Advanced Therapy for Hepatitis C E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 590

Veröffentlichungsjahr: 2011

Beschreibung

Hepatologists, both researchers and front-line clinicians, now have a comprehensive guide to the treatment of patients with the HCV virus. This book focuses mainly on the efficacy and clinical use of antiviral therapies, including the use of antivirals post liver transplantation and in the presence of the HIV virus. It also explores the foundations for understanding antiviral therapies in HCV, such as the complex pharmacology and mechanisms of antiviral drugs. A 'late-breaking information' chapter contains the most recent trial results on the latest antiviral drugs.

Leseprobe

Contents

Cover

Title Page

Copyright

Contributors

Preface

Section I: Foundations for Understanding Antiviral Therapies in HCV

Chapter 1: HCV Replication

Introduction

The HCV Genome

Models to Study HCV Replication

The HCV Virion and Entry

HCV Replication

Chapter 2: Hepatitis C Virus Genotypes

Introduction

HCV Genotypes and Subtypes

Intergenotypic Recombinants

Quasispecies

Genotype-Specific Effects of HCV Proteins on Response to Antiviral Therapy

Insulin Resistance

Steatosis

Conclusion

Chapter 3: Immune Responses to HCV: Implications for Therapy

Introduction

Humoral Immune Responses to HCV

Adaptive Cellular Immune Responses to HCV

HCV and Immune Evasion: Mechanisms of Viral Persistence

Immune Responses to HCV: Implications for HCV Therapy

Concluding Remarks

Chapter 4: Mechanisms of Action of Antiviral Drugs: The Interferons

Introduction

Hepatocyte Response to HCV Infection

Recognition of HCV by RIG-I and TLR-3

Activation and Antagonism of HCV Recognition Pathways

Interferon-stimulated Genes and HCV Control

ISGs and Prediction of Treatment Outcome in CHC Infection

IL28B and its Prediction to CHC Treatment Response and Spontaneous Clearance

Concluding Remarks

Chapter 5: Pharmacology and Mechanisms of Action of Antiviral Drugs: Ribavirin Analogs

Introduction

Ribavirin

Ribavirin Analogs

Levovirin

Taribavirin

Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitors

Summary

Chapter 6: Pharmacology and Mechanisms of Action of Antiviral Drugs: Polymerase Inhibitors

Introduction

Nucleoside Inhibitors

Non-Nucleoside Inhibitors

Perspectives

Acknowledgments

Chapter 7: Pharmacology and Mechanisms of Action of Antiviral Drugs: Protease Inhibitors

Introduction

Need for Improved Anti-HCV Therapies

Design of NS3 Protease Inhibitor Ciluprevir/BILN 2061: First Anti-HCV Proof-of-Concept in Humans

NS3 Protease Inhibitors in Clinical Development

Challenges and Future Directions

Acknowledgments

Chapter 8: Measuring Antiviral Responses

HCV RNA Level Measurement

Response-Guided Therapy with Current Standard-of-Care

Response-Guided Therapy with Future Standard-of-Care

Section II: Efficacy and Clinical Use of Antiviral Therapies

Chapter 9: Genotype 1: Standard Treatment

Goals of Therapy

Duration of Treatment

Deriving the Regimen

Monitoring Treatment Response

Medication Dosing

Side Effects and Dose Adjustments

Identifying Candidates for Therapy

The Future

Chapter 10: Individually Tailored Treatment Strategies in Treatment-naïve Chronic Hepatitis C Genotype 1 Patients

Introduction

Assessment of Baseline Viral Load

Viral Kinetics during Therapy: When to Measure HCV-RNA in Serum

Is it Possible to Further Individualize Treatment Beyond the Time Points of Weeks 4, 12, and 24?

Impact of Ribavirin in Individualized Treatment Strategies for Chronic Hepatitis C Genotype 1 Infection

Patients in Whom Shorter Treatment Durations May Not Be Considered

Impact of IL28B Genotype on Treatment Individualization

Summary

Chapter 11: Genotype 1 Relapsers and Non-responders

The Burden of Failures of Anti-HCV Therapy

Management of Patients Who Failed to Respond to Antiviral Therapy

Emerging Treatments

Conclusion

Chapter 12: Standard Therapy for Genotypes 2/3

Introduction

Peginterferon Alfa and Ribavirin for Genotype 2 or 3

Differences in Response to Treatment between HCV Genotype 2 and 3 Infections

Relevance of a Rapid Virological Response in HCV Genotype 2 or 3

Peg-IFN Formulations, Ribavirin Dose, and Response to Treatment

Albinterferon in the Treatment of Hepatitis C Genotype 2 or 3

Duration of Treatment in Hepatitis C Genotype 2 or 3

Treatment of Patients with Advanced Liver Disease

Predictors of Treatment Response: Other Factors to Consider

Long-Term Outcome of Peg-IFN Alfa and RBV Treatment

Summary and Conclusions

Chapter 13: Altered Dosage or Durations of Current Antiviral Therapy for HCV Genotypes 2 and 3

Background

Variable Length of Antiviral Therapy

Studies Randomizing Patients at Baseline to Short or Standard Treatment Duration

Conquering HCV Clearance in All Patients

Optimal Treatment Duration for Non-RVR Patients

Dosages of Peginterferon Alfa

Dosages of Ribavirin

Conclusions

Chapter 14: Genotypes 2 and 3 Relapse and Non-response

Background

Why Do Patients Infected with Genotypes 2 and 3 Not Have SVR?

Preventing Treatment Failure in G2/G3 Patients

Management of Relapse or Non-response to Antiviral Therapy

Management of Relapse in Patients Who Underwent Short Treatment Regimens

Future Therapies for Patients Who Experienced a Treatment Failure

Acknowledgments

Chapter 15: Hepatitis C Genotype 4 Therapy: Progress and Challenges

HCV Genotype 4: Shifting Epidemiology

Treatment of Chronic HCV-4 Naïve Patients

Duration of Chronic HCV-4 Therapy

The Efficacy of Different Pegylated Interferon Formulations in HCV-4 Infections

HCV-4 Therapy in Special Populations

Emerging New Regimen for Treatment of Chronic HCV-4

Personalizing Chronic HCV-4 Therapy

Conclusions and Future Prospects

Chapter 16: Antivirals in Acute Hepatitis C

Diagnosis of Acute Hepatitis C

Prevention of HCV Transmission

Treatment of Acute Hepatitis C

Timing of Therapy

Duration of Therapy

Is Ribavirin Needed to Treat Acute Hepatitis C?

