GLoBAL ANTIVIRAL JoURNAL - IHL Press

GAJ 

Gl o b a l 

An t i v i r a l 

Jo u r n a l 

December 9-13, 2007 

The Westin Maui 

Lahaina, Hawaii 

Final Program and Abstract Book 

This program is sponsored by Emory University School of Medicine 

GAJ 

Volume 3, Supplement 2

GAJ 

Gl o b a l 

An t i v i r a l 

Jo u r n a l 

Aims and Scope 

Global Antiviral Journal publishes peer-reviewed original works related to international efforts to advance antiviral 

discovery and development, including full-length articles and short papers, as well as solicited review articles, conference 

reports, letters and book reviews. Occasional supplements contain conference abstracts presentations and/or posters 

from international meetings in the fields of virology and antiviral research. The scope of the journal encompasses 

chemistry and biological advances in the fundamental and clinical study of antiviral diseases and their treatment. Areas 

covered include HIV, hepatitis B, hepatitis C and emerging viruses, co-infections, vaccines, animal models, pharmacology, 

microbicides, alternative therapies, viral dynamics and resistance issues. 

The journal is published online by IHL Press at www.ihlpress.com/gaj.html. 

All printed supplements are also made available online. 

Publication Policy 

Global Antiviral Journal publishes only original, documented research of high scientific quality, following accepted ethical 

standards of research. Submission of a manuscript signifies that it has been neither copyrighted, published, nor submitted 

or accepted for publication elsewhere. 

Editor-in-Chief 

Raymond F. Schinazi, Emory University School of Medicine and Veterans Affairs Medical Center, Department of Pediatrics, 

Medical Research 151H, 1670 Clairmont Road, Decatur, Georgia, 30033, USA 

Editorial Office 

IHL Press, 26 Bailey Road, Arlington, MA, 02476, USA 

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Copyright 

© 2007 IHL Press. All rights reserved. 

No part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, 

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Notice 

No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products 

liability, negligence or otherwise, or from any use or operation of any methods, products, diagnoses, drug dosages, 

instructions or ideas contained in the material herein. 

ISSN (print): 1556-9047 

ISSN (online): 1556-9055

December 9-13, 2007 • The Westin Maui • Lahaina, Hawaii 

Final Program 

and Abstract Book 

This program is sponsored by Emory University School of Medicine 

HEP DART 2007 - Frontiers in Drug Development for Viral Hepatitis 

i

Table of Contents 

Corporate Supporters .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv 

Continuing Medical Education............................................................... v 

Scholarships .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v 

Conference Committees .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi 

Special Events........................................................................... vii 

Scientific Program .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 

Abstracts.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 

Page 

Sunday, December 9 

State of the Art Lectures.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 

Monday, December 10 

Advances in New Models for HBV and HCV..........................................5 

Tuesday, December 11 

Diagnosis and New Treatments for HBV and HCV....................................19 

Hepatitis B Foundation Symposium: 

Early Detection and Management of Cirrhosis and Hepatocellular Carcinoma..........39 

Immunology & Pathogenesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 

Wednesday, December 12 

New Therapeutic Approaches.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 

Resistance to Antiviral Agents.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 

Co-infection with HIV and Other Viruses .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 

Thursday, December 13 

Pharmacology and Drug Interactions of HBV and HCV Therapeutics.. . . . . . . . . . . . . . . . . 105 

Optimizing the Outcome of Therapeutics for HBV and HCV .. . . . . . . . . . . . . . . . . . . . . . . . 113 

Late Breaker Session .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 

Author Index........................................................................... 133 


iii

Support for HEP DART 2007 was provided by: 

Platinum Support 

Gold Support 

Silver Support 

Bronze Support 

Anadys Pharmaceuticals Inc. • GlaxoSmithKline • GlaxoSmithKline R&D 

Merchant & Gould LLC • Romark Laboratories, L.C. • Samchully Pharmaceutical Co., Ltd. 

Additional Support 

ACLIRES International Ltd • Department of Veterans Affairs 

Genelabs Technologies, Inc. • GlobeImmune, Inc. • Novartis Vaccines and Diagnostics, Inc. 

Raymond F. Schinazi & Family Foundation • RFS Pharma, LLC 

William H. Prusoff Foundation 

iv Global Antiviral Journal Volume 3, Supplement 2

Continuing Medical Education 

HEP DART 2007 is sponsored by Emory University School of Medicine. 

Accreditation Statement 

The Emory University School of Medicine is accredited by the ACCME to provide continuing medical 

education for physicians. 

Continuing Medical Education 

The Emory University School of Medicine designates this educational activity for a maximum of 

23.5 AMA PRA Category 1 Credits. Physicians should only claim credit commensurate with the 

extent of their participation in the activity. 

Conference Objectives 

The Scientific Committee has designed the Conference program to ensure that the delegates achieve 

the following objectives: 

• Understand the role of viral targets in the drug development and discovery process 

• Identify the next generation of inhibitors of viral hepatitis 

• Assess the impact of resistance and treatment failure in the drug development and 

discovery process 

• Increase awareness of the clinical impact of antiviral agents 

• Understand the consequences of co-infection with HIV on the management of patients 

• Assess the role of vaccines and therapeutic vaccines in future therapies for viral hepatitis 

Scholarships 

Conference scholarships were provided for post-doctoral fellows, nurses, assistant professors, 

underrepresented minorities, and residents of developing countries. 

2007 Recipients 

Cafer Eroglu, Ondokuz Mayis University, Turkey 

Pablo Gastaminza, The Scripps Research Institute, USA 

Jason Grebely, National University of New South Wales, Australia 

Gustine Liu-Young, Yale University School of Medicine, USA 

Severine Margeridon-Thermet, Stanford University, USA 

Saneyuki Ujino, Chiba Institute of Technology, Japan 


v

Organizing Committee 

Eugene Schiff, Chair 

University of Miami, USA 

Raymond F. Schinazi, Chair 

Emory University/Veteran Affairs Medical Center, USA 

Robert Murphy, Co-chair 

Northwestern University, USA 

Charles Rice, Co-chair 

The Rockefeller University, USA 

Emeritus Chair and Founding Member 

Jean-Pierre Sommadossi 

Idenix Pharmaceuticals, USA 

Miriam Alter 

University of Texas Medical Branch at Galveston, USA 

Scientific Advisory Committee 

Stanley M. Lemon 


Mithat Bozdayi 

Ankara University, Turkey 

Ralf Bartenschlager 

University of Heidelberg, Germany 

Frank V. Chisari 

The Scripps Research Institute, USA 

Jules Dienstag 

Harvard School of Medicine, USA 

Michael W. Fried 

University of North Carolina at Chapel Hill, USA 

Robert Gish 

California Pacific Medical Center, USA 

Allison Jilbert 

University of Adelaide, Australia 

Stephen Locarnini 

Victorian Infectious Diseases Reference Laboratory, Australia 

Anna Lok 

University of Michigan Medical Center, USA 

Michael P. Manns 

Hannover Medical School, Germany 

John G. McHutchison 

Duke University, USA 

Masao Omata 

University of Tokyo, Japan 

Thierry Poynard 

Groupe Hospitalier Pitié-Salpêtrière, France 

Lorne Tyrrell 

University of Alberta, Canada 

Brent E. Korba 

Georgetown University, USA 

vi Global Antiviral Journal Volume 3, Supplement 2

Special Events 

Satellite Symposium 

Toward Curative Therapies for Hepatitis C 

Sunday, December 9, 2007 

9:00 

 

Welcome Reception 


18:00 

Lanai Pool Deck, Westin Maui 

 

Poster Session Reception 

Tuesday, December 11, 2007 

16:30 

Haleakala Ballroom 

 

Gala Party 

Wednesday, December 12, 2007 

19:30 

Aloha Pavilion, Westin Maui 

Conference Secretariat 

1631 Phoenix Boulevard, Suite 4 

College Park, Georgia 30349 USA 

Telephone: +1 770 997 2484 

Facsimile: +1 770 997 2488 

E-mail: info@informedhorizons.com 

Website: www.informedhorizons.com 


vii

HEP DART 2007 

Frontiers in Drug Development for Viral Hepatitis 

Scientific Program 


9:00 Satellite Symposium: Toward Curative Therapies for Hepatitis C 

Abstract 

12:10 Satellite Symposium Lunch 

HEP DART 2007 Opening Session 

15:00 Opening Remarks 

Raymond F. Schinazi 


Chairs: Robert Murphy 

Charles Rice 



15:15 State of the Art Lecture 

The Changing Global Epidemiology of HBV and HCV 01 

Kevin Fenton 

Centers for Disease Control and Prevention, USA 


New Insights into Hepatitis B Pathogenesis and Resistance 02 



18:00 Welcome Reception 

Monday, December 10, 2007 

Chairs: Raymond F. Schinazi 

Robert Murphy 



8:00 Presentation of William H. Prusoff HEP DART Lifetime Achievement Award 



Robert Murphy 


8:10 William H. Prusoff HEP DART Lifetime Achievement Award 

A Pre-Mortem Lookback Investigation of One’s Life in Research: 03 

The Payoffs of a Persistent Patient 

Harvey Alter 

National Institutes of Health, USA 


New Insights into the HCV Replication Cycle: Lessons Learned from Novel Culture Systems 04 

Ralf Bartenschlager 


Advances in New Models for HBV and HCV 

9:20 HCV Cell Culture Systems and the Development of Antiviral Therapeutics 05 

T. Jake Liang National Institutes of Health, USA 

9:40 Role of Negative Regulatory Signals in the Pathogenesis of HCV 06 

Arash Grakoui 

Emory Vaccine Center, USA 

10:00 A Model System for Hepatitis B and C Virus Co-infection 07 

Volker Brass 

University of Freiburg, Germany 


ix

Abstract 

10:20 Special Lecture 

Insights into the Impact of HBV and HCV Viral Dynamics on Antiviral Therapy 08 

Alan S. Perelson 

Los Alamos National Laboratories, USA 

10:40 Panel Discussion 

11:05 Break 

Oral Abstract Presentations 

Chairs: Thierry Poynard 




11:30 Interferon Modulation of Cellular microRNAs as a Novel Antiviral Mechanism 09 

Irene Pedersen 

University of California, San Diego, USA 

11:40 DNA Microarray Analysis of the NS3 and NS5B Genes from Hepatitis C Genotype 1a 10 

Infected Patients 

Gustine Liu-Young 

Yale University, USA 

11:50 Assessment of Both Virological Response at Week 4 and at Week 12 Optimizes Prediciton of 11 

Treatment Outcome in Patients with Chronic Hepatitis C Treated with Peginterferon Alfa-2B 

plus Ribavirin 

Michelle Martinot-Peignoux INSERM U-773-CRB3, France 

12:00 Week 24 is the Optimal Time Point for Predicting Outcomes at 2 Years with Telbivudine 12 

Rajender Reddy 

University of Pennsylvania School of Medicine, USA 

12:10 Lunch 

Monday Evening Session 

The Business of Hepatitis Antivirals 

Chairs: Abel De La Rosa 

Patrick Higgins 

Pharmasset, Inc., USA 


17:00 The Business of Hepatitis Antivirals: Bridging Diverse Interests 

Patrick Higgins 



Adam Cutler 

Sandra Lehrman 

Dennis Liotta 

Richard Smith 

Frank Zavrl 

Canaccord Adams, USA 

Merck Research Laboratories, USA 

Emory University, USA 

JP Morgan Securities, Inc., USA 

Adage Capital, USA 

Tuesday, December 11, 2007 

Diagnosis and New Treatments for HBV and HCV 

Chairs: Ralf Bartenschlager 


Charles Rice 



HCV Replicons vs. Infectious Virus Systems in Drug Discovery 18 

Stanley M. Lemon 



The Size of the Viral Inoculum Determines the Kinetics, Magnitude and Outcome 19 

of Hepatitis B Virus Infection 

Frank V. Chisari 

Scripps Research Institute, USA 

x Global Antiviral Journal Volume 3, Supplement 2

Abstract 

9:00 Population Genetics of HBV and HCV Quasispecies: Implications for Drug Development 20 

and Drug-resistance Testing 

Robert W. Shafer 

Stanford University, USA 

9:20 Nitazoxanide: A Potent Antiviral Agent against HCV and HBV 21 



9:40 Clinical Implications of Combined Intra-cellular and Cellular Evolution of HCV Resistance 22 

during Direct Anti-HCV Treatment 

Avidan U. Neumann 

Bar-Ilan University, Israel 


10:30 Break 


Chairs: George Lau 

Adrian DiBisceglie 

Queen Mary Hospital, Hong Kong 

Saint Louis University School of Medicine, USA 

11:00 Phase 2 Studies with Albinterferon Alfa-2b (alb-IFN) Dosed q4wk Provide Insights 23 

into Dose Selection for Future Studies 

Ira Jacobson 

Weill Medical College of Cornell University, USA 

11:10 Robust Antiviral Efficacy of a “Finger-loop” Allosteric Inhibitor of the HCV Polymerase 24 

in HCV Infected Chimpanzees 

Raffaele De Francesco 

I.R.B.M. “P Angeletti,” Italy 

11:20 Preclinical Development of the Amphipathic DNA Polymer REP 9AC for the Treatment 25 

of HBV Infection 

Andrew Vaillant 

REPLICor Inc., Canada 

11:30 Inhibition of Hepatitis C Virus Replication by Octadecyloxypropyl-(S)-HPMPA 26 

in Genotype 1B and 2A Replicons 

Karl Y. Hostetler 

University of California, San Diego, USA 

11:40 FKBP8 Plays a Crucial Role in the Replication of Hepatitis C Virus 27 

Yoshiharu Matsuura 

Osaka University, Japan 

11:50 Initial Recommendations for HCV Drug Resistance Analysis: A Consensus Statement 28 

from the HCV Drug Resistance Advisory Group 

Kai Lin 

Novartis Institutes for Biomedical Research, Inc., USA 

12:00 Lunch 

Hepatitis B Foundation Symposium: Early Detection and Management of Cirrhosis 

and Hepatocellular Carcinoma 

Chairs: Timothy Block 

Hepatitis B Foundation and Drexel University 

Medical College, USA 

Harvey Alter 


13:30 Introduction: Early Detection and Management of Cirrhosis and Hepatocellular Carcinoma 

Timothy Block 

Hepatitis B Foundation and Drexel University 

Medical College, USA 


xi

Abstract 

13:40 Hepatocellular Carcinoma: Why Early Diagnosis is Needed 39 

Adrian M. DiBisceglie 

Saint Louis University Liver Center, USA 

13:55 Assessment of Fibrosis: Non Biopsy Methods to Determine Hepatic Fibrosis 40 

Nezam H. Afdhal 

Harvard Medical School, USA 

14:10 Screening Fibrosis: “La Révolution Française” 41 

Thierry Poynard 


14:25 Treatment of HCC over the Past Decade: The Experience of over Four Thousand Patients 42 

in Japan 

Masao Omata 


14:40 Use of Pre-emptive Nucleoside Analogue Therapy for Hepatitis B Reactivation 43 

after Chemotherapy 

George K. K. Lau 

Queen Mary Hospital, Hong Kong 


16:30 Poster Session 

Tuesday Evening Session 

Immunology & Pathogenesis 

Chairs Lawrence M. Blatt 

Michael Fried 

Alios BioPharma, Inc., USA 



Cellular Immune Responses against Hepatitis C in Acute and Chronic Infection 44 

Margaret J. Koziel 


18:30 Broadly Neutralizing Antibodies to HCV: Definition of Epitopes and Antiviral Activity 45 

In Vitro and In Vivo 

Mansun Law 

The Scripps Research Institute, USA 

18:50 Innate Immune Studies in Hepatitis B: Novel Studies in Pathogenesis and Treatment 46 

Kumar Visvanathan 

Monash Institute of Medical Research, Australia 

19:10 Early Steps in the Replicative Cycle of Hepatitis B Virus 47 

Dieter Glebe 

University of Giessen, Germany 


Wednesday, December 12, 2007 

New Therapeutic Approaches I 

Chairs: Stanley M. Lemon 

Karen Anderson 


Yale University School of Medicine, USA 


HCV Entry Pathways and Implications for the Development of Entry Inhibitors 53 

Matthew J. Evans 


8:30 The Role of CD81 and Scavenger Receptor Class B Member 1 in HCV Entry 54 

Peter Balfe 

University of Birmingham, UK 

8:50 Lipid Droplet is an Important Organelle for Production of Infectious Hepatitis C Virus 55 

Kunitada Shimotohno 

Keio University, Japan 

xii Global Antiviral Journal Volume 3, Supplement 2

Abstract 

9:10 The Role of Cyclophilins and Cyclophilin Inhibitors in the Replication of HCV 56 

Rafael Crabbé 

Debiopharm S.A., Switzerland 

Oral Abstract Presentation 

9:30 HCV Infection Increases Claudin-1 and Claudin-7 Expression by Inducing Cirrhosis 57 

Andras Kiss 

Semmelweis Medical University, Hungary 


10:05 Break 

New Therapeutic Approaches II 

Chairs: Yves Benhamou 

Marion Peters 

Hôpital Pitié-Salpêtrière, France 

University of California, San Francisco, USA 


10:30 Development of Novel Hyperglycosylated Type 1 Interferons: A Strategy to Improve PK 58 

Performance without Loss of Biological Potency 

Lawrence M. Blatt 

Alios BioPharma, Inc., USA 

10:40 Bioinformatics Resources Supporting the Analysis of Hepatitis C Virus 59 

Elliot J. Lefkowitz 

University of Alabama at Birmingham, USA 

Invited Presentations 

10:50 A Combination of Direct Antiviral Compounds can Achieve Sustained Viral Response 60 

in Hepatitis C Virus-infected Chimpanzees 

David B. Olsen 

Merck Research Laboratories, USA 

11:10 Potent Antiviral Activity of the Nucleoside HCV Inhibitor, R7128, in Prior 61 

IFN Non-responders 



11:30 R1626 Demonstrates Synergistic Antiviral Effect in Combination with Peginterferon Alfa-2a 62 

[40 KD], with or without Ribavirin, and High Barrier to Resistance Development 

David Nelson 

University of Florida, USA 

11:50 Tenofovir DF (TDF) Showed Superior Antiviral Efficacy to Adefovir Dipivoxil (ADV) 63 

at 48 Weeks in Two Pivotal, Randomized, Double-Blind, Studies for the Treatment of 

HBeAg-Negative (-) and HBeAg-Positive (+) Chronic Hepatitis B (CHB): 

Study 102 and Study 103 

Patrick Marcellin 

Hôpital Beaujon, France 


12:35 Lunch 

wednesday Evening Session 

Resistance to Antiviral Agents 

Chairs: Patrick Marcellin 


Hôpital Beaujon, France 



Navigating Dangerous Complexities: Antiviral Strategies for Clinical Trials for Hepatitis C 

Yves Benhamou 

Hôpital Pitié-Salpêtrière, France 

16:30 Overcoming HCV Treatment-resistant Characteristics 77 

Michael W. Fried 



xiii

Abstract 

16:50 Genetic and Structural Variability of the Hepatitis C Viral Polymerase, NS5B: Implications 78 

for Resistance to Inhibitors 

Philip C. Simister 

CNRS, France 


Co-infection with HIV and Other Viruses 

Chairs: Robert Gish 

California Pacific Medical Center, USA 

Masao Omata 


17:30 Treatment of HBV/HIV Co-infections 88 

Marion Peters 

University of California, San Francisco, USA 

17:50 Treatment of HCV in HIV-infected Individuals 89 

Zelalem Temesgen 

Mayo Clinic, USA 

18:10 HCV/HIV Co-infections: Challenges from the Perspective of TAG 90 

Tracy Swan 

Treatment Action Group (TAG), USA 


20:00 Gala Party 

Thursday, December 13, 2007 

Pharmacology and Drug Interactions of HBV and HCV Therapeutics 

Chairs: Dennis Liotta 

Emory University, USA 

Nezam H. Afdhal 



STAT-C: Role in HCV Treatment Algorithm in 2008 



8:30 Preclinical Drug Metabolism in Viral Hepatitis Drug Discovery 97 

Adrian S. Ray 

Gilead Sciences, Inc., USA 

8:50 Frontiers of Clinical Pharmacology in Hepatitis Drug Development 98 

Charles W. Flexner 

Johns Hopkins University, USA 

9:10 Special Lecture 

Current and Future: HCV Maintenance Therapy 99 

Karen L. Lindsay 

University of Southern California, USA 

9:30 Break 

Late Breaker Abstracts 

Chairs: Margaret J. Koziel 




10:00 State of the Art Lecture - Part I 

Long Term Follow-up of HCV Patients Completing Peg-IFN Plus Ribavirin Treatment - 115 

Is There a Cure? 



xiv Global Antiviral Journal Volume 3, Supplement 2

Abstract 

10:20 State of the Art Lecture - Part II 

Delta Hepatitis: Treatment Options for a Largely Neglected Disease 116 



10:40 Biochemical Mechanism of Entecavir as an Antiviral Polymerase Inhibitor 117 

Karen S. Anderson 

Yale University School of Medicine, USA 

11:00 Advancing RNAi-based Therapeutic from Discovery to Clinic 118 

Robert Gish 

California Pacific Medical Research Institute, USA 

Catherine Pachuk 

Nucleonics Inc., USA 


11:50 Reframing the Knowledge and Looking Beyond the Horizon 

Eugene Schiff 

University of Miami, USA 

12:10 Closing Remarks 




xv

Abstracts 

page session title and author abstract 

1 State of the Art Lectures 

3 The Changing Global Epidemiology of HBV and HCV 01 

K Fenton 

3 New Insights into Hepatitis B Pathogenesis and Resistance 02 

S Locarnini 

5 Advances in New Models for HBV and HCV 

7 A Pre-mortem Lookback Investigation of One's Life in Research: 03 

The Payoffs of a Persistent Patient 

H Alter 

8 New Insights into the HCV Replication Cycle: Lessons Learned from 04 

Novel Culture Systems 

R Bartenschlager 

8 HCV Cell Culture Systems and the Development of Antiviral Therapeutics 05 

TJ Liang 

9 Role of Negative Regulatory Signals in the Pathogenesis of HCV 06 

A Grakoui 

10 A Model System for Hepatitis B and C Virus Co-infection 07 

V Brass 

10 Insights into the Impact of HBV and HCV Viral Dynamics on Antiviral Therapy 08 

AS Perelson 

11 Interferon Modulation of Cellular MicroRNAs as a Novel Antiviral Mechanism 09 

IM Pedersen 

11 DNA Microarray Analysis of the NS3 and NS5B Genes from Hepatitis C 10 

Genotype 1a Infected Patients 

G Liu-Young 

12 Assessment of Both Virological Response at Week 4 and at Week 12 11 

Optimizes Prediction of Treatment Outcome in Patients with Chronic 

Hepatitis C Treated with Peginterferon Alfa-2b Plus Ribavirin 

M Martinot-Peignoux 

13 Week 24 is the Optimal Time Point for Predicting Outcomes at 2 Years 12 

with Telbivudine 

KR Reddy 

14 Green Fluorescence Based Assays for Hepatitis C Virus Replication and 13 

Infectivity in Cell Culture 

S Dash 


xvii


15 Development of Mouse Model for Hepatitis C Virus Replication 14 

S Dash 

16 A Cell-Based, Miniaturized Infection System to Identify Bioactive Molecules 15 

Against Hepatitis C Virus 

P Gastaminza 

16 Generation of HCV E2 Specific Neutralizing Monoclonal Antibody 16 

SJ Park 

17 A Mutation within the Hepatitis C Virus NS3 Helicase Domain Promotes 17 

Virion Assembly in Cultured Hepatoma Cells 

M Yi 

19 Diagnosis and New Treatments for HBV and HCV 

21 HCV Replicons vs. Infectious Virus Systems in Drug Discovery 18 

SM Lemon 

21 The Size of the Viral Inoculum Determines the Kinetics, Magnitude and 19 

Outcome of Hepatitis B Virus Infection 

FV Chisari 

22 Population Genetics of HBV and HCV Quasispecies: Implications for Drug 20 

Development and Drug-Resistance Testing 

RW Shafer 

23 Nitazoxanide: A Potent Antiviral Agent Against HCV and HBV 21 

BE Korba 

24 Clinical Implications of Combined Intra-Cellular and Cellular Evolution of 22 

HCV Resistance during Direct Anti-HCV Treatment 

AU Neumann 

25 Phase 2 Studies with Albinterferon alfa-2b (alb-IFN) Dosed q4wk Provide 23 

Insights into Dose Selection for Future Studies 

I Jacobson 

25 Robust Antiviral Efficacy of a “Finger-loop” Allosteric Inhibitor of the HCV 24 

Polymerase in HCV Infected Chimpanzees 

R De Francesco 

26 Preclinical Development of the Amphipathic DNA Polymer REP 9AC for the 25 

Treatment of HBV Infection 

A Vaillant 

27 Inhibition of Hepatitis C Virus Replication by Octadecyloxypropyl -(S)-HPMPA 26 


KY Hostetler 

xviii Global Antiviral Journal Volume 3, Supplement 2


28 FKBP8 Plays a Crucial Role in the Replication of Hepatitis C Virus 27 

Y Matsuura 

29 Initial Recommendations for HCV Drug Resistance Analysis: A Consensus 28 

Statement from the HCV Drug Resistance Advisory Group 

K Lin 

30 Early Biochemical and Virological Response of Clevudine Therapy in Patients 29 

with HBV Associated Liver Cirrhosis 

KW Chung 

30 Clevudine was Superior to Lamivudine in the Patients with HBeAg(+) 30 

Chronic Hepatitis B 

GK Lau 

31 Clevudine Monotherapy Showed Rapid Viral and Biochemical Response in 31 

Chronic Hepatitis B Patients with Cirrhosis 

CH Lee 

32 Phase I Evaluation of a Novel Oral HCVp7 Inhibitor, BIT225, in Healthy 32 

Volunteers 

CA Luscombe 

32 Novel Mechanism of Action of Clevudine Triphosphate: Evidence for 33 

Non-competitive Inhibition of Hepatitis B Virus DNA Polymerase 

E Murakami 

33 Mechanistic Characterization of Potent Small Molecule HCV Inhibitors that 34 

Target NS5A 

A Sandrasagra 

34 GSK949614; a Novel and Potent Thumb Site Inhibitor of the HCV NS5B 35 

Polymerase 

PA Thommes 

35 Pyrrolo[1,2-b]pyridazin-2-ones Show Promising In Vitro Antiviral Activity 36 

Against HCV NS5B Polymerase 

CV Tran 

36 4-(1,1-Dioxo-1,4-dihydro-1λ 6 -benzo[1,4]thiazin-3-yl)-5-hydroxy-2H- 37 

pyridazin-3-ones are a Novel Series of Molecules which Inhibit the 

HCV NS5B Polymerase 

CV Tran 

37 Interference of Hsp90 Activity by Hsp90 Inhibitor Suppresses Hepatitis 38 

C Virus (HCV) Replication 

S Ujino 


xix


39 Hepatitis B Foundation Symposium: 

Early Detection and Management of Cirrhosis and Hepatocellular Carcinoma 

41 Hepatocellular Carcinoma: Why Early Diagnosis is Needed 39 

AM Di Bisceglie 

41 Assessment of Fibrosis: Non Biopsy Methods to Determine Hepatic Fibrosis 40 

NH Afdhal 

43 Screening Fibrosis: “La Révolution Française” 41 

T Poynard 

44 Treatment of HCC Over the Past Decade: The Experience of Over Four 42 

Thousand Patients in Japan 

M Omata 

45 Use of Pre-emptive Nucleoside Analogue Therapy for Hepatitis B Reactivation 43 

after Chemotherapy 

GKK Lau 

47 Immunology & Pathogenesis 

49 Cellular Immune Responses Against Hepatitis C in Acute and Chronic Infection 44 

MJ Koziel 

49 Broadly Neutralizing Antibodies to HCV: Definition of Epitopes and 45 

Antiviral Activity In Vitro and In Vivo 

M Law 

50 Innate Immune Studies in Hepatitis B: Novel Studies in Pathogenesis 46 

and Treatment 

K Visvanathan 

51 Early Steps in the Replicative Cycle of Hepatitis B Virus 47 

D Glebe 

52 Therapeutic Vaccination Against Chronic Hepatitis C Virus Infection: 48 

Towards a Proof-of-concept in Man? 

CS Klade 

53 Amelioration of Metabolic Disturbances and Oxidative Stress in 49 

Hepatitis C Viral Infection by FK506 (Tacrolimus) 

K Koike 

53 Poor Costimulatory Effects of CD 137 in Patients with Hepatocellular 50 

Carcinoma 

JW Shin 

54 Studies on a Therapeutic Vaccine by Modulating Host Dentritic Cells 51 

in Viral Hepatitis B Patients 

YM Wen 

xx Global Antiviral Journal Volume 3, Supplement 2


55 HBsAg-HBIg Complex Modulates Antigen Presentation of Dendritic Cells 52 

from Chronic Hepatitis B Patients 

B Zheng 

57 New Therapeutic Approaches 

59 HCV Entry Pathways and Implications for the Development of 53 

Entry Inhibitors 

MJ Evans 

59 The Role of CD81 and Scavenger Receptor Class B Member I in HCV Entry 54 

P Balfe 

60 Lipid Droplet is an Important Organelle for Production of Infectious 55 

Hepatitis C Virus 

K Shimotohno 

61 The Role of Cyclophilins and Cyclophilin Inhibitors in the Replication of HCV 56 

R Crabbé 

62 HCV Infection Increases Claudin-1 and Claudin-7 Expression by Inducing 57 

Cirrhosis 

A Kiss 

63 Development of Novel Hyperglycosylated Type 1 Interferons: A Strategy to 58 

Improve PK Performance Without Loss of Biological Potency 

LM Blatt 

64 Bioinformatics Resources Supporting the Analysis of Hepatitis C Virus 59 

EJ Lefkowitz 

65 A Combination of Direct Antiviral Compounds Can Achieve Sustained Viral 60 

Response in Hepatitis C Virus-Infected Chimpanzees 

DB Olsen 

65 Potent Antiviral Activity of the Nucleoside HCV Inhibitor, R7128, in Prior 61 

IFN Non-responders 

JG McHutchison 

66 R1626 Demonstrates Synergistic Antiviral Effect in Combination with 62 

Peginterferon Alfa-2a [40KD], with or without Ribavirin, and High Barrier 

to Resistance Development 

D Nelson 

67 Tenofovir DF (TDF) Showed Superior Antiviral Efficacy to Adefovir 63 

Dipivoxil (ADV) at 48 Weeks in Two Pivotal, Randomized, Double-Blind, 

Studies for the Treatment of HBeAg-Negative (-) and HBeAg-Positive (+) 

Chronic Hepatitis B (CHB): Study 102 and Study 103 

P Marcellin 


xxi


68 Sequence Sections of the 5‘-Non-Coding Region of the Hepatitis C-Virus 64 

RNA Genome at the DNA Level: Their Diverse Forms of Composition 

RH Dennin 

69 A Novel Anti-viral Compound, BIT225, Inhibits Bovine Diarrhea Virus 65 

(BVDV) In Vitro, and has Synergistic Antiviral Activity in Combination with 

Recombinant Interferon Alpha-2b (rIFNα−2b) and Ribavirin 

G Ewart 

70 Identification of Therapeutic Targets in Hepatitis B Virus (HBV) Associated 66 

Hepatocellular Carcinoma (HCC) 

MA Feitelson 

71 A Phase 1, Randomized, Blinded, Placebo-controlled, Single-dose, Dose- 67 

escalation Study of PEG-Interferon lambda (PEG-rIL-29) in Healthy Subjects 

DF Hausman 

72 Influence of Laboratory Parameters on Appearance and Course of Bleeding in 68 

Patients with Portal Hypertension and Liver Cirrhosis 

L Husova 

73 Structural Studies of the Hepatitis C Virus NS5A Protein 69 

R Love 

73 Dose Selection of Albinterferon Alfa-2b (alb-IFN) for a Phase 3 Clinical 70 

Program 


74 Efficacy of Cationic Lipid-DNA Complexes (CLDC) on Hepatitis B Virus in 71 

Transgenic Mice 

JD Morrey 

75 Role of Stomatin in the Assembly of Hepatitis C Virus RNA Replicase 72 

Complex 

J-W Oh 

76 Antiviral Activity of Amino Acid Derivatives of Monascus Pigment in 73 

Hepatitis C Virus Replicating Cells 

J-W Oh 

77 Silymarin Displays Anti-viral, Anti-inflammatory, and Immunomodulatory 74 

Effects Towards Hepatitis C Virus 

S Polyak 

77 Construction and Applications of a Liver-specific Lentivirus Vector with 75 

Host Range Determined by the Envelope Proteins of HBV 

J Taylor 

xxii Global Antiviral Journal Volume 3, Supplement 2


78 Designed Zinc Finger Proteins Bind Duck Hepatitis B Virus Enhancer 76 

DNA and Decrease Production of Viral RNA, Proteins and Progeny In Vitro 

KA Zimmerman 

81 Resistance to Antiviral Agents 

83 Overcoming HCV Treatment-resistant Characteristics 77 

MW Fried 

84 Genetic and Structural Variability of the Hepatitis C Viral Polymerase, NS5B: 78 

Implications for Resistance to Inhibitors 

PC Simister 

85 Selection of Multidrug Resistant Hepatitis B Virus Following Sequential 79 

Monotherapy and Combination Therapy 

A Ayres 

86 Multidrug Resistance and Cross-resistance Pathways in HBV as a 80 

Consequence of Treatment Failure 

A Ayres 

87 Antiviral Effect of Nucleoside Analogs and Interferon on a Novel 81 

Infectious Cloned Hepatitis C Virus Containing the S282T Mutation in the 

NS5B RNA Polymerase 

L Bassit 

88 Understanding the Molecular Basis of HBV Drug Resistance by Molecular 82 

Modeling 

CK Chu 

89 High Genetic Barrier to HCV Resistance Presented by PSI-6130 83 

A De La Rosa 

90 Determination of Lamivudine, Adefovir and Entecavir Resistance in Acute 84 

and Chronic Hepatitis B Virus Infections 

C Eroglu 

90 Databases for Antiviral Resistance Monitoring in Hepatitis B 85 

S Locarnini 

91 Combination Chemotherapy with Nucleos(t)ide Analogue Reverse 86 

Transcriptase Inhibitors (NRTI) May Suppress Multidrug-resistant HBV: 

Using In Vitro Assays to Identify Optimal NRTI Combinations and Doses 

T Sozzi 

92 Relative Replication Capacity and Antiviral Cross-Resistance Phenotypes 87 

of Multidrug-resistant Hepatitis B Virus Mutants Implicated in 

Non-response to Sequential Treatment with Adefovir, Lamivudine 

and Entecavir 

T Sozzi 


xxiii


95 Co-infection with HIV and Other Viruses 

97 Treatment of HBV/HIV Co-infections 88 

MG Peters 

97 Treatment of HCV in HIV-infected Individuals 89 

Z Temesgen 

98 HCV/HIV Co-infections: Challenges from the Perspective of TAG 90 

T Swan 

99 Tolerability of Darunavir/Ritonavir Versus Lopinavir/Ritonavir in 91 

Lopinavir/Ritonavir-naïve, Treatment-experienced, Hepatitis B or C 

Co-infected Patients in TITAN 

YK Dayaram 

100 The Effect of Lopinavir/Ritonavir on HIV/HCV Co-Infected Individuals 92 

CA Smith 

100 Pharmacokinetics of TMC125 in HIV-negative Volunteers with Mild or 93 

Moderate Hepatic Impairment 

F Tomaka 

101 Tolerability of Once-daily Darunavir/r versus Lopinavir/r in 94 

Treatment-naïve, Patients Co-infected with Hepatitis B and/or C in the 

ARTEMIS Trial 

F Tomaka 

102 Pharmacokinetics of Multiple-Dose Darunavir in Combination with 95 

Low-Dose Ritonavir in Individuals with Impaired Hepatic Function 

F Tomaka 

103 TMC125 Safety and Tolerability in Treatment-Experienced Hepatitis B 96 

or C Co-infected Patients in DUET-1 and DUET-2 

F Tomaka 

105 Pharmacology and Drug Interactions of HBV and HCV Therapeutics 

107 Preclinical Drug Metabolism in Viral Hepatitis Drug Discovery 97 

AS Ray 

107 Frontiers of Clinical Pharmacology in Hepatitis Drug Development 98 

CW Flexner 

109 Current and Future: HCV Maintenance Therapy 99 

KL Lindsay 

110 The Role of Pgp-driven Efflux on the Potency of HCV Replication Inhibitors 100 

F McPhee 

xxiv Global Antiviral Journal Volume 3, Supplement 2


111 Pharmacologic Evaluation of Novel Small Molecule HCV Inhibitors 101 

Affecting NS5A-dependent Functions 

CR Wobbe 

113 Optimizing the Outcome of Therapeutics for HBV and HCV 

115 Measuring and Mapping Partnership Integration for Optimizing 102 

Assessment and Treatment of Hepatitis C 

G Butt 

115 Development of a Novel Mouse Model to Evaluate Single and 103 

Combination Therapy Against Hepatitis B Virus 

MA Feitelson 

116 Association of Pretreatment Serum Interferon-γ-inducible Protein 10 104 

(Ip-10) Levels with Sustained Virological Response to Peginterferon Plus 

Ribavirin Therapy for Chronic Hepatitis C (CHC) with Virus Genotype 1 

Infection 

M Fukuda 

117 Optimizing Uptake and Response to Treatment of Hepatitis C Virus 105 

(HCV) Infection in Injection Drug Users (IDUs): A Novel Model 

Incorporating Multidisciplinary Care, Directly Observed Therapy and 

Peer-support 

J Grebely 

118 Efficacy of Pegylated Interferon Alpha-2a and Ribavirin Treatment in 106 

Chronic Hepatitis C Patients Depends on Various Baseline Parameters 

and Early Viral Kinetics 

P Husa 

119 Peginterferon Alpha-2a and Ribavirin Combination Therapy for Chronic 107 

Hepatitis C in Patients with Hemophilia – Preliminary Report 

HJ Kim 

120 Changes of IFN-inducible Gene (IP-10, PKR, MxA) Expressions During 108 

Pegylated Interferon Alfa-2b Plus Ribavirin Therapy in HCV Genotype 1 

Infected Japanese Patients 

R Nakao 

121 Baseline Characteristics and Early Virologic Response to Telbivudine Predict 109 

2‐Year Outcomes for Patients with HBeAg-negative Chronic Hepatitis B 

T Poynard 

122 Predictors of Sustained Viral Response in the Retreatment of Previous 110 

Interferon/Ribavirin Nonresponders: Results from the EPIC 3 Program 

T Poynard 

123 Addition of Lamivudine to Patients with Chronic Hepatitis B who are 111 

Viremic on Adefovir Dipivoxil Lowers Plasma HBV DNA Levels 

J Ryan 


xxv


123 A Systematic Review of the Effectiveness of Pegylated Interferon, 112 

Lamivudine, Adefovir and Entecavir for the Treatment of Hepatitis B 

G Woo 

124 Safety Recommendations for Laboratory Values in Specific Product 113 

Characteristics (SPC) of Peginterferon Alfa-2a (PEG) and Ribavirin (RBV) - 

What Happens Under Real Life Conditions? 

E Zehnter 

125 Standard of Medical Care for Patients with Chronic Hepatitis C (cHC) and 114 

Liver Cirrhosis in Germany - A Status Report 

E Zehnter 

127 Late Breaker Session 

129 Long Term Follow-Up of HCV Patients Completing Peg-IFN Plus Ribavirin 115 

Treatment – Is There a Cure? 

MP Manns 

130 Delta Hepatitis: Treatment Options for a Largely Neglected Disease 116 

MP Manns 

130 Biochemical Mechanism of Entecavir as an Antiviral Polymerase Inhibitor 117 

KS Anderson 

131 Advancing RNAi-based Therapeutic from Discovery to Clinic 118 

C Pachuk and R Gish 

131 Ultra-deep Pyrosequencing of HBV Quasispecies in Nucleoside Analog 119 

Treated and Untreated Patients 

S Margeridon-Thermet 

xxvi Global Antiviral Journal Volume 3, Supplement 2

State of the Art Lectures 

HEP DART 2007 - Frontiers in Drug Development for Viral Hepatitis 1

ABSTRACT 01 

The Changing Global 

Epidemiology of HBV and HCV 

K Fenton 

Centers for Disease Control and Prevention, Atlanta, USA 

Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) 

infections continue to have a severe and pervasive 

impact on the health of millions around the world. 

Today, an estimated 350 million persons are living 

with chronic HBV and there are currently 500-700,000 

HBV-related deaths/year. There are an estimated 

120-180 million individuals living with chronic HCV 

with approximately 370,000 HCV-related deaths/ 

year. Both infections have a disproportionate 

and increasing burden in developing countries. 

Transmission modes for HBV include perinatal, 

parenteral, sexual transmission; and persons infected 

as newborns and young children have highest risk 

of chronic disease. For HCV, healthcare exposures 

are particularly problematic in developing countries, 

whereas injecting drug use are among the major 

determinants in developed country settings. 

Preventing HBV and HCV in the United States and 

globally remains challenging. Despite some success of 

infant vaccination programs - by 2005, 80% of WHO 

Member States had introduced HBV vaccination 

programs and an estimated 55% of the world's 

children less than 1 year of age had received 3 doses 

of HepB – much remains to be done as coverage varies 

by WHO region. However, progress is being observed 

as a result of global advocacy, decreasing vaccine 

prices, and availability of resources to the poorest 

countries. Vaccinating high risk adults is also a major 

public health priority and barriers to adult HBV 

vaccination include: Fiscal concerns; cost of vaccine; 

reimbursement for supply and delivery; provider 

practices; time constraints; and patient acceptance. 

For HCV, prevention strategies include protecting 

the blood and tissue supply; promoting safe injection 

practices; assuring infection control in health care 

settings; promoting HCV screening for persons at 

risk; and referring for care and treatment to stop 

liver disease and transmission. However the global 

challenges to prevention include the high incidence 

of infection among IDUs; the tremendous unmet 

need for HCV counseling and harm reduction; higher 

prevalence of infection; lower rate of viral clearance; 

lower rates of response to treatment; greater risk of 

liver cancer. 

This presentation will provide an overview of the 

evolving global epidemiology of HBV and HCV 

infections; current prevention and control strategies; 

and reflect upon recent developments and future 

plans related to preventing HBV and HCV in the 

United States. 

ABSTRACT 02 

New Insights into Hepatitis B 

Pathogenesis and Resistance 

S Locarnini 

Head, R&MD, VIDRL and Director, WHO Collaborating 

Centre for Virus Reference and Research, Melbourne, 

Victoria, 3051, Australia 

The hepatitis B virus (HBV) has developed a number of 

strategies to ensure its persistence in the infected host. 

The production of excess hepatitis B surface antigen 

(HBsAg), the expression of hepatitis Be antigen 

(HBeAg) and the use of a viral minichromosome (in 

which can be found the covalently closed circular [ccc] 

DNA), a transcriptional template which guarantees 

virus production with minimal interference from 

the host’s immune response. The pathogenesis of 

hepatitis B is the outcome of the interplay between 

HBV, the hepatocyte and the immune response, 

since under normal circumstances, the virus is not 

cytopathic. The liver damage of chronic hepatitis 

B (CHB) is the result of the host’s cellular immune 

response to HBV-infected hepatocytes as part of the 

“immune clearance” phase of the disease. Thus, the 

clinical presentations and natural history of CHB are 


mediated though complex interactions between the 

virus and host immune response. Both the innate and 

adaptive branches of the host immune response need 

to be considered as a coordinated and dynamic line of 

attack, rather than as consecutive and independent 

entities. Both established and potential roles for 

many components of the innate immune system have 

been identified. In particular, important interactions 

between HBeAg, HBV and Toll-like receptors (TLR-2), 

Kupffer cells, natural killer T-cells, and dendritic 

cells have been described (see Visvanathan, K. et al 

2007 Hepatology;45:102). Achieving clearance of 

HBV appears to require initial viral suppression and 

recruitment of effector cells by the innate immune 

system, along with adequate antigen presentation 

to and activation of the adaptive immune response 

arm. With the increasing challenge of antiviral drug 

resistance to nucleos(t)ide analogues, a greater 

understanding of the immunological mechanisms 

involved in CHB has the potential to identify new 

therapeutic targets in order to eventually control this 

challenging viral infection of man. 

4 Global Antiviral Journal Volume 3, Supplement 2

Advances in New Models 

for HBV and HCV 


Abstract 03 

A Pre-mortem Lookback 

Investigation of One's Life 

in Research: The Payoffs of a 

Persistent Patient 

H Alter 


Harvey J Alter, MD, serves as Chief, Infectious 

Disease Section and Associate Director for Research, 

Department of Transfusion Medicine, Clinical Center, 

NIH. Dr. Alter was coinvestigator in the discovery 

of the Australia antigen and principal investigator 

in the first study to biophysically characterize that 

antigen. He was principal investigator in prospective 

studies that identified the clinical entity non-A, 

non-B (NANB) hepatitis and was the first to prove 

NANB was a transmissible agent and to establish the 

chimpanzee model for study of this disease. Dr. Alter 

collaborated in a series of studies that defined the 

natural history of NANB/HCV infection and proved 

its frequent progression to chronic hepatitis and 

cirrhosis, a prophetic concept at that time. 

Dr. Alter was principal investigator in sequential 

prospective studies of transfusion-associated 

hepatitis that established the inordinate risk of paid 

donor and HBsAg positive blood, that demonstrated 

the risk of ALT and anti-HBc positive blood, and that 

were instrumental in influencing a national blood 

policy that mandated HBsAg and surrogate assay 

screening. These unique prospective studies have 

documented the progressive decline of TAH incidence 

from 33% in the 1960s to near zero in 1997 and have 

sequentially established the efficacy of various donor 

screening interventions. 

studies that defined the size, buoyant density and 

infectivity titer of the NANB/HCV agent, that defined 

replication characteristics and mutation rates, and 

that characterized neutralizing antibody responses. 

Dr. Alter was principal investigator in a large cohort 

study to define the modes of transmission and clinical 

relevance of asymptomatic HCV infection and first to 

suggest HCV may be spread by cocaine snorting. He 

was senior investigator in a study that characterized 

the HCV quasispecies early in infection and related 

the complexity and diversity of the quasispecies to 

the severity of clinical outcome. 

For these studies, Dr. Alter has been awarded the 

DHEW Superior Service Award and Distinguished 

Service Medal, the latter the highest award conferred 

to persons in the Public Health Service. Dr. Alter 

was corecipient of the Landsteiner Prize, the most 

prestigious award of the American Association 

of Blood Banks. For his cumulative research 

accomplishments, Dr. Alter was elected to fellowship 

in the American Association of Physicians and is the 

year 2000 recipient of the Clinical Lasker Award. In 

2001, Dr. Alter was elected to the National Academy 

of Sciences and in 2002 to the Institute of Medicine. 

He was also elected to Master status in the American 

College of Physicians. In 2004, he was recipient of 

the First International Medal for Science given by 

INSERM, the French counterpart to NIH and recipient 

of the American College of Physicians Award for 

Outstanding Work in Science as Related to Medicine. 

Following the cloning of HCV, Dr. Alter conducted the 

key study that established HCV as the major cause 

of transfusion-associated hepatitis and proved the 

clinical efficacy of anti-HCV screening assays. He was 

a collaborator in a series of human and chimpanzee 


Abstract 04 

New Insights into the HCV 

Replication Cycle: Lessons 

Learned from Novel Culture 

Systems 

R Bartenschlager 

Department for Molecular Virology, University of 

Heidelberg, Im Neuenheimer Feld 345, Heidelberg, 

Germany 

Hepatitis C viruses (HCV) comprise a group of 

positive-strand RNA viruses that together with 

the flaviviruses and the pestiviruses belong to the 

Flaviviridae family. As a major cause of acute and 

chronic liver disease worldwide for which no selective 

therapy exists, HCV has received much attention. 

The HCV genome encodes a single polyprotein that 

is cleaved into 10 different products. To most of 

these proteins distinct functions could be ascribed 

and the 3D X-ray crystal structures have been solved 

for several viral enzymes that are prime targets for 

antiviral therapy, especially the NS3 protease and the 

NS5B RNA-dependent RNA polymerase (RdRp). 

With the availability of cell culture model systems, 

in particular HCV replicons and the HCVcc system, 

new insights into the molecular and cellular 

mechanisms underlying HCV entry, replication, 

assembly and egress have been gained. For instance, 

novel molecules involved in viral entry have been 

identified and a complex picture emerges how the 

virus productively infects a cell. Likewise, new and 

surprising insights have been gained how HCV 

particles assemble. It turned out that lipid droplets 

play a very important role for virion morphogenesis, 

and that NS5A appears to be a key regulator of the 

different steps of the viral replication cycle. Finally, 

high-throughput screening methods have been used 

to identify host cell factors that contribute to HCV 

replication. Prominent examples are cyclophilins that 

appear to activate NS5B RdRp activity, and h-VAPA as 

well as FBL-2 interacting with NS5A and contributing 

to RNA replication. The common denominator is that 

HCV usurps multiple cellular processes, including 

lipid metabolism to achieve efficient RNA replication, 

virus assembly and virion infectivity. 

Abstract 05 

HCV Cell Culture Systems and 

the Development of Antiviral 

Therapeutics 

TJ Liang 

Liver Diseases Branch, NIDDK, NIH, USA 

Hepatitis C virus (HCV) is a leading cause of morbidity 

and mortality worldwide. The pathogenesis of hepatitis 

C and effects of HCV gene expression on infected cells 

remain unclear in vivo. Efforts to establish cell culture 

and animal models for hepatitis C are critical for the 

understanding of the virus and the disease it causes, 

as well as for developing antiviral therapeutics. 

We recently showed the production of HCV particles 

by using a DNA expression plasmid containing fulllength 

HCV cDNA flanked by self-cleaving ribozymes. 

This construct also contains the secreted alkaline 

phosphatase gene to monitor the expression from this 

construct and to normalize transfection efficiency 

and to control for effects of culture conditions, such 

as anti-viral testing. We produced HCV particles of 

various genotypes including 1a (H77), 1b (CG1b) 

and 2a (J6 and JFH-1) in the HCV-ribozyme system. 

The constructs also contain the secreted alkaline 

phosphatase gene to control for transfection 

efficiency and effects of culture conditions. After 

transfection into Huh7-derived cell line Huh7.5.1, 

continuous HCV replication and secretion were 

confirmed by detection of HCV RNA and core antigen 

in the culture medium. HCV replication levels of 

strains H77, CG1b and J6 were comparable, whereas 

the JFH-1 strain replicates at a substantially higher 

level than the other strains. Interferon-alfa treatment 


can substantially supporess HCV replication in this 

model. To evaluate the infectivity in vitro, the culture 

medium of JFH-1-transfected cells were inoculated 

to naïve Huh7.5.1 cells. HCV proteins were detected 

by immunofluorescence 3 days after inoculation. To 

evaluate the infectivity in vivo, the culture medium 

from HCV genotype 1b-transfected cells was 

inoculated into a chimpanzee and caused a typical 

course of HCV infection. The HCV 1b propagated in 

vitro and in vivo had identical sequences as the HCV 

genomic cDNA used for cell culture transfection. 

Current treatment of chronic hepatitis C based 

on combination of peginterferon and ribavirin is 

only effective in about half of the patients and is 

accompanied by substantial side effects. Developing 

new classes of drugs against HCV is crucial. A new class 

of HCV inhibitors (amphipathic DNA polymers) that 

are active in the viral entry step will be presented. Its 

potential mechanism of action and value in dissecting 

the molecular pathway of HCV entry will be discussed. 

The development of culture systems for production 

of various HCV genotypes provides a valuable tool 

not only to study the replication and pathogenesis of 

HCV but also to screen for antivirals. 

Abstract 06 

Role of Negative Regulatory Signals 

in the Pathogenesis of HCV 

A Grakoui 

Emory Vaccine Center, USA 

The adaptive immune response relies upon controlled 

activation of antigen specific T lymphocytes which 

are critical for maintaining immunity to pathogens 

as well as tolerance to host tissues. The recently 

described PD-1/PD-L1 pathway is important for both 

responsibilities of the adaptive immune response. 

PD-1 (programmed death 1) is a co-inhibitory receptor 

expressed by T cells whose engagement by its ligands 

PD-L1 and PD-L2 results in decreased proliferation 

and cytokine production by antigen specific T cells. 

Studies in the most well established murine model 

of chronic infection, lymphochoriomeningitis virus 

(LCMV), first demonstrated the exciting possibility 

that functional blockade of the PD-1/PD-L1 pathway 

could reverse functionally impaired antigen specific 

T cells. And indeed, subsequent studies in HIV 

showed that PD-1 expression on HIV-specific CD8 + T 

cells was upregulated and that PD-1 expression levels 

correlated both with impaired capacity for cytokine 

production and proliferation as well as viral load. 

We hypothesized that this important co-inhibitory 

pathway may contribute to hepatitis C virus (HCV) 

persistence in humans and established a large patient 

cohort in which to study the negative regulation of 

the anti-HCV immune response. Since there is no 

vaccine available to prevent HCV infection and current 

treatment regimens yield a sustained viral response 

of less than 50%, elucidating the points at which 

host immunity fails to abrogate viremia is critical for 

the development of immune interventions that may 

augment the host response to HCV. To this end, we 

have found that HCV specific CD8 + T cells found in 

the peripheral blood of patients with persistent HCV 

infection express high levels of the co-inhibitory 

receptor, PD-1. These cells have impaired proliferative 

capacity that can be reversed by PD-1/PD-L1 blockade. 

Importantly, intrahepatic HCV-specific cells not only 

express high levels of PD-1, but also low levels of 

the IL-7 receptor (CD127), characteristic findings of 

functionally exhausted T cells. These phenotypically 

exhausted cells are compartmentalized to the liver, 

the site of active viral replication, suggesting that 

repeated exposure to viral antigen may contribute to 

regulation of PD-1 expression. The functional reversal 

of T cell exhaustion by PD-1/PD-L1 blockade holds 

promise for a possible new therapeutic intervention 

that will revive dysfunctional T cells in persistent 

viral infection. 


Abstract 07 

A Model System for Hepatitis B 

and C Virus Co-infection 

P Bellecave 1 , J Gouttenoire 1 , M Gajer 2 , V Brass 2 , 

G Koutsoudakis 3 , HE Blum 2 , M Nassal 2 , 

R Bartenschlager 3 , and D Moradpour 1 

1 Division of Gastroenterology and Hepatology, Centre 

Hospitalier Universitaire Vaudois, University of Lausanne, 

CH-1011 Lausanne, Switzerland; 2 Department of 

Medicine II, University of Freiburg, D-79106 Freiburg, 

Germany; 3 Department of Molecular Virology, University 

of Heidelberg, D-69120 Heidelberg, Germany 

Co-infection with hepatitis B virus (HBV) and 

hepatitis C virus (HCV) has been associated with 

severe liver disease and frequent progression to 

liver cirrhosis and hepatocellular carcinoma. Clinical 

evidence suggests reciprocal replicative suppression 

of the two viruses (‘viral interference’). However, due 

to the lack of appropriate model systems virtually 

nothing is known about molecular interactions 

between HBV and HCV. On this background, the aim 

of this study was to develop a model system to study 

HBV and HCV co-infection. 

A tetracycline-regulated gene expression system was 

used to generate stable Huh-7 cell lines inducibly 

replicating HBV. These cell lines and control Huh-7 

cell lines inducibly expressing the green fluorescent 

protein (GFP) were transfected with selectable HCV 

replicons or infected with cell culture-derived HCV 

(HCVcc). 

Three successive transfection and selection steps 

allowed the establishment of Huh-7 cells inducibly 

replicating HBV and constitutively replicating 

subgenomic HCV RNA. In these cell lines, it is now 

possible to regulate the expression of HBV proteins, 

HBV genome replication, and infectious HBV particle 

formation by the concentration of tetracycline in 

the cell culture medium while HCV proteins are 

expressed and HCV RNA replicated in a constitutive 

fashion. In a series of proof-of-principle experiments, 

this system was used to assess the antiviral effects of 

interferon-alpha and of specific inhibitors of the HBV 

polymerase as well as the HCV serine protease and 

NS5B polymerase. These experiments demonstrated 

that HBV and HCV replication can be selectively 

inhibited in cell lines harboring both viruses and 

that the presence or absence of replicating HBV does 

not significantly alter HCV RNA replication or the 

response of HCV to interferon-alpha. Preliminary 

immunofluorescence and confocal laser scanning 

microscopy data do not show colocalization between 

HBV and HCV proteins. In addition, HBV replication 

does not significantly alter the characteristics of 

HCV replication complexes. These initial results 

demonstrate that HBV and HCV can replicate in 

the same cell. Studies using HCVcc to infect HBVinducible 

cell lines are currently in progress to validate 

and extend these findings. 

In conclusion, we have successfully established and 

are currently exploiting a novel model system to 

investigate interactions between HBV and HCV. 

Understanding such interactions at the molecular 

level should yield new insights into the pathogenesis 

and clinical management of HBV-HCV co-infection. 

Abstract 08 

Insights into the Impact of HBV 

and HCV Viral Dynamics on 

Antiviral Therapy 

AS Perelson 

Theoretical Biology and Biophysics, Los Alamos National 

Laboratory, Los Alamos, NM 87545, USA 

Much of the modeling of HCV and HBV infection 

and treatment has centered around a simple model 

introduced by Neumann et al. in 1998 to model the 

first 14`days of treatment of HCV infection with daily 

interferon. While this model has been very successful 

it makes a number of simplifying assumptions. Here 

I shall show how the Neumann et al model has been 

improved so as to be able to incorporate proliferation 

of both uninfected and infected hepatocytes as 


well time-varying changes in drug efficacy that is 

particularly relevant to once-weekly treatment with 

pegylated interferon. Using these new versions of 

the model I shall show how a variety of patterns of 

viral decline under therapy that have been called 

biphasic, triphasic, and stepwise declines as well as 

viral rebound can be explained. 

that IFNβ treatment leads to a significant reduction 

in the expression of the liver-specific miR-122, a 

miR that has been previously shown to be essential 

for HCV replication. Therefore, our findings strongly 

support the notion that mammalian organisms too, 

via the interferon system, utilize cellular miRs to 

combat viral infections. 

Abstract 09 

Interferon Modulation of Cellular 

MicroRNAs as a Novel Antiviral 

Mechanism 

IM Pedersen 1 , G Cheng 3 , S Wieland 3 , S Volonia 4 , 

CM Croce 4 , FV Chisari 3 , and M David 1,2 

1 Department of Molecular Biology, Division of Biological 

Sciences; 2 Moores Cancer Center, University of California 

San Diego, La Jolla, CA 92093, USA; 3 Division of 

Experimental Pathology, The Scripps Research Institute, 

La Jolla, CA 92037, USA; 4 Department of Molecular 

Virology, Immunology & Medical Genetics, Ohio State 

University, Columbus, OH 43210, USA 

RNA interference through non-coding microRNAs 

(miRs) represents a vital component of the innate 

antiviral immune response in plants and invertebrate 

animals, however, a role for cellular miRs in the 

defense against viral infection in mammalian 

organisms has thus far remained elusive. We show 

now that interferon beta (IFNβ) rapidly modulates 

the expression of numerous cellular miRs, and 

that 8 of these IFNβ-induced miRs have sequencepredicted 

targets within the hepatitis C virus (HCV) 

genomic RNA. Introduction of synthetic miR-mimics 

corresponding to these IFNβ-induced miRs reproduces 

the antiviral effects of IFNβ on HCV replication and 

infection, whereas neutralization of these antiviral 

miRs with anti-miRs reduces the antiviral effects of 

IFNβ against HCV. Single nucleotide changes in the 

target seed sequences of miR-196 and miR-448 in the 

HCV genome render miRs-196 and -448 ineffective 

against HCV replication, however, introduction of 

compensatory nucleotide substitutions in the miRs 

restores their efficacy. Furthermore, we demonstrate 

Abstract 10 

DNA Microarray Analysis of 

the NS3 and NS5B Genes from 

Hepatitis C Genotype 1a Infected 

Patients 

G Liu-Young 1 , J Chiarella 1 , J Stapleton 2 , 

G Garcia-Tsao 1 , J Grebely 3 , B Conway 3 , K Dieckhaus 4 , 

PK Barua 1 and MJ Kozal 1 

1 Yale University and the VA CT Healthcare System, 

Connecticut, USA; 2 University of Iowa, Iowa, USA; 

3 University of British Columbia, Vancouver, BC, Canada; 

4 University of Connecticut, Connecticut, USA 

Background: The HCV coding region for new 

targets of HCV therapy encompass >5Kb (NS3, 

NS4A, NS5A and NS5B) of the viral genome, and 

investigations of drug resistance at one or more sites 

require extensive sequencing. The genetic diversity 

of major HCV genotypes further complicates assay 

development. New assays that can rapidly and 

accurately provide the sequence of target genes from 

major HCV genotypes could greatly facilitate drug 

resistance investigations. 

Methods: The entire HCV 1a genome and the NS3, 

NS4A, NS5A and NS5B genes from HCV genotypes 

1b, 2a, 2b, and 3a, have been placed on a customdesigned 

microarray. In addition, the microarray 

has arrays to detect minority variants containing 

drug resistance mutations in NS3, NS5A and NS5B 

for genotypes 1a and 1b. RT-PCR was performed 

to amplify the NS3 and NS5B genes from HCV 1ainfected 

patients. Amplicons were hybridized to the 

microarray and the sequences were analyzed to detect 


polymorphisms/mutations. A consensus for 1a NS3 

and NS5B sequences was determined by analyzing 

data from HCV databases and the interrogated 

samples. 

Results: 129 unique HCV NS3 gene sequences from 

treatment naïve individuals (region of interest: codons 

32 to 173) were analyzed by microarray and standard 

sequencing and 56.8% of nucleotide (nt) and 42% of 

amino acid (aa) positions were polymorphic; of 109 

unique NS5B gene sequences (region of interest: 

codons 230 to 484) analyzed by microarray, 69.3% 

of nt and 29.8% of aa positions were polymorphic. 

Positions in NS3 gene associated with drug resistance 

to VX-950 and SCH503034 (codons 36, 54, 155, 156, 

168, and 170) and positions in NS5B associated with 

drug resistance to NM283 and HCV-796 (codons 

282 and 316) remained highly conserved and no 

resistance-associated aa changes were identified by 

microarray. The microarray determined the sequence 

of major HCV protease and polymerase inhibitor 

positions in 99.2% and 96.4% of samples respectively 

(range 95-100% depending on codon interrogated). 

Conclusions: The HCV microarray determined 

the sequence of a large portion of the HCV 1a genome 

in a single hybridization experiment, including the 

major NS3 protease and NS5B polymerase inhibitor 

resistance mutation sites. In this microarray survey 

of viral variants from HCV-infected therapy-naïve 

subjects, the NS3 and NS5B genes were highly 

polymorphic in nature. Further studies using specific 

arrays and Ultra Deep sequencing to detect very 

low levels of minor HCV variants will be required 

to determine if minor resistant variants pre-exist in 

patients’ samples. 

Abstract 11 

Assessment of Both Virological 

Response at Week 4 and at Week 

12 Optimizes Prediction of 

Treatment Outcome in Patients 

with Chronic Hepatitis C Treated 

with Peginterferon Alfa-2b Plus 

Ribavirin 

M Martinot-Peignoux, M-P Ripault, S Maylin, 

R Moucari, N Boyer, N Giuily, C Castelnau, and 

P Marcellin 

INSERM, U-773, Centre de Recherche Biomédicale 

Bichat-Beaujon CRB3; Université Paris VII, and Service 

d’Hépatologie, Hôpital Beaujon, Clichy, France 

Pretreatment viral load (VL) is an important predictor 

for treatment outcome in patients with chronic 

hepatitis C. Zeuzem et al. (AASLD 2006) proposed 

400 000 IU/ml as the optimal cut-off to best 

discriminate low and high VL, based on the 

probability to achieve SVR in patients treated with 

PEG-IFN+RBV. 

We aimed to analyze the positive predictive value 

(PPV) of this cut-off value at baseline, the PPV of 

rapid virological response at week 4 (RVR4) and the 

negative predictive value (NPV) of non early virological 

response at week 12 (non-EVR12), in patients treated 

with PEG-IFN alpha 2b+RBV. 

Patients-Methods: 408 patients (221 naïve; 

187 non-naïve) consecutively treated with standard 

schedule of PEG-IFN alpha-2b+RBV, were included. 

Serum HCV RNA was measured at baseline, week 4, 

week 12, end of treatment and 6 months after the 

end of treatment with the quantitative VERSANT R 

HCV 3.0 Assay (bDNA). Samples below the limit of 

quantification were tested with VERSANT R HCV RNA 

Qualitative Assay (TMA) (Siemens Medical Solution 

Diagnostic). SVR was defined as undetectable serum 

HCV RNA by TMA at end of 6 months post-treatment 


follow-up. At treatment initiation PPV was defined 

with a cut-off set up at ≤ 400 10 3 IU/ml; at week 4 

the PPV was defined as TMA undetectable or ≥ 2 log 

drop baseline VL; at week 12 the NPV was defined 

as TMA detectable or < 2 log drop baseline viral 

load. A logistic regression analysis was performed 

to identify factors independently associated with 

RVR. The characteristics included were: baseline 

VL (≤vs>400x10 3 IU/ml), HCV genotype (1, 2, 3, 

4, 5), gender, age (≤vs>45y), histology grade (A0- 

1≤vs>A2-3), fibrosis stage (F0-2≤vs>F3-4) assessed 

with Metavir score, serum ALT, pretreatment status. 

Results: At baseline PPV of VL ≤ 400 000IU/ml 

were: 63%, 76%, 27% in naïve, relapsers and Nonresponders, 

respectively. At week 4 the PPV were: 

96% or 78%, 100% or 70%, 100 or 50% for TMA 

undetectable or 2 log drop, in naïve, relapsers and 

Non-responders, respectively. 

At week 12 the NPV were: 86% or 97%, 64% or 75%, 91 

or 93% for TMA undetectable or 2 log drop, in naïve, 

relapsers and Non-responders, respectively. Factors 

significantly associated with RVR odds ratio (95%CI) 

were genotypes 2-3: 6.5(6.3.4-12.2)(p

RESULTS: Youden indices derived from ROC curves 

increased from 0.26–0.34 at Week 4 to 0.41–0.55 at 

Week 32 in HBeAg-positive patients. Associated HBV 

DNA threshold values decreased from 5.1–5.6 log 10 

copies/mL at Week 4 to 2.7–3.1 logs at Week 24. In 

HBeAg-negative patients, at Week 24 Youden indices 

were maximized (0.34–0.49) and HBV DNA threshold 

values were minimized (2.5 log 10 

copies/mL). 

At Weeks 8 and 12, the predictive sensitivity of HBV 

DNA level is low, and specificity is high. At Weeks 24 

and 32, sensitivity is substantially higher, consistent 

with the marked increase in PCR-negativity at those 

points. NPV increased from Weeks 4 through 32 for all 

outcomes. Although the sensitivity of PCR-negativity 

for predicting no resistance increased from 0.63 to 

0.69 (HBeAg-positive) or from 0.87 to 0.97 (HBeAgnegative) 

from Weeks 24–32, specificity decreased 

from 0.78 to 0.76 (HBeAg-positive), or from 0.62 

to 0.46 (HBeAg-negative), indicating a decreased 

ability to assess resistance risk. Multivariate analyses 

of baseline HBV DNA, ALT, BMI, age, Ishak fibrosis 

score, Knodell HAI score, gender, and HBV genotype 

indicated that baseline HBV DNA level was the 

strongest predictor of virologic outcome at Week 24 

in both HBeAg-positive and negative patients. 

CONCLUSIONS: Week 24 is the best time point 

to assess initial response to telbivudine therapy, 

as it provides a favorable balance of sensitivity and 

specificity for identifying patients at risk of negative 

outcomes at 2 years. Thus clinical decisions to either 

continue or modify therapy can be made as early as 

Week 24. 

Abstract 13 

Green Fluorescence Based Assays 

for Hepatitis C Virus Replication 

and Infectivity in Cell Culture 

S Hazari, RF Garry, T Wakita, and S Dash 

Department of Pathology and Lab Medicine, Microbiology 

and Immunology, Tulane University Health Sciences 

Center, New Orleans, