrophobic pocket, 509

Index [HIV/AIDS] mutations and drug hypersusceptibility, 514– 515 NNRTI binding site, 509–511 NNRTIs, 505–521 non-nucleoside inhibitor binding pocket (NNIBT), 509 NRTIs, 485–504 pyrophosphorolytic removal of chain terminator, 514– 515 resistance (see also HIV/AIDS, drug resistance), 497–499, 508–516 RNAse H, 505 structure, 509–513 YMDD-motif, 506 reverse transcriptase polymerase chain reaction (RT-PCR), 398, 403, 450–453 reverse transcription, 383, 386, 505– 506 rifampin, 404, 412 ritonavir (ABT-538, RTV, Norvir2), 401, 412, 534–538, 541, 544– 548 ritonavir-boosted regimen/ pharmacokinetic enhancement, 412, 534, 541, 545–547 RNA (see also HIV/AIDS, viral load), 383–384, 389–391, 398–405, 505–506, 510 RNAse H (see also HIV/AIDS, reverse transcriptase), 505 RT (see Reverse transcriptase) RT-PCR (reverse transcriptase polymerase chain reaction), 398, 403, 450–453 RTV (see Ritonavir, Norvir2) saquinavir (Ro 31–8959, SQV, Fortovase2, Invirase2), 401, 529–539, 541 screening assays/tests, 434–440 seroconversion, 434, 438, 439, 445

735 [HIV/AIDS] serological testing (see also HIV/ AIDS, immunoassays), 435– 445 seroprevalence, 379 set-point (viral), 387, 393, 405 sexually transmitted disease (STD), 373–374, 559 side effects of antiretroviral therapy/treatment (see Adverse drug effects/ events) simian immunodeﬁciency virus (SIV), 380, 381, 393, 394, 511 spermicides (see also HIV/AIDS, microbicides), 374 SQV (see Saquinavir) stavudine (d4T, Zerit2), 401, 485– 487, 485–490, 496–499, 540 structure of the virion, 383, 384 structured treatment interruption (STI; see also HIV/AIDS, antiretroviral therapy/ treatment), 572–574 subtypes, 380–382, 460–462, 468– 472 Sustiva2 (see also Efavirenz, EFV), 401, 598 T-20 (enfuvirtide, fuzeon), 401, 557, 560–562 bioavailability, 562 mechanism of action, 560–561 T-cell mediated immunity, 396– 398 3TC (lamivudine, Epivir2; Combivir2, Trizivir2, Zefﬁx2), 401, 409, 485–499, 514–515, 537–540 TDF (see Tenofovir Disoproxil Fumarate) tenofovir disoproxil fumarate (TDF, Viread2), 401, 406, 409, 485–487, 491–492 testing accuracy, 445, 459–460 alternative strategies, 445

736 [HIV/AIDS] AMPLICOR HIV-1 MONITOR2, 450–451, 460, 462, 463 Antivirogram2, 465, 468–469 assay sensitivity, 434, 436, 438– 440, 444, 459–461 assay variability, 459, 455, 461, 468 branched chain DNA assay (b-DNA), 447–448 competitive reverse transcription PCR assay, 451–453 detection limit, 439, 460, 464, 459, 463 detection of genetic variants, 462 drug level monitoring, 468–469, 472, 542 drug resistance/susceptibility, 464–472, 497–499 dynamic range, 463, 455 emergency situations, 440 enzyme-linked immunosorbent assay (ELISA), 435–441 false-negative results, 438–439 false-positive results, 435, 439 from saliva, 445 from urine, 445 GENChec2, 465–466 GeneSeq2, 465, 466 Gen-Probe HIV-12 viral load assay, 453–454, 462 gold standard, 440, 460 home brew assays for viral drug resistance, 466 indeterminate results, 438, 441, 444, 442 LCx HIV RNA assay2, 451– 453 molecular beacons, 455, 456 NASBA (nucleic acid sequencebased ampliﬁcation) assay, 447, 448–450 nucleic acid based sequence ampliﬁcation (NASBA) assay, 447, 448–450

Index [HIV/AIDS] NucliSens2 HIV-1 QT test, 449, 463, 447–450 PCR (polymerase chain reaction), 450–467 Phenoscript2, 465, 470 PhenoSense2, 465, 469–470 predictive value, 441, 457, 458 real-time PCR assay, 454–457 scorpion probes, 455, 456 TaqMan technology, 455–456 TMA (transcription mediated ampliﬁcation) assay, 453, 447, 454 transcription mediated ampliﬁcation (TMA) assay, 453, 447, 454 TRUGENE2, 466–466 VERSANT2 HIV-1 protease resistance assay, 465, 467 VERSANT2 HIV-1 RNA assay, 460–461, 463, 465, 467 VERSANT2 HIV-1 RT resistance assay, 465, 467 viral load assays, 445–463 ViroSeq2, 465–466 VirutalPhenotype2, 465, 467– 468 western blot, 434–435, 440–444 therapeutic drug monitoring in HIV infection, 468–469, 472, 542 therapy (see Antiretroviral therapy/treatment) thymus in HIV infection, 395 TM (see Transmembrane glycoprotein) topical microbicides, 558–559 transmembrane glycoprotein (TM; see also HIV/AIDS, gp41), 384, 386 transmission, 373–380, 433–434, 457, 464, 516, 542, 559 treatment (see Antiretroviral therapy/treatment)

Index [HIV/AIDS] treatment interruptions (see Antiretroviral therapy/ treatment) triple therapy (see Antiretroviral therapy/treatment) Trizivir2 (azidothymidine + lamivudine + abacavir, AZT + 3TC + ABC), 401, 409, 487 tropism, 383, 387–390, 392–395, 565–568 CCR5-tropic (R-5 tropic, M-tropic) viruses, 565 CXCR4-tropic (R4-tropic, T-tropic) viruses, 565 macrophage tropic viruses (M-tropic, CCR5-tropic, R-5 tropic), 565 switch, 567 T-cell tropic viruses (T-tropic, CXCR4-tropic, R4-tropic), 565 tuberculosis, 404, 372 UNAIDS (see Joint United Nations Program on HIV/AIDS), 376, 379 uncoating, 557, 564 U.S. Centers for Disease Control and Prevention (CDC), 369– 370, 441 vaccine, 399 variants, 462, 464 VERSANT2 HIV-1 RNA assay, 460–461, 463, 465, 467 Videx2, Videx-EC2 (see also Didanosine, ddI), 401, 487 Viracept2 (nelﬁnavir, NFV), 401, 537–540 viral attachment, 382–386, 566 viral dynamics, 389, 394, 408 viral fusion, 383, 386, 556–557, 560– 562, 566 inhibitors (see also Antiviral agents), 405, 557–562 viral latency, 398, 407, 555, 573

737 [HIV/AIDS] viral load (VL; see also HIV/AIDS, RNA), 372, 387, 391, 399– 409, 433–434, 445–465, 469, 524, 533–534, 544, 567– 574 viral load assays, 445–463 accuracy, 445, 459–460 AMPLICOR HIV-1 MONITOR2, 450–451, 460, 462, 463 branched chain DNA assay (b-DNA), 447–448 competitive reverse transcription PCR assay, 451–453 detection of genetic variants, 462 dynamic range, 463, 455 Gen-Probe HIV-12 viral load assay, 453–454, 462 LCx HIV RNA assay2, 451–453 linearity, 459, 461–462 NASBA (nucleic acid sequencebased ampliﬁcation) assay, 447, 448–450 nucleic acid sequence-based ampliﬁcation (NASBA) assay, 447, 448–450 NucliSens2 HIV-1 QT test, 449, 463, 447–450 precision, 459 real-time PCR assays, 454–457 reproducibility, 461 sensitivity, 459–461 signal ampliﬁcation methods, 446–447 speciﬁcity, 459 target ampliﬁcation methods transcription mediated ampliﬁcation (TMA) assay, 447, 453–454 VERSANT2 HIV-1 RNA assay, 460–461, 463, 465, 467 Viramune2 (see also Nevirapine, NVP), 401, 598 Viread2 (see also Tenofovir disoproxil fumarate, TDF), 401, 487

738 [HIV/AIDS] viremia (see also HIV/AIDS, viral load), 387–389, 395–402, 408, 434, 524, 533–536, 544, 573 blips, 408, 458 virological failure (see Antiretroviral therapy/ treatment) virus elimination, 400 VL (see Viral load) western blot, 398, 440–444, 434– 435 women, 376, 377 WWW (World Wide Web), 413, 465, 437 zalcitabine (ddC, HIVID2), 401, 409, 485–489, 498–499, 534 ZDV (zidovudine, azidothymidine, AZT; Retrovir2, Combivir2, Trizivir2), 401, 409, 485–499, 512–515, 533–534, 537, 540 Zefﬁx2 (see Lamivudine, 3TC, Epivir2) 2 Zerit (see also Stavudine, d4T), 401, 487 Ziagen2 (Abacavir, ABC; Trizivir2), 401, 409, 487 zidovudine (ZDV, azidothymidine, AZT; Retrovir2, Combivir2, Trizivir2), 401, 409, 485–499, 512–515, 533–534, 537, 540 zoster, 371 HIV entry inhibitors, 557, 560–562, 565–569 HIVID2 (see also Zalcitabine, ddC), 401, 487 HMPV (human metapneumo virus), 16 Hodgkin’s disease (HD), 635, 642 HPS (hanta virus pulmonary syndrome), 7 HPV (human papillomavirus; see Papillomavirus), 227–258 HSIL (high grade squamous intraepithelial lesions), 235, 236, 239, 247 HSV (see Herpes simplex virus)

Index HSV helicase-primase inhibitors (aminothiazolylphenyl derivatives), 177 HTLV-1 (human T lymphotropic virus type 1), 3, 8 HTLV-2 (human T lymphotropic virus type 2), 3, 8 Human cytomegalovirus (HCMV; see Cytomegalovirus) Human herpes virus 6 (HHV-6), 3, 9, 659–705 aciclovir (ACV), 684 acquired immunodeﬁciency syndrome (AIDS), 660 acute/primary infection, 682 adults, 667 AIDS (acquired immunodeﬁciency syndrome), 660 ampligen [poly(I):poly C12 (U)], 685 biology, 664 CD46, 665 children, 666 chronic persistent infection, 682 cidofovir, 686 clinical pathology/manifestation, 665–669 diagnosis, 682–684 effect on cardiovascular system, 669 effect on central nervous system, 668–669 effect on lymphatic and hematopoietic system, 667– 668 epidemiology, 678–679 exanthema subitum, 660, 666 fever, 666 foscarnet, 685 ganciclovir (GCV), 684–685 genomic organization, 661–663 GS isolate/strain, 660 b-herpesvirus subfamily, 661 herpes virus, 660 highly active antiretroviral therapy (HAART), 685

Index [Human herpes virus 6 (HHV-6)] interferon, 686 Kikuchi-Fujimoto’s disease (KFD), 667 kutapressin (KU), 686 lymphadenopathy, 666 lymphoma, 667 lymphoproliferative disease, 666 molecular biology, 661–666 morphology, 661 multiple sclerosis (MS), 660 pharyngitits, 666 rash, 666 reactivation of infection, 668, 682 receptor, 665 salivary gland, 665 transfer factor, 686 transplant recipients, 667 treatment/therapy, 684–686 U1102 isolate/strain, 659 valaciclovir, 686 Z-29 isolate/strain, 659 zidovudine, 685 Human herpes virus 7 (HHV-7), 3, 9, 659–705 aciclovir (ACV), 684–685 acquired immunodeﬁciency syndrome (AIDS), 660 ampligen [poly(I):poly C12 (U)], 686 biology, 669–670 CD4, 669 chronic fatigue syndrome, 660 cidofovir, 686 clinical pathology/manifestation, 670–672 diagnosis, 682–684 effect on central nervous system, 672 effect on lymphatic and hematopoietic system, 671 effect on skin, 671 epidemiology, 678–680 exanthema subitum, 671

739 [Human herpes virus 7 (HHV-7)] foscarnet, 685 ganciclovir (GCV), 684–685 genomic organization, 661–663 b-herpesvirus subfamily, 661 herpes virus, 660 highly active antiretroviral therapy (HAART), 685 interferon, 686 Kawasaki’s disease, 672 kutapressin (KU), 686 lymphoma, 671 lymphoproliferative disorders/ disease, 671 morphology, 661 systemic effects, 670–671 therapy/treatment, 684–686 transfer factor, 686 transplant recipients, 672 treatment/therapy, 684–686 valaciclovir, 686 zidovudine, 685 Human herpes virus 8 (HHV-8), 4, 9, 659–705 aciclovir (ACV), 684–685 acquired immunodeﬁciency syndrome (AIDS), 660, 674– 675 ampligen [poly(I):poly C12 (U)], 686 biology, 672–676 Castleman’s disease, 672, 677 cidofovir, 686 clinical pathology/manifestation, 674–678 cytokines, 673 diagnosis, 674, 678, 682–684 epidemiology, 680–682 foscarnet, 685 ganciclovir (GCV), 684–685 genomic organization, 661–663 g-herpesvirus subfamily, 670 herpes virus, 660 highly active antiretroviral therapy (HAART), 685 IL-6, 677

740 [Human herpes virus 8 (HHV-8)] immunosuppressed/ immunocompromised patients, 674, 677–678 interferon, 686 Kaposi’s sarcoma (KS), 660, 674– 675 Kaposi’s sarcoma-associated herpes virus (KSHV), 660 KS (Kaposi’s sarcoma), 672–673 KSHV (Kaposi’s sarcomaassociated herpes virus), 660 kutapressin (KU), 686 lymphadenopathy, 677 lymphoma, 673, 675–676 lymphoproliferative disease/ disorder, 677 morphology, 661 MS (multiple sclerosis), 660 multiple sclerosis (MS), 660 rhadinovirus, 660 seroprevalence, 680–681 transfer factor, 686 transmission, 681–682 transplant recipients/patients, 678 treatment/therapy, 684–686 tumor, 673, 677 zidovudine, 685 Human immunodeﬁciency virus (HIV; see also HIV/AIDS), 3, 20, 228–235, 287, 369–586, 589 clades/subtypes/genetic diversity, 8, 380–382, 438–439, 458, 460–462, 468–472, 526–527, 541–542, 559 structure of the virion, 383, 384 type 1 (HIV-1), 297, 380–381, 398, 434, 438–440, 443–472 type 2 (HIV-2), 380–381, 398, 434, 438–444, 455, 511 Human metapneumo virus (hMPV), 16 Human papillomavirus (HPV; see also Papillomavirus), 227–258 Human T-cell leukemia virus (HTLV), 8

Index Human T-cell lymphotropic virus (HTLV), 3, 8 Hyperimmune globulin, 603 ICAM-1, soluble, (BIRR 4), 147, 152– 154 IDV (see Indinavir) IL-12 (interleukin 12), 350–351, 557, 571–572 IL-2 (interleukin 2), 350, 557, 571 Imiquimod (1-(2-methylpropyl)-1Himidazol[4, 5-c]quinolin-4amine; Aldara2, Zartra2), 238 Immune globulin (Ig), 107–109, 207– 209, 271 Immune response modiﬁer, 351–352 Immunotherapy, 642 IMPDH inhibitors, 109–112 Indinavir (IDV, Crixivan2), 401, 536– 538, 540–541, 545 Infectious mononucleosis, 628–635 Inﬂuenza virus, 4, 13–15, 20, 32, 3990 amantadine, 57–61 antivirals (see also Antiviral agents), 57–67 Asian ﬂu, 72 children, 47, 48, 52, 54–56, 60, 6166, 67, 71 clinical manifestation, 54–56 diagnosis, 40, 54–57 elderly, 40, 47, 54–56, 66, 68 epidemiology, 46–48, 72–73 evolution, 69, 48–50 hemagglutinin, 41–44, 67 Hong Kong ﬂu, 72 host range, 50–52, 55 ion channel, 42–43, 58–60 M2, 57–61 blockers, 42–43, 58–60 molecular biology, 41–46 neuraminidase, 41–44, 61–67 inhibitors, 41–44, 61–63 oseltamivir (Tamiﬂu2), 58, 61–67 pandemic management, 72–73 pathogenesis, 52–54

Index [Influenza virus] prophylaxis and treatment, 6067 Relenza2, 61–67 replication, 41–46 reservoirs, 46–52 resistance, 59–60, 64–65 rimantadine, 57–61 sialic acid, 58, 62 Spanish ﬂu, 72 structure, 13–15, 41–46 subtypes, 41–46, 57 Tamiﬂu2, 61–67 transmission, 52–54 treatment, 60–61, 63–64, 66–67 vaccine, 67–71 zanamivir (Relenza2), 58, 61–67 Integrase inhibitors, 557, 562–564 Interferon, 11, 117–118, 145–148, 155– 157, 269, 285–289, 317–318, 557, 570–572, 635, 686 a11, 155–157, 286, 318, 322–327, 337 a-2a, 328–329 a-2b, 324, 329–331 pegylated/peginterferon, 327–340 Intraepithelial neoplasia, 228 Intravenous immunoglobulin (IVIG), 599, 602 Invirase2 (saquinavir, SQV; see also Fortovase2), 401, 532–534 Ipratropium, 148 IVIG (intravenous immunoglobulin), 599, 602 Jaundice, 259, 267, 268, 277, 278, 295, 297, 598, 630 Kaletra2 (lopinavir/ritonavir, LPV/ r), 401, 409, 412, 536–538, 541 Kaposi’s sarcoma associated herpes virus (KSHV; see also Human herpesvirus 8, HHV-8), 9, 10, 660 Kaposi’s sarcoma (KS), 4, 9, 369, 372, 390, 660, 672–675

741 Kawasaki’s disease, 672 Keratitis, 165 Kikuchi-Fujimoto’s disease (KFD), 667 Korean hemorrhagic fever, 7 KS (Kaposi’s sarcoma), 4, 9, 660, 672– 675 KSHV (Kaposi’s sarcoma associated herpes virus; see also Human herpesvirus 8, HHV-8), 4, 9– 10, 660 Kutapressin (KU), 686 Lamivudine (3TC; Epivir2, Zefﬁx2; Combivir2, Trizivir2), 284– 289, 401, 409, 485–499, 514– 515, 537–540 Lassa virus, 7 LAV (lymphadenopathy-associated virus), 8 L-dT (telbivudine), 288 Lentivirinae, 8, 381 L-Fd4C, 287, 288 L-FMAU (clevudine), 287, 288 Liver failure, 278, 296, 297 Liver ﬁbrosis, 297, 318, 319, 323 Liver transplantation, 284 Lopinavir (LPV, ABT-378), 536–538, 540–541, 544–545, 548 Lopinavir/ritonavir (LPV/r, Kaletra2), 401, 406, 412, 536– 538, 540–541, 544–548 Lovastatin, 116 Low grade squamous intraepithelial lesions (LSIL), 235, 236 LPV/r (see Lopinavir/ritonavir, Kaletra2) LY253963, 112 Lymphadenopathy, 388, 392, 629, 634, 637, 666, 677 Lymphadenopathy-associated virus (LAV; see also Human immunodeﬁciency virus, HIV), 8 Lymphatic and hematopoietic system disease, 671, 667–668

742 Lymphoma, 3, 4, 624, 634, 635, 642, 667–668, 671, 673, 675–676 Lymphoproliferative disease/ disorder, 634–635, 666, 671, 677 Malaise, 55 Marburg virus, 6 Meningitis/meningo-encephalitis, 4, 15, 17, 388 Metapneumo virus, 4, 16, 20 a-Methyl-1-adamantane methylamine hydrochloride (rimantadine), 57–60 Methylenecyclopropane nucleoside analogs, 491 60 -(R-60 -C-Methyleneplanocin) A, 112 Metoclopramide, 269 Microbicides, 374, 516, 558–559 Microcephaly, 598 MIV-150, 508 Mizoribine (4-carbamoyl-1-b-Dribofuranosylimidazolium-5olate), 111 Mononegavirales, 18 Mononucleosis, 587 Multiple sclerosis (MS), 660 Myalgia, 54, 388 Myelopathy, 8 Myocarditis, 56, 630 9238X, 113 N-[5-(aminosulfonyl)-4-methyl-1,3thiazole-2-yl]-N-methyl-2-[4(2-pyridinyl)phenyl]acetamide (BAY 57-1293, HSV helicaseprimase inhibitor), 177 Naphtalene sulfonate polymer (see PRO 2000) Naproxene, 148 Nasopharyngeal carcinoma (NPC), 636–639 Nelﬁnavir (NFV, Viracept2), 401, 537–541, 543–545, 547–548 Neonatal herpes, 166, 180–181 Nephropathia epidemica, 3, 7

Index Neplanocin, 113 Neurodegenerative disease, 18 Neuropsychiatric disease, 4 Neutropenia, 601 Nevirapine (NVP, Viramune2), 401, 506–509, 512–516 New variant-Creutzfeldt-Jakob disease (nv-CJD), 18, 19 NFV (see Nelﬁnavir) Nipah virus, 4, 16, 20 NMSO3, 116 NNRTIs (non-nucleoside reverse transcriptase inhibitors), 401, 497–499, 505–521 NNY-RANTES, 567 Non-A, non-B hepatitis (see also Hepatitis C virus), 3, 10, 11 Non-Hodgkin’s lymphoma (see also Lymphoma), 10 Non-nucleoside reverse transcriptase inhibitors (NNRTIs), 401, 497– 499, 505–521 Non-steroidal antiinﬂammatory drugs (NSAID), 148 5’-Noraristeromycin, 113 Norovirus, 2 Norvir2 (see also Ritonavir, RTV), 401, 536 Norwalk virus, 2 Norwalk-like virus, 2 NPC (nasopharyngeal carcinoma), 636–639 NRTI phosphorylation, 493–495 NRTIs (nucleoside reverse transcriptase inhibitors), 401, 485–504 NtRTIs (nucleotide reverse transcriptase inhibitors), 401, 485–504 Nucleoside/nucleotide analogs, 109– 113, 238, 284–289, 485–504, 628, 643 Nucleoside reverse transcriptase inhibitors (NRTIs), 401, 485– 504

Index Nucleotide reverse transcriptase inhibitors (NtRTIs), 401 nv-CJD (new variant-CreutzfeldtJakob disease), 4, 18, 19 NVP (see Nevirapine), 401 Oncovirinae, 8 Ophthalmic zoster, 203 Opportunistic disease/infections, 382, 395, 404, 412 Oral hairy leukoplakia, 632 Orthomyxoviridae, 13 Orthopoxviridae, 17 Oseltamivir [3R,4R, 5S-4-acetamido5-amino-3-(1-ethylpropoxyl)1-cyclohexene-1-carboxylic acid; Tamiﬂu2], 58, 61–67 Otitis media, 56, 70, 55, 98, 140 PALA [N-(phosphonoacetyl)-Laspartate], 112 Palivizumab, 107 Papillomavirus, 227–258 anal cancer, 233 anogenital warts, 232 antigen-presenting cells (APC), 242 atypical squamous cells of undetermined signiﬁcance (ASCUS), 236 capsid, 229, 241, 243 carcinoma, 232, 234 carcinoma in situ, 234 cervical cancer, 228, 233, 235, 245 cervical intraepithelial neoplasia (CIN), 233, 236 cervix, 227, 234, 237 CIN (cervical intraepithelial neoplasia), 233, 236 common warts, 228, 231 condyloma, 228, 232, 233, 236 CTL (cytotoxic T-lymphocytes), 240, 241, 245, 246 cytology, 230, 236, 237 cytotoxic T-lymphocytes (CTL), 240, 241, 245, 246 diagnosis, 230, 232, 235–236

743 [Papillomavirus] Digene Hybrid Capture2, 237 E6, 229, 240–243 E7, 230, 240–243, 245 epidemiology, 228 epidermodysplasia verruciformis (EV), 228, 231, 239, 232 genital warts, 232, 233, 245 guidelines, 237 high grade squamous intraepithelial lesions (HSIL), 235, 236, 239, 247 high risk, 227, 233 HIV-positive patients, 231, 233, 235 HPV typing, 237 HPV6, 227, 228, 233 HPV11, 227, 228, 233 HPV16, 227, 228, 233, 234, 239– 247 HPV18, 227, 228, 233, 234, 246 HSIL (high grade squamous intraepithelial lesions), 235, 236, 239, 247 immune response, 239–248 intraepithelial neoplasia, 228 L1, 241, 243, 244 L2, 243, 245 low grade squamous intraepithelial lesions (LSIL), 235, 236 low risk, 227, 233 LSIL (low grade squamous intraepithelial lesions), 235, 236 major histocompatibility complex (MHC), 240, 243 MHC, 240, 243 neutralizing antibody, 241, 244 Pap smear, 235, 238 Papanicolaou, 230, 234 pathology, 231–235 plantar warts, 228, 232 prophylactic vaccine, 243, 244 screening, 228, 230, 234, 235, 236, 237

744 [Papillomavirus] SIL (squamous intraepithelial lesions), 233, 235, 241, 245, 247 therapeutic vaccine, 243, 244 transmission, 228 transplant patients, 231, 235 treatment, 237–239 50 -ﬂuorouracil, 238 imiquimod, 238 interferon, 238 laser, 238 LEEP, 238 podophyllin, 238 triage, 236, 237 vaccine, 241–248 DNA, 244, 246, 248 fusion protein, 244, 245 peptide, 244, 245, 246, 248 VLP (virus-like particles), 241– 245, 248 virus-like particles (VLP), 241–245, 248 vulvar intraepithelial neoplasia (VIN), 233 wart, 227, 228, 229, 237, 245 Paramyxoviridae, 12, 16, 112 Parotid carcinoma, 640 Parvoviridae, 6 Parvovirus B19, 3, 6 Patients/risk groups adolescents EBV, 629 hepatitis A virus, 269 HIV infection, 370–371, 404 adults CMV, 587 EBV, 629 hepatitis A virus, 269 hepatitis B virus, 278 HHV-6/-7/-8, 667 HIV infection, 370–371, 375–377, 404 inﬂuenza, 48 respiratory syncytial virus, 91– 92, 98

Index [Patients/risk groups] varicella zoster virus, 196 asthma rhino virus, 140 children CMV, 588 EBV, 629 hepatitis A virus, 269–270 hepatitis B virus, 278, 281 HHV-6/-7/-8, 666 HIV infection, 375–378, 402 inﬂuenza, 47–48, 52–71 papillomavirus, 227 respiratory syncytial virus, 93, 99 rhino virus, 139–140 varicella zoster virus, 194, 208 chronic heart disease respiratory syncytial virus, 91, 108–110 clotting factor disorders hepatitis A virus, 269 COPD rhino virus, 140 cystic ﬁbrosis respiratory syncytial virus, 91, 109 rhino virus, 140 drug users hepatitis A virus, 269 HIV infection, 374, 377 elderly EBV, 629, 632 inﬂuenza, 40, 47, 55, 66–68 respiratory syncytial virus, 100, 105–106 varicella zoster virus, 195–201, 204 fetus herpes simplex virus, 180 HIV infection, 374 health care workers hepatitis B virus, 283 HIV infection, 375 inﬂuenza, 69 hemodialysis hepatitis B virus, 283

Index [Patients/risk groups] hepatitis hepatitis B virus, 277 HIV-infection, 545 hepatocellular carcinoma (HCC), hepatitis B virus, 281, 285 HIV, hepatitis B virus, 287 immunocompromised cytomegalovirus, 594–607 Epstein-Barr-virus, 624, 634, 641– 642 hepatitis C virus, 304, 340 HHV-6/-7/-8, 667, 672, 674, 677– 678 respiratory syncytial virus, 109, 110, 111 varicella zoster virus, 195–198, 201, 207, 371 immunocompromised/HIV Epstein-Barr-virus, 634, 635 herpes simplex virus, 172, 181, 371, 441, 559 HHV-6/-7/-8, 660, 674–675 papillomavirus, 231, 233, 235 varicella zoster virus, 206 infants HIV infection, 374, 457 inﬂuenza, 55 respiratory syncytial virus, 92, 93, 105, 108, 110, 111 rhino virus, 140 liver failure, hepatitis B virus, 278 liver transplantation, hepatitis B virus, 284 men, hepatitis A virus, 269 mothers, hepatitis C virus, 304 mother-to-child transmission, HIV, 374, 457, 516 neonate (see also Patients/risk groups, newborns) cytomegalovirus, 587 herpes simplex virus, 171–172, 180–181 nephrotoxicity, Hepatitis B Virus, 287

745 [Patients/risk groups] newborns (see also Patients/risk groups, neonates) hepatitis C virus, 304 hepatitis B virus, 278, 281, 283 pregnancy cytomegalovirus, 597, 598 herpes simplex virus, 171–172, 180 HIV, 374, 402, 439 transplant patients/recipients (see also Patients/risk groups, immunocompromised) cytomegalovirus, 594–607 Epstein-Barr-virus, 624, 634, 641– 642 HHV-6/-7/-8, 667, 672, 678 respiratory syncytial virus, 110, 111 women herpes simplex virus, 174 HIV/AIDS, 376–377 papillomavirus, 228, 233 Penciclovir, 175–176, 211 Peptide T, 557 Pericarditis, 56 Peripheral neuropathy or radiculopathy, 388 PFA (see Phosphonoformic acid, foscarnet) Phenyldicarboxylic acid (see BRI2923) Phosphonoformic acid (foscarnet, PFA), 506 PI (see Protease inhibitors, PRI) Picornaviridae, 16–17 Pirodavir, 145 Plantar warts, 228, 232 Pleconaril (VP63843), (3-[3,5dimethyl-4-[[3-(3-methyl-5isoxazolyl)propyl]oxy]phenyl]-5(triﬂuoromethyl)-1,2,4oxadiazole), 149–152, 265 PMEA (adefovir), 491, 492 PMPA (tenofovir), 485, 491, 492

746 Pneumocystis carinii infection, 369, 372, 390 Pneumonia, 68, 56, 99, 110, 196, 369– 372, 404, 604, 607 Pneumonitis, 3, 9 PNU-140690 (see Tipranavir) Podophyllin, 238 Polyanionic compounds, 558 POM (polyoxometalates), 114 Porphyria cutanea tarda, 279 Postherpetic neuralgia, 212–214 PRI (see Protease inhibitors, PI) Primary hepatocellular carcinoma, 295 Primate immunodeﬁciency viruses, 380 Prion, 4, 18 PRO 140, 557, 567 PRO 2000, 557, 559 PRO 542, 556–558 Prodrugs, 491, 494–497, 547 Progressive generalized lymphadenopathy (PGL), 370, 390 Protease inhibitors (PI, PRI), 401, 404– 406, 409–412, 469, 523–554 Puumala virus, 7, 3 Pyrazofurin [3-(b-D-ribofuranosyl)-4hydroxypyrazole-5carboxamide], 112, 113 R61837, 265 RD3–0028 [1,4-dehydro-2,3bonzodithiin], 116 Reactive airway disease, 99, 140 Relenza2 (Zanamivir), 60–61 Reoviridae, 5 Rescriptor2 (see also Delavirdine, DLV), 401, 598 Resiquimod, 176 RespiGam2, 107 Respiratory syncytial virus (RSV), 91–138 antivirals, 107–118 antibodies, 107–109

Index [Respiratory syncytial virus (RSV)] chemotherapeutic agents, 109– 117 interferons, 117–118 attachment inhibitors, 114 children, 93, 99 chronic heart disease, 91, 108, 110 cystic ﬁbrosis, 91, 109 diagnosis, 100–101 economic impact, 91–92 elderly, 100, 105, 106 epidemiology, 93 F-protein inhibitors, 114–116 immunocompromised, 109 IMPDH, 109 infants, 110, 92, 111, 93, 105, 108 inosine monophosphate dehydrogenase, 109 molecular biology, 93–97 nosocomial infection, 93 pathogenesis, 97–100 replication, 96–97 ribavirin, 110–111 S-adenosyl homocysteine hydrolase, 109–113 SAH, 109–113 transplant patients, 110, 111 vaccine, 102–107 Retinitis, 598 Retroviridae, 8, 380–386 Retrovir2 (zidovudine, ZDV, azidothymidine, AZT; Combivir2, Trizivir2), 401, 409, 487 Reye’s syndrome, 56, 70, 196 RFI 641, 115 Rhinovirus, 139–164 3C protease, 142, 149, 154 asthma, 140 canyon, 142 common cold, 139 COPD, 140 cystic ﬁbrosis, 140 diagnosis, 144 economic impact, 141 epidemiology, 139–140

Index [Rhinovirus] hydrophobic pocket, 142, 150 interferon, 155–157 interferon, intranasal, 145–147, 155–156 non-steroidal antiinﬂammatory drugs (NSAID), 148 pathogenesis, 139–143 pro-inﬂammatory mediators, 142–143 pleconaril, 149–152 adverse effects, 151 prophylaxis, 145, 146 replication, 141, 142, 154 resistance, 151–152 ruprintrivir (AG7088), 147, 154–155 transmission, 141 Tremacamra2, 152–154 Ribavirin (1-b-D-ribofuranosyl-1,2,4triazole-3-carboxamide; Cotronak2, Rebetol2, Virazole2, Vilona2, Viramid2, Virazide2), 11, 110–111, 325, 331–340 1-b-D-ribofuranosyl-1,2,4-triazole-3carboxamide, 110, 111 Ribozymes, 348, 557, 570–571 Rimantadine (a-methyl-1adamantane methylamine hydrochloride; Flumadine2), 57–60 Ritonavir (ABT-538, RTV, Norvir2), 401, 412, 534–538, 541, 544–548 Rituximab, 635 Ro 31–8959 (see also Saquinavir, SQV), 532, 539 Roseola infantum, 3, 9 Rotavirus, 3, 5, 20 RSV (respiratory syncytial virus), 91– 138 RTV (see Ritonavir, Norvir2) Ruprintrivir (AG 7088), 147–149, 154– 155, 265 RWJ-270201, 63 S-1360, 557, 562

747 Sabia virus, 3, 7 Sapovirus, 2 Sapporo virus, 3 Sapporo-like virus, 2 Saquinavir (Ro 31–8959, SQV, Fortovase2, Invirase2), 401, 529–539, 541 SCH38057, 265 SCH-C, 557, 567 Scrapie, 18 SDS NIM811, 557, 564 Sexually transmitted disease (STD), 373–374, 559 SIL (squamous intraepithelial lesions), 233, 235, 241, 245, 247 Simian immunodeﬁciency virus (SIV), 8, 380–381, 393–394, 511 Sin nombre virus, 3, 7, 20 Single chain antibodies (SFv), 570 Sinusitis, 140 SIV (simian immunodeﬁciency virus), 8 Small round structure virus (SRSV), 3 Soluble CD4 (sCD4), 556–558 Splenomegaly, 629 Squamous intraepithelial lesions (SIL), 233, 235, 241, 245, 247 SQV (see Saquinavir) SRSV (small round structure virus), 3 Stavudine (d4T, Zerit2), 401, 485– 487, 485–490, 496–499, 540 Steroids, 633 Sustiva2 (see also Efavirenz, EFV), 401, 598 Synergis2, 107 T1249, 557, 560–561 T-134, 568 T-140, 568 T-20 (enfuvirtide, Fuzeon2), 557, 560–562 T-22, 557, 568 TAK-779, 557, 567 Tamiﬂu2 (Oseltamivir), 58, 60–61 T-cell lymphoma (see also Lymphoma), 640

748 3TC (2’,3’-dideoxy-3’-thiacytidine, lamivudine, Epivir2; Combivir2, Trizivir2, Zefﬁx2), 287, 401, 409, 485– 499, 514–515, 537–540 TDF (tenofovir disoproxil fumarate), 401, 406, 409, 485–487, 491–492 Tenofovir (PMPA), 485, 491, 492 Tenofovir disoproxil fumarate (TDF, Viread2), 401, 406, 409, 485– 487, 491–492 Thrombocytopenia, 630 TIBO (tetrahydroimidazobenzodiazepinone), 512 Tipranavir (U-140690, PNU-140690), 547 TMC120, 508 TMC125, 508 Topical microbicides, 558–559 Topoisomerase I inhibitors, 557 Topotecan, 557 TPMV (tupaia paramyxovirus), 16 Tracheobronchitis, 55 Transfer factor, 686 Transfusion transmitted virus (TTV), 4, 12 Transmissible bovine spongiform encephalopathy, 18 Tremacamra2 (soluble ICAM-1), 147, 152–154, 264 Trizivir2 (azidothymidine + lamivudine + abacavir, AZT + 3TC + ABC), 401, 409, 487 Tropical spastic paraparesis (TSP), 8 TSAO (tertbutyldimethylsilylspiroaminooxathioledioxidethymines), 512 TTV (transfusion transmitted virus), 4, 12 Tuberculosis, 404, 372 Tumor, 631, 636–640, 644, 673, 677 Tupaia paramyxovirus (TPMV), 16 U-140690 (see Tipranavir) UC781, 513–514, 516

Index Vaccines Epstein-Barr virus (EBV), 641– 642 hepatitis A virus (HAV), 269–271 adverse effects, 270 formalin-inactivated, 269 HAVRIXTM, 269–270 Vaqta2, 269–270 hepatitis B virus (HBV), 280–283 hepatitis C virus (HCV), 352 hepatitis E virus (HEV), 12 herpes simplex virus (HSV), 173– 174 DNA, 174 glycoproteins (dD2, gD2), 173 live virus, 174 human papillomavirus (HPV) 243– 248 DNA, 244, 246, 248 fusion protein, 244, 245 peptide, 244, 245, 246, 248 virus-like particles (VLP), 241– 245, 248 inﬂuenza virus, 67–71 live-attenuated, 70–71 subunit, 67–68 respiratory syncytial virus (RSV), 102–107 DNA, 107 glycoprotein, 105, 106 live virus, 104–105 recombinant, 105 subunit, 105–107 rotavirus, 5 varicella zoster virus (VZV), 208– 209 live attenuated virus, 208 Vaccinia virus (VV), 17 Valaciclovir, 175–176, 210–211, 686 Valganciclovir, 592 Variceal hemorrhage, 297 Varicella (chickenpox), 193 Varicella zoster virus (VZV), 193226 analgesics, 211 anticonvulsants, 211

Index [Varicella zoster virus (VZV)] antivirals (see also Antiviral agents), nucleoside analogs, 210–212 central nervous system complications, 196 chickenpox (varicella), 196–198 corticosteroids, 212 diagnosis, 206–207 embryopathy, 197 epidemiology, 193–194, 214–215 immunocompromised host, 195– 198, 201 latency, 195, 196 pathogenesis, 195–206 postherpetic neuralgia (PHN), 211– 214 treatment, 211–214 pregnancy, 196–197 prevention, 207–209 primary infection, 196–198, 200 sensory ganglia, 195–206 shingles (see also Zoster), 193 transmission, 193–194 treatment, 207–215 immunocompromised, 207, 208, 215 PHN, postherpetic neuralgia, 212–214 zoster, 209–212 tricyclic antidepressants, 211 vaccine, 208–209 varicella (chickenpox), 193 zoster, 198–206 Bell’s palsy, 212 clinical manifestations, 204 dermatome, 197–203 elderly, 195–201, 204 ophthalmic, 203 Vasculitis, 279 Videx2, Videx-EC (see also Didanosine, ddI), 401, 487 Viracept2 (nelﬁnavir, NFV), 401, 537–540

749 Viramune2 (nevirapine, NVP), 401, 598 Virazole2 (ribavirin), 110–111 Viread2 (see also Tenofovir disoproxil fumarate, TDF), 401, 487 VP14637, 115 VP63843 (pleconaril), 149–152, 265 Vulvar intraepithelial neoplasia (VIN), 233 VV (see Vaccinia virus) VX497, 112 VZV (varicella zoster virus), 193– 226 Wart, 227–229, 237, 245 West Nile encephalitis virus (WN virus), 4, 17, 20 Whitewater Arroyo virus, 4, 7 WIN52035, 265 WN virus (West Nile encephalitis virus), 4, 17 Zalcitabine (ddC, HIVID2), 401, 409, 485–489, 498–499, 534 Zanamivir (see Relenza2), 58, 61– 67 Zartra2 (see Imiquimod) ZDV (see Zidovudine) Zefﬁx2 (see Lamivudine, 3TC; other trade name: Epivir2), 284, 287–289 Zerit2 (see also Stavudine, d4T), 401, 487 Ziagen2 (abacavir, ABC; Trizivir2), 401, 409, 487 Zidovudine (ZDV; azidothymidine, AZT; Retrovir2, Combivir2, Trizivir2), 401, 409, 485–499, 512–515, 533–534, 537, 540, 635, 685 Zintevir (see AR177) Zoster (see Herpes zoster)

About the Editors

¨ BSAMEN-WAIGMANN is Vice President, Head of AntiHELGA RU infective Research, Bayer HealthCare, Wuppertal, and Professor of Biochemistry, University of Frankfurt, Frankfurt, Germany. The author, coauthor, or coeditor of numerous professional publications including Antivirals Against AIDS (Marcel Dekker, Inc.), Dr. Ru¨bsamen-Waigmann served on the boards of Gesellschaft Deutscher Chemiker, the German AIDS Society, and the German Society of Virology. She is also a member of the European Research Advisory Board. She received the Diploma (1971) and Ph.D. degree (1973) in chemistry from the University of Mu¨nster, Germany, and the Habilitation in biochemistry and virology (1983) from the University of Frankfurt, Germany. KARL DERES is Senior Scientist, Bayer HealthCare, Wuppertal, Germany. He is the author or coauthor of several original papers, reviews, and abstracts. Dr. Deres is a member of the Gesellschaft Deutscher Chemiker, Vereinigung fu¨r Allgemeine und Angewandte Mikrobiologie, and Verein zur Fo¨rderung der Fachhochschule Remagen. Dr. Deres received the Diploma (1989) in microbiology and Ph.D. degree (1992) in immunochemistry from the University of Tu¨bingen, Germany 751

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About the Editors

and a fellowship of the Studienstiftung des Deutschen Volkes (1989– 1992). GUY HEWLETT is Principal Scientist, Bayer HealthCare, Wuppertal, Germany. The author, coauthor, or coeditor of several book chapters, reviews, and abstracts, he is a member of the American Society of Microbiology. Dr. Hewlett received the B.S. degree (1969) in biological science from the University of East Anglia, Norwich, England, and the Ph.D. degree (1972) from the University of London, England. REINHOLD WELKER is Senior Scientist, Bayer HealthCare, Wuppertal, Germany. The author or coauthor of several professional publications, Dr. Welker is a member of the German Society of Virology and the International Society of Antiviral Research. Dr. Welker studied molecular biology at the State University of New York at Stony Brook, New York, and graduated from the Medical School of the University of Tu¨bingen, Germany (1993). He received his doctorate degree (1997) from the German Cancer Research Center Heidelberg, Germany, and a Feodor Lynen Fellowship (1999–2001) from the Alexander von Humboldt Foundation.