Conclusions

Chapter 17: Antivirals in Cirrhosis and Portal Hypertension

Introduction

Compensated Cirrhosis

Decompensated Cirrhosis

Maintenance Interferon

Side Effects

Conclusions

Chapter 18: Treatment of Recurrent Hepatitis C Following Liver Transplantation

Introduction

Timing of Antiviral Therapy

Tolerability of Current Antiviral Therapy

Efficacy of Current Antiviral Therapy for Established Chronic Hepatitis

Future Antiviral Strategies

Summary

Chapter 19: Antiviral Treatment in Chronic Hepatitis C Virus Infection with Extrahepatic Manifestations

Introduction

HCV-Associated Mixed Cryoglobulinemia Vasculitis

HCV-Associated B-Cell Non-Hodgkin Lymphoma

Other HCV-Associated Extrahepatic Manifestations

Conclusion

Chapter 20: Cytopenias: How they Limit Therapy and Potential Correction

Mechanisms by which Interferon Contributes to Cytopenia

Mechanisms by Which Ribavirin Contributes to Anemia

Management of Neutropenia

Management of Thrombocytopenia

Management of Anemia

The Importance of Assessing Response During Therapy

Disclosures of Conflicts of Interest

Chapter 21: The Problem of Insulin Resistance and its Effect on Therapy

Introduction

Defining Insulin Resistance

Insulin Signaling

Molecular Mechanisms of Insulin Resistance in CHC

Host Factors Influencing IR

Effect of IR on Treatment Response

Management Strategies for CHC in the Setting of IR

Summary

Chapter 22: HIV and Hepatitis C Co-infection

Global Prevalence of HIV/HCV Co-Infection

Natural History of HCV in HIV Co-infection

Diagnosis and Monitoring of HCV in HIV Infection

Treatment of HIV-HCV Co-infection

Management of Treatment Non-Response and End-Stage Liver Disease

New Directions in Therapy

Chapter 23: HCV and Racial Differences

Introduction

Epidemiology

Treatment

Summary

Chapter 24: HCV and the Pediatric Population

Epidemiology and Natural History

Clinical Trials: Past, Present, and Future

Special Treatment Considerations for Pediatric Populations

Chapter 25: New Horizons: IL28, Direct-acting Antiviral Therapy for HCV

Introduction

NS3/4A Protease Inhibitors

NS5B Polymerase Inhibitors

NS5B Nucleos(t)ide Inhibitors

NS5B Non-Nucleos(t)ide Inhibitors

NS5A Inhibitors

Cyclophilin Inhibitors

Other DAA Targets

Other Novel Therapeutic Approaches

Drug Resistance

Combination Strategies

IL28B Polymorphism

Unresolved Issues

Summary and Conclusions

Authors’ Declaration of Personal Interests

Index

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Library of Congress Cataloging-in-Publication Data

Advanced therapy for hepatitis C / edited by Geoffrey W. McCaughan, John G. McHutchison, Jean-Michel Pawlotsky. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-1-4051-8745-9 (hardcover : alk. paper) ISBN-10: 1-4051-8745-X (hardcover : alk. paper) ISBN-13: 978-1-4443-4631-2 (ePDF) ISBN-13: 978-1-4443-4634-3 (Wiley Online Library) [etc.] 1. Hepatitis C–Treatment. 2. Antiviral agents. I. McCaughan, Geoffrey W. II. McHutchison, J. G. III. Pawlotsky, Jean-Michel. [DNLM: 1. Hepatitis C–therapy. 2. Antiviral Agents–therapeutic use. WC 536] RC848.H425A38 2012 616.3′62306–dc23 2011016561

A catalogue record for this book is available from the British Library.

This book is published in the following electronic formats: ePDF 9781444346312; Wiley Online Library 9781444346343; ePub 9781444346329; Mobi 9781444346336

Contributors

Nezam H. Afdhal MD Beth Israel Deaconess Medical Center Boston, MA, USA

Angelo Andriulli MD Gastroenterology Department IRRCS Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy

Martin Baril PhD Research Associate Institut de Recherche en Immunologie et Cancérologie (IRIC) Montréal, Québec, Canada

Michael R. Beard PhD Head, Hepatitis C Virus Research Laboratory School of Molecular and Biomedical Science The University of Adelaide & Center for Cancer Biology, SA Pathology Adelaide, SA, Australia

Thomas Berg MD Professor Department of Gastroenterology and Rheumatology Division of Hepatology University of Leipzig Leipzig, Germany

David G. Bowen MBBS, PhD Sydney Medical School, University of Sydney Royal Prince Alfred Hospital Sydney, NSW, Australia

Patrice Cacoub MD, PhD Professor Department of Internal Medicine Groupe Hospitalier Pitié-Salpêtrière Université Pierre et Marie Curie Paris, France

Laurent Chatel-Chaix PhD Post-doctoral Fellow Institut de Recherche en Immunologie et Cancérologie (IRIC) Montréal, Québec, Canada

Grace M. Chee PharmD Hepatology Department Cedars-Sinai Medical Center Los Angeles, CA, USA

Stéphane Chevaliez PharmD, PhD National Reference Center for Viral Hepatitis B, C and delta Department of Virology & INSERM U955 Hôpital Henri Mondor Université Paris-Est Créteil, France

Lotte Coelmont PhD Laboratory of Virology and Chemotherapy Rega Institute for Medical Research University of Leuven Leuven, Belgium

Antonio Craxì, MD Full Professor of Gastroenterology Sezione di Gastroenterologia, Di.Bi.M.I.S. Policlinico Paolo Giaccone University of Palermo Palermo, Italy

Leen Delang PhD Laboratory of Virology and Chemotherapy Rega Institute for Medical Research University of Leuven Leuven, Belgium

Gregory J. Dore BSc, MBBS, MPH, PhD, FRACP Professor and Head, Viral Hepatitis Clinical Research Program National Centre in HIV Epidemiology and Clinical Research The University of New South Wales; Infectious Diseases Physician St Vincent's Hospital Sydney, NSW, Australia

Mark W. Douglas BSc (Med)(Hons), MBBS (Hons), PhD, FRACP Senior Lecturer, Hepatology and Virology Storr Liver Unit, Westmead Millennium Institute Sydney Emerging Infections and Biosecurity Institute University of Sydney Sydney, NSW, Australia

Xavier Forns MD, PhD Liver Senior Specialist Liver Unit, Hospital Clinic IDIBAPS and Ciberehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) Barcelona, Spain

Mathy Froeyen PhD Assistant Professor of Pharmacy Laboratory of Medicinal Chemistry Rega Institute for Medical Research University of Leuven Leuven, Belgium

Ed Gane MB ChB, MD, FRACP Associate Professor and Hepatologist New Zealand Liver Transplant Unit Auckland City Hospital Auckland, New Zealand

Jacob George MBBS (Hons), FRACP, PhD Director of Gastroenterology and Hepatic Services Storr Liver Unit, Westmead Millenium Institute University of Sydney Sydney, NSW, Australia

Rebekah G. Gross MD Assistant Professor of Medicine Division of Gastroenterology and Hepatology Weill Cornell Medical College New York, NY, USA

Piet Herdewijn PhD Professor of Pharmacy Laboratory of Medicinal Chemistry Rega Institute for Medical Research University of Leuven Leuven, Belgium

Ira M. Jacobson MD Vincent Astor Professor of Medicine Chief, Division of Gastroenterology and Hepatology Division of Gastroenterology and Hepatology Weill Cornell Medical College New York, NY, USA

Sanaa M. Kamal MD, PhD Department of Gastroenterology and Liver Disease Ain Shams Faculty of Medicine Cairo, Egypt; Department of Gastroenterology Tufts School of Medicine Boston, MA, USA

Daniel Lamarre PhD Full Professor, Department of Medicine Faculty of Medicine Institute for Research in Immunology and Cancer (IRIC) Université de Montréal Montréal, Québec, Canada

Alessandra Mangia MD Liver Unit IRRCS Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy

Diarmuid S. Manning MB, BCh Beth Israel Deaconess Medical Center Harvard Medical School Boston, MA, USA

Stella Martínez MD Liver Unit, Hospital Clinic IDIBAPS and Ciberehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) Barcelona, Spain

Gail V. Matthews MBChB, MRCP (UK), FRACP, PhD Clinical Academic National Centre in HIV Epidemiology and Clinical Research University of New South Wales Sydney, NSW, Australia

Geoffrey W. McCaughan MBBS, PhD, FRACP Head of Liver Immunobiology Program Centenary Research Institute A.W. Morrow Professor of Medicine Director A.W. Morrow GE/Liver Center Director Australian Liver Transplant Unit Royal Prince Alfred Hospital University of Sydney Sydney, NSW, Australia

John G. McHutchison, MD Senior Vice President, Liver Disease Therapeutics Gilead Sciences, Inc. Foster City, CA, USA

Leonardo Mottola PhD Liver Unit IRRCS Casa Sollievo della Sofferenza San Giovanni Rotondo, Italy

Andrew J. Muir MD MHS Director, Gastroenterology/Hepatology Research Duke Clinical Research Institute Duke University Medical Center Durham, NC, USA

Johan Neyts PhD Professor of Virology Laboratory of Virology and Chemotherapy Rega Institute for Medical Research University of Leuven Leuven, Belgium

Venessa Pattullo MBBS, FRACP Storr Liver Unit, Westmead Millennium Institute University of Sydney, Sydney, NSW, Australia; Division of Gastroenterology Toronto Western Hospital University Health Network, University of Toronto Toronto, Ontario, Canada

Jean-Michel Pawlotsky MD, PhD Director, French National Reference Center for Viral Hepatitis B, C and delta Head, Department of Virology, Bacteriology, and Hygiene INSERM U955 Hôpital Henri Mondor Université Paris Est Créteil, France

Salvatore Petta MD, PhD Sezione di Gastroenterologia, Di.Bi.M.I.S. Policlinico Paolo Giaccone University of Palermo Palermo, Italy

Fred Poordad MD Associate Professor of Medicine David Geffen School of Medicine at UCLA; Chief, Hepatology and Liver Transplantation Cedars-Sinai Medical Center Los Angeles, CA, USA

Scott A. Read MSc Storr Liver Unit, Westmead Millennium Institute University of Sydney Sydney, NSW, Australia

Jose María Sánchez-Tapias MD, PhD Senior Consultant Liver Unit, Hospital Clinic IDIBAPS and Ciberehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) Barcelona, Spain

Kathleen B. Schwarz MD Director, Pediatric Liver Center Division of Pediatric Gastroenterology and Nutrition Professor of Pediatrics Johns Hopkins University School of Medicine Baltimore, MD, USA

Mitchell L. Shiffman MD Director Liver Institute of Virginia Bon Secours Virginia Health System Richmond and Newport News, VA, USA

Benjamin Terrier MD Department of Internal Medicine Groupe Hospitalier Pitié-Salpêtrière Université Paris 6 Pierre et Marie Curie Paris, France

Alexander J. Thompson MD, PhD St. Vincent's Hospital Melbourne, University of Melbourne, Melbourne, VIC, Australia; Victorian Infectious Diseases Reference Laboratory (VIDRL), North Melbourne, VIC, Australia; Department of Gastroenterology and Duke Clinical Research Institute Duke University Durham, NC, USA

Edmund Tse MBBS, FRACP School of Molecular and Biomedical Science The University of Adelaide and the Center for Cancer Biology SA Pathology Adelaide, SA, Australia

Heiner Wedemeyer MD Professor Department of Gastroenterology, Hepatology and Endocrinology Medizinische Hochschule Hannover Hannover, Germany

Johannes Wiegand MD Private Lecturer Department of Gastroenterology and Rheumatology Division of Hepatology University of Leipzig Leipzig, Germany

Kenneth Yan MBBS, Mmed (Clin Epi), FRACP Conjoint Associate Lecturer St George Clinical School Faculty of Medicine University of New South Wales Sydney, NSW, Australia

Amany Zekry MBBS, PhD, FRACP Department of Gastroenterology and Hepatology Clinical School of Medicine St George Hospital Sydney, NSW, Australia

Preface

Hepatitis C virus results in chronic liver disease in over 170 million people worldwide. This book arrives at a watershed in the history of antiviral treatment of the hepatitis C virus. It is the beginning of the end of non-specific antiviral approaches via interferon-based therapies. From now on the field will be dominated by the arrival of HCV-specific direct antiviral agents. Initially these agents will still require interferon and ribavirin but already clinical trials are under way that do not include either of these agents.

This publication outlines the current standard of care up until this time and includes therapeutic approaches to wide patient groups. We believe that the structure of the book will remain relevant for future editions as the new therapies are gradually rolled out across these patient groups, as well as across an increasing number of countries.

G.W.M. J.G.M. J-M.P.

I

Foundations for Understanding Antiviral Therapies in HCV

1

HCV Replication

Michael R. Beard

School of Molecular and Biomedical Science, University of Adelaide, and Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia

Introduction

Hepatitis C virus (HCV) is classified in the Hepacivirus genus within the family Flaviviridae and is the leading cause of chronic hepatitis and liver disease related morbidity worldwide. With an estimated 170 million people infected worldwide and the ability of the virus to establish a chronic infection in approximately 70% of cases, it is not surprising that HCV represents a major cause of global suffering and morbidity and a burden to many public health systems. Chronic HCV infection is often associated with development of serious liver disease, including cirrhosis, liver failure, and hepatocellular carcinoma. Accordingly, a thorough understanding of the life cycle and molecular biology of HCV and its interaction with the host are essential in the development of treatment and vaccine strategies. Although these studies have been hampered by the lack of a small-animal model and, until recently, a lack of a tissue culture system that accurately reflects the life cycle of HCV, significant progress has been made in the understanding of HCV molecular biology and pathogenesis. In this chapter we discuss recent advances in models to study HCV replication and the HCV life cycle.

The HCV Genome

HCV possesses a 9.6 kb single-stranded, positive-sense RNA genome composed of a 5′ UTR (untranslated region), a long open-reading frame (ORF) encoding a polyprotein of approximately 3000 amino acids, and a 3′ UTR (Figure 1.1). The polyprotein can be divided into three segments based on the functional aspects of the proteins: the NH2 terminal region comprises the structural proteins (core, E1, and E2); a central region consists of two proteins (p7 and NS2) that are not involved in HCV replication or are structural components of the virus, but probably play a role in virion morphogenesis; and the COOH-terminal proteins (NS3, NS4A, NS4B, NS5A, and NS5B) that are required for HCV replication (Figure 1.1). A detailed description and function of the HCV proteins can be found in an excellent review from Moradpour and colleagues [1]. After release of the HCV genome into the cytoplasm the genome is exposed to the host cellular machinery for translation of the viral polyprotein. The 5′ and 3′ UTRs are highly conserved and critical to viral genome replication and translation of the viral polyprotein. The 5′ UTR is approximately 341 nucleotides long and contains a highly structured RNA element known as the internal ribosome entry site (IRES) that is recognized by the cellular 40S ribosomal subunit to initiate translation of the RNA genome in a cap-independent manner. The importance of the secondary and tertiary structure of the IRES domain for initiation of translation has been demonstrated by mutational analysis. However, the primary sequence, particularly in stem-loop IIId and IIIe, is also critical for efficient HCV IRES activity [2,3]. Recently, the structural nature of HCV IRES interactions with the 40S ribosomal subunit and the eIF3 complex has been revealed by cryo-electron microscopy [4,5]. Preceding the IRES at the extreme 5′ end are elements required for viral replication that overlap partially with the IRES region (domain II), leading to speculation that this region is involved in regulation of a viral translation to replication switch [6]. Consistent with this speculation is the recent observation that a short highly conserved RNA segment at the 5′ end of the HCV genome binds a liver-specific microRNA, miR-122, that is required for efficient HCV replication in cultured hepatoma cells [7]. However, in the HCV-infected liver no correlation was noted between miR-122 abundance and levels of HCV RNA in the liver or serum, which suggests that the impact of miR-122 may be less prominent in vivo than in vitro [8]. miR-122 may impact HCV replication indirectly through stimulation of translation of the viral polyprotein by enhancing association of ribosomes with the IRES [9]. Although more work is required to determine its role in the HCV life cycle and pathogenesis, modulation of miR-122 expression and activity presents as an attractive target for future therapy.

Figure 1.1 Genomic organization and polyprotein processing of the HCV genome. The HCV genome consists of a positive-stranded RNA genome that is flanked by 5′ and 3′ UTRs of highly ordered secondary structure. The polyprotein is cleaved by either host- or viral-encoded proteases (depicted by triangles) to liberate the mature structural and non-structural proteins.

Like the 5′ UTR, the 3′ UTR of the HCV genome contains a high degree of secondary structure. This region is 200–300 nucleotides in length and is comprised of three major elements involved in replication: (i) a variable region (30–50 nucleotides), which directly follows the NS5B stop codon; (ii) a polyuridine (U/C) tract (20–200 nucleotides); and (iii) a highly conserved region (98 nucleotides), known as the 3′ X region, which forms a three stem-loop structure [10,11,12]. Mutational analysis has revealed that the poly-U/C tract and the 3′ X region play a more important role than the variable region in the synthesis of negative-strand RNA [13].

Models to Study HCV Replication

HCV Replicons

The development of the subgenomic HCV replicon system, first reported in 1999, significantly enabled the study of HCV replication in cultured cells for the first time [14]. Replicons represent autonomously replicating HCV RNAs, and typically contain an in-frame insertion of a selectable antibiotic marker (e.g., neomycin phosphotransferase: G418) within the amino terminal HCV core sequence, followed downstream by a heterologous IRES from encephalomyocarditis virus (EMCV), a picornavirus, to drive internal translation of the downstream HCV open reading frame (NS2 to NS5B) (Figure 1.2). The minimal requirements for a viable HCV replicon are HCV-derived 5′ and 3′ termini and the non-structural proteins (NS3 to NS5B) that form the replication complex, however, replication-competent HCV replicons encoding the complete HCV polyprotein are viable [15,16]. Transfection of Huh-7 (hepatoma-derived) cells with synthetically derived transcripts followed by selection with G418 results in the establishment of cell lines that harbor autonomous replication of the virus. HCV RNA isolated from cell lines under antibiotic selection often contains cell-culture adapted mutations that greatly enhance replication, although the molecular basis for this increased replication is unclear [15,17,18]. These adaptive mutations often map to the NS5A protein and may potentially influence phosphorylation, resulting in a hypophosphorylated state and increased replication. Adaptive mutations have also been mapped to NS3, NS4A, NS4B, and NS5B. Interestingly, these replication-adaptive cell-culture mutations have been shown to reduce in vivo infectivity in chimpanzees, highlighting the adaptive nature of these viruses derived from cell culture [19]. HCV replicons are not restricted to Huh-7 cells, and other cell lines such as HeLa and cells of murine origin have also yielded selected clones of replicating HCV, highlighting that HCV replication is not restricted to liver-derived cells of human origin [20,21].

Figure 1.2 Schematic diagram of the organization of the (a) genomic and (b) subgenomic HCV replicons. In both cases the HCV IRES drives expression of the neomycin phosphotransferase gene while the EMCV IRES drives expression of the HCV proteins.

The antibiotic selection process not only selects for HCV genomes with high replication capacity but also clones of Huh-7 cells that are highly permissive for HCV infection. One such cell line, Huh-7.5, has been “cured” of HCV by treatment with low doses of interferon-α, is hyperpermissive for HCV replication [22], and is clearly enriched for factors that promote replication and/or defects in innate viral sensing pathways. For example, Huh-7.5 cells have a spontaneous knockout of the dsRNA cellular sensing protein RIG-1 and do not mount a robust antiviral response to viral infection that allows for HCV replication. This highlights the importance of innate immune sensing in HCV infection and is consistent with the ability of the HCV NS3/4A protein to cleave IPS-1, which is integral to the innate immune RIG-I pathway [23].

HCV replicons have been valuable tools for studying numerous aspects of the HCV life cycle and interaction with the host cell. However, their major limitation has been inability to produce infectious virus particles even when the complete complement of HCV proteins is expressed, for reasons that are not entirely clear [15,16,22]. The original replicon concept has undergone evolution and replicons are now available that contain various markers (e.g., GFP, luciferase) that allow quantitative assessment of HCV replication and have been useful in high-throughput screening of antiviral compounds.

Productive Viral Infection in Cell Culture

The recent identification in 2005 of a cloned HCV genome (genotype 2a), known as JFH-1, that is capable of initiating high-level replication in cell culture and production of infectious virus particles represents a major breakthrough in the pursuit of a cell-culture model for HCV [24,25]. In contrast to HCV replicon systems, transfection of Huh-7 cells with RNA synthesized in vitro from the cloned JFH-1 cDNA genome and a related genotype 2a chimera, FL-J6/JFH replicate efficiently in Huh-7 cells without the need for cell-culture adaptive mutations. Moreover, virus particles produced by these cells are infectious in chimpanzees and can be serially passaged in vivo [25] while the FL-J6/JFH virus can infect mice containing human liver grafts [24]. Interestingly, virus produced in vivo has a lower buoyant density than virus produced in cell culture, suggesting association with low-density lipids [26]. This system represents a major advance in the study of virus-host interactions and the virus life cycle, all in the context of replicating virus. Similarly, the highly adapted genotype 1a HCV isolate known as H77-S (derived from the H77 isolate) [27] is also capable of instigating HCV RNA replication and production of infectious virus particles [28]. This represents another breakthrough in the generation of tools to study the HCV life cycle, particularly because this genotype is more prevalent worldwide and is associated with more significant liver disease. Intragenotypic and intergenotypic chimeras of HCV that contain the non-structural protein-encoding regions of JFH-1 and the structural protein-encoding regions of other HCV genomes may help define regions of structural proteins that influence the efficiency of virus particle synthesis and secretion [29]. This relatively new cell-culture model system will be invaluable in the study of many aspects of virus-host interaction, including viral entry, and assembly and release, which were previously inaccessible to manipulation.

The HCV Virion and Entry

The relatively low levels of HCV in plasma samples have hampered visualization of viral particles; however, virus-like particles have been identified by electron microscopy, which has indicated that infectious HCV virions are roughly spherical particles of diameter 55–65 nm with fine projections of approximately 6 nm ([25] and references therein). The major protein constituents of the host-derived lipid bilayer envelope are the highly glycosylated HCV envelope glycoproteins E1 and E2 that surround the viral nucleocapsid, composed of many copies of the HCV core protein and the genomic HCV RNA (Figure 1.3). HCV from serum and plasma fractionates with a wide range of buoyant densities that can be attributed to association of the virus with lipoproteins, in particular apolipoprotein-B (Apo-B) and apolipoprotein-E (Apo-E), which are components of host low density lipoprotein (LDL, Apo-B) and very low density lipoprotein (VLDL, Apo-B, Apo-E) and suggest a close association with circulating LDL/VLDL [26]. The physiological association of HCV with LDL/VLDL remains unexplained mechanistically; however, it could be involved in viral uptake (see below), or alternatively the association of Apo-B with HCV virions may indicate a role for the hepatic LDL/VLDL secretory pathway in release of the virus.

Figure 1.3 The HCV particle. The RNA genome is encapsidated by the icosahedral nucleocapsid consisting of the core protein. The nucleocapsid is enveloped by a host-derived spherical lipid bilayer that is enriched with heterodimers of the envelope glycoproteins E1 and E2.

Hepatocytes are the main target for infection with HCV; however, identification of the cellular receptors responsible for HCV entry has proven difficult due to the lack of appropriate model systems. However, using a combination of HCV pseudotyped particles (HCVpp) [30,31] and cell-culture-derived HCV (HCVcc) [25], the complement of HCV receptors now seems complete.

The 25 kDa tetraspanin molecule CD81 and the human scavenger receptor class B type I (SR-BI) both bind HCV E2 and are necessary but not sufficient for HCV entry [32]. For example, CD81 ectopic expression in hepatocyte-derived cell lines that are negative for CD81 confers susceptibility to HCVpp and HCVcc; however, expression of both factors in non-hepatocyte-derived cell lines does not concur infectivity [24,30]. Clearly additional hepatocyte factors are required for HCV entry. Using an interactive cloning and expression approach, the tight junction protein claudin-1 (CLDN1) was recently identified as an HCV co-receptor [33]. CLDN1 was found to be essential for entry into hepatic cells and rendered non-hepatic cells susceptible to infection. However, despite the identification of CD81, SR-B1, and CLDN1 as essential HCV entry co-factors, a number of human cell lines and those of non-primate origin remained resistant to HCV infection, suggesting an additional entry factor. Using a cyclic lentivirus-based screen of a cDNA library derived from a highly HCV-permissive hepatocarcinoma cell line (Huh-7.5) for genes that render the non-permissive CD81+, SR-BI+ 293T cell line infectable with HCVpp, the remaining crucial factor was recently identified as occludin (OCLN), also a tight junction protein [34]. Although expression of all four entry factors (CD81, SR-B1, CLDN1, OCLN) renders mouse cell lines susceptible to HCVpp infection, these cells could not support HCVcc infection. This is not surprising given past reports of inefficient replication of HCV RNA in mouse cell lines, and suggests that specific hepatocyte factors are crucial for efficient HCV replication. The identification of CD81 and OCLN as the minimal human-specific entry factors (HCV can bind to murine SR-B1 and CLDN1) not only significantly advances our understanding of the molecular mechanisms of HCV entry but also provides important steps for the development of a mouse model of HCV infection and provides an attractive target for the development of novel antiviral strategies.

Other molecules have been suggested to be involved in HCV entry. The association of HCV virions in serum with LDL and VLDL suggests that the LDL receptor (LDLR) may be an attractive candidate receptor. However, its precise role remains to be determined [26,35]. LDLR is not sufficient itself for entry and it does not bind directly to HCV E2 [30]. Together with the glycosaminoglycans, the LDLR in concert with other cell-surface proteins may serve to collect HCV at the cell surface and facilitate binding with receptors crucial for HCV entry. Consistent with this, a role has been proposed for L-SIGN and DC-SIGN in HCV attachment although they do not seem to mediate cell entry of HCV and their role is unclear [36].

The precise molecular events underlying HCV binding and entry are not well understood. However, HCV binding to the cell surface is thought to occur in a stepwise process by binding to several receptors followed by transfer to the tight junction proteins CLDN1 and OCLN that may facilitate cellular uptake (Figure 1.4). Similar to other flaviviruses, HCV entry is thought to be mediated by clathrin-mediated endocytosis with delivery of the nucleocapsid from the endosome in a pH-dependent manner [37,38,39]. Furthermore, the E1 and E2 proteins are class II fusion proteins that result from the production of a fusion pore in the endosome membrane that facilitates genome release to the cytoplasm [40].

Figure 1.4 Model for HCV entry. HCV particles associated with LDL and VLDL are thought to be tethered to the hepatocyte surface by the LDL-R and GAGs and subsequent stepwise interactions with CD81 and SR-B1. HCV is transferred to the tight junction proteins OCLN and CLDN-1 from where virus enters the cell by endocytosis. Release of the HCV core containing the RNA is mediated by fusion of the E1 and E2 proteins with the endosome. The relative roles and the spatial distributions of each of the HCV receptors remain to be determined.

HCV Replication

The HCV replication process is summarized in Figure 1.5. After translation of the HCV proteins from the positive-sense RNA genome by direct interaction of the host 40S ribosomal subunit with the IRES within the 5′ UTR of the genome, HCV replication begins. This IRES-directed translation is cap-independent and enables virus translation/replication to continue even after host cell cap-dependent translation has been shut down in response to viral infection.

Figure 1.5 Lifecycle of HCV. 1, Virus binding and internalization; 2, release of HCV RNA and translation of viral proteins; 3, association of HCV proteins in ER and formation of the replication complex in association with lipid droplets, replication of HCV RNA; 5, virion maturation and packaging of RNA; 6, release of virions.

Similar to other positive-stranded viruses, HCV is believed to replicate in association with intracellular membranes in a complex called the membranous web, although the exact details of this association are not well understood. It is thought that the association predominantly with endoplasmic reticulum (ER) membranes may provide support for the organization of the replication complex, compartmentalization of the viral products, concentration of lipid constituents important for replication, and protection of the viral RNA from host-mediated innate immune defenses. This membranous web was first noticed in cultured cells harboring HCV replicons and contains detectable concentrations of the non-structural proteins NS3, NS4A, NS4B, NS5A, and NS5B, and is very similar to sponge-like inclusions noted in liver tissue from HCV-infected chimpanzees [41--44]. Expression of NS4B alone induces the formation of the membranous web, and recent work has shown that membrane association is facilitated by amino acids 40 to 69 of the N-terminal region of NS4B [45].

The phosphorylation status of NS5A appears to be a determinant of HCV RNA replication with mutations that reduce hyperphosphorylation of NS5A dramatically enhancing HCV RNA replication [46,47]. In this manner, hyperphosphorylation of NS5A may induce a switch from genome replication to viral protein translation. NS5A also interacts with several host proteins that may be important in HCV replication through formation of the replication complex or facilitating assembly. NS5A interacts with the SNARE-like vesicle-associated membrane host proteins, VAP-A and VAP-B [48]. NS5A also interacts with geranylgeranylated F-box protein, FBL2, which is essential for replication and seems to be part of the replication complex [49]. How this interaction contributes to replication is unclear but it may help anchor the replicase complex to membranes. Its involvement in the replication process highlights the close interaction between HCV replication and the host cholesterol biosynthetic pathway [50]. Another host factor, cyclophilin B, has also been implicated in HCV replication through interaction with NS5B and stabilization of RNA binding, and was originally discovered through the ability of the powerful immunosuppressive drug cyclosporin A (CsA) to inhibit HCV replication [51]. However, more recent work suggests that cyclophilin A plays a critical role in cleavage of NS5A/5B and assembly of the replication complex [52,53]. CsA analogs are currently being developed as antivirals against HCV [54].

The precise details of the HCV RNA replication process are still unclear but comparison with other flaviviruses suggests that the positive-stranded genome serves as a template for the synthesis of negative-strand RNA. Components of the membrane-bound replication complex associate with the 3′ end of the positive strand of the genome, with NS5B at the catalytic core, and initiate de novo synthesis of negative-strand RNA. These two strands remain base-paired, which results in the formation of a double-stranded RNA molecule that is copied multiple times by semiconservative replication by the RNA-dependent RNA polymerase (RdRp) NS5B to generate multiple progeny, positive-strand viral RNA genomes. Importantly, the NS5B RdRp has no proofreading capacity and as such is error prone. This lack of proofreading ability results in the generation of many different but closely related genomes, often referred to as quasispecies. This genetic diversity is ideally suited to escape of immune control and is a significant factor in the generation of antiviral resistance to select antiviral agents. While a proportion of new positive-strand genomes serve as templates for viral protein translation, others associate with the core protein and form dimers within core-protein-enriched nucleocapsids. The association of the core protein with cytoplasmic lipid droplets has emerged as a critical determinant of nucleocapsid and infectious viral particle assembly. It is thought that the core uses this platform to recruit replication complexes and associated new genomes from closely associated ER-derived “lipid-droplet-associated membranes” in the assembly process [55,56]. Core particles may then become enveloped via budding through the ER where viral glycoproteins (E1/E2 heterodimers) become embedded. Little is known about the process of viral particle egress except that particles change in their biophysical properties (increased density) upon exit. Recent studies have indicated that the processes of HCV particle assembly, maturation, and secretion are dependent upon the machinery involved in the assembly and secretion of VLDL by hepatocytes [57].

The development and use of in vitro cell-culture model systems described above has been and continues to be fundamental in dissecting the stages of HCV replication and identification of viral-host interactions at the molecular level (Figure 1.5). While these studies are important for our understanding of HCV biology, they also provide specific targets for the development of novel therapeutics designed to completely eradicate HCV infection across all genotypes. Current therapies for HCV focus on modulation of the host immune response. However, with a greater understanding of HCV replication and host interactions, we are currently in a phase of developing therapeutics that directly target various stages of the HCV life cycle. Drugs targeting HCV entry and fusion, viral helicase, and polymerase or protease function are all under clinical investigation, with some showing exceptional promise. These targeted therapies when used in combination with the current therapeutic regime of Peg-IFN alfa-2 and ribavirin will provide the foundation for systemic eradication of HCV in infected persons. Furthermore, defining novel host factors essential for HCV replication and a greater understanding of the immunological correlates of immunity to HCV will provide the cornerstone for further development of novel therapeutics to combat HCV infection.

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2

Hepatitis C Virus Genotypes

Scott A. Read and Mark W. Douglas

Storr Liver Unit, Westmead Millennium Institute, University of Sydney at Westmead Hospital, NSW, Australia

Introduction

The hepatitis C virus (HCV) was cloned by Choo et al. in 1989 [1]. Like most RNA viruses, the genome exhibits a high degree of genetic variability. Thus, it soon became evident that a unified classification scheme was required and a consensus paper was published in 1994 [2]. This scheme identified 6 HCV genotypes and 11 subtypes, based on phylogenetic analysis of the NS5B region, and is still used today (Figure 2.1). More than 75 confirmed and provisional subtypes of HCV have now been identified, which are still grouped into 6 major genotypes (Table 2.1).

HCV genetic variability is seen at all levels of classification: between genotypes, within genotypes (subtypes), and within the infected individual (quasispecies). HCV genotypes differ from each other at the nucleotide level by over 30%; subtypes by 20–25%; and quasispecies by up to 10% [4,5]. This degree of polymorphism results from the error-prone viral RNA polymerase and many years of divergent evolution within different human populations. The resulting HCV genotypes have unique distributions, but all share similar gene arrangement, viral replication, and the ability to establish persistent infection. This chapter will focus on genetic differences among HCV genotypes and genotype-specific differences in disease pathogenesis and treatment response.

HCV Genotypes and Subtypes

Although HCV genotypes differ by up to one third overall, sequence divergence is not uniform across the genome. The most conserved region is the 5′ untranslated region (UTR), where RNA secondary structure forms the internal ribosomal entry site (IRES) that initiates replication [6]. In contrast, the most diverse genes are for the envelope glycoproteins E1 and E2, which under immune pressure have evolved nine times faster than the 5′ UTR [7].

Each HCV genotype contains distinct subtypes, the geographic distributions of which reflect local social and medical practices, as well as host genetics. Contaminated blood products and needle sharing have allowed widespread dissemination of genotypes 1a, 1b, and 3a, which together account for over 80% of HCV infections worldwide [3]. Focal outbreaks have led to high local prevalence of certain subtypes, such as subtype 4a, which accounts for over 50% of HCV cases in Egypt. This followed mass parenteral antischistosomal therapy (PAT) administration in the latter half of the twentieth century [8]. In contrast, geographic isolation and/or inefficient sexual transmission routes in parts of South-East Asia have kept genotype 6 subtypes geographically restricted.

Figure 2.1 Evolutionary tree of the major subtypes of HCV. Subtypes can be differentiated by their distribution, risk groups and epidemiology. IDU, injecting drug user. Reproduced from [3], with permission from the Society for General Microbiology.

Intergenotypic Recombinants

With 3% of the world's population infected with an ever-increasing number of HCV genotypes and subtypes [9], the emergence of intergenotypic recombinants was virtually inevitable. During co-infection with multiple genotypes, subtypes, or strains of HCV, template switching can occur during replication, resulting in a recombinant virus containing two (or more) parental portions in its genome. This process is not site-specific and has been reported for the E1, E2, NS2, NS3, and NS5A genes [10,11,12,13]. Recently Sentandreu et al. estimated that intrasubtype recombination occurs in over 10% of HCV infected patients [11], but the rate of recombination between genotypes is probably much less, due to their divergent sequences.

Table 2.1 Defined HCV genotypes and their representative subtypes.

GenotypeAssigned subtypesa1a, b, c, d, e, f, g, h, i, j, k, l, m2a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r3a, b, c, d, e, f, g, h, i, k, l4a, b, c, d, e, f, g, h, k, l, m, n, o, p, q, r, s, t5a6a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p, q, r, s, t, u7aa Bold indicates confirmed subtype designation demonstrated by the coding sequences of two or more variants that are epidemiologically independent. Source: http://hcv.lanl.gov/content/sequence/HCV/classification/genotable.html

Quasispecies

The HCV RNA polymerase lacks proofreading ability, resulting in a high frequency of point mutations. The production of 1010 to 1012 new viral genomes per day [14], with a mutation rate of 1.5–2.0 ×10−3 sites per genome per year [15], results in a “swarm” of different HCV quasispecies in chronically infected individuals. Host immunity exerts the main selection pressure in vivo, resulting in mutants that replicate efficiently and evade the adaptive and innate immune responses. Genetic diversity is also thought to play a role in antiviral resistance as patients with higher pre-treatment quasispecies diversity are less likely to respond to interferon and ribavarin [16].

Genotype-Specific Effects of HCV Proteins on Response to Antiviral Therapy

The effectiveness of interferon-based therapy depends on HCV genotype [17]; less than 50% of patients with genotype 1 infection obtain a sustained virologic response (SVR) on current treatment regimens, compared to 75% or 80% with genotypes 2 and 3 [18,19]. Interactions have been observed between HCV E2, core, NS3/4A, and NS5A proteins and the innate immune system, so diversity among these proteins may influence treatment response.

The HCV E2 protein contains a 12 amino acid sequence that closely mimics the double-stranded RNA protein kinase (PKR) autophosphorylation site and translation initiation factor 2 (eIF2) phosphorylation site, termed the PKR-eIF2a phosphorylation homology domain (PePHD) [20]. It has been suggested that the degree of E2-PKR homology may correlate with resistance to treatment [20], but other studies have produced conflicting results [21,22]. Larger studies are thus required.

Core protein has been shown in vitro to up-regulate suppressor of cytokine signaling 3 (SOCS3), a key negative regulator of the interferon-stimulated JAK-STAT signaling pathway [23]. Hepatic expression of SOCS3 is higher in patients who do not respond to interferon-based therapy [24] and SOCS3 levels are higher in genotype 1 infection than genotype 2, consistent with genotype-specific variations in treatment response [24].

The NS3/4A complex forms a viral protease that cleaves non-structural proteins from the HCV polyprotein [25]. NS3/4A also cleaves the signaling molecules TRIF and Cardif, ablating pattern recognition receptor (PRR) signaling from Toll-like receptor-3 (TLR3) and RIG-I, respectively [26,27]. Franco et al. compared NS3 protease activity between 12 HCV isolates and showed up to a six-fold variation, with the highest activity in genotype 1 isolates [28].

NS5A is the most promiscuous HCV protein, interacting with key components of metabolic, growth, and immune signaling cascades [29]. NS5A interacts with PKR to prevent autophosphorylation and subsequent eIF2 phosphorylation, in part via a 40 amino acid segment in the C-terminal domain called the interferon sensitivity-determining region (ISDR) [30]. Enomoto et al. showed that genotype 1b strains with mutations in this region responded better to treatment [31]. Reports vary [32,33], but a meta-analysis found a positive correlation between the number of ISDR mutations and response to therapy [34]. NS5A is a highly immunogenic protein, so Simmonds has proposed a balance between optimal function and immune evasion [3]. NS5A from genotype 2 and 3 isolates may be more immunogenic than genotypes 1 and 4, so under increased selection pressure it becomes less efficient at suppressing innate immunity, resulting in better treatment outcomes.

Elevated hepatic basal interferon-stimulated genes (ISG) expression is seen in patients who do not respond to interferon therapy, blunting their interferon response [17,35]. Interestingly, pre-treatment ISG levels are higher in genotype 1 and 4 infections than genotype 2 and 3, perhaps contributing to genotype-specific response rates [17].

Insulin Resistance

HCV genotype 1 and 4 infections are associated with insulin resistance [36,37], which reduces the response to interferon-based therapy [38], as discussed in Chapter 23 and reviewed recently [39]. Differential effects of genotypes 1 and 3 core on insulin signaling have been observed [40], and may partially explain genotype-specific differences in treatment response.

Steatosis

HCV affects lipid metabolism in a genotype-specific manner, with a strong association between genotype 3 and steatosis [41,42]. Genotype 3-associated steatosis appears to be a direct viral effect, as it occurs in the absence of other metabolic risk factors [43,44], correlates with increased viral load [41,45], and improves following successful antiviral therapy [46,47,48]. In contrast, patients infected with non-3 genotypes develop steatosis in association with HCV-induced insulin resistance and other metabolic abnormalities [45,49].

Conclusion

The HCV genome, although highly diverse overall, remains constrained by its host in many key areas. The error-prone RNA polymerase has allowed HCV to diversify among populations, resulting in various genotypes and subtypes, and to adapt quickly to an individual host through quasispecies diversity. Hypervariable regions in immunogenic proteins allow the virus to escape the host immune system, while regions essential for viral structure and replication remain conserved.

The complex interactions between HCV and its human host vary among genotypes, providing significant challenges for interferon-based therapies. A number of specific HCV polymerase and protease inhibitors are in phase III clinical trials, but they are rarely effective against all genotypes, and resistance emerges rapidly [50]. It is therefore likely that a multifaceted treatment approach is required to eradicate this virus, which has demonstrated the ability to securely embed itself within the liver interactome.

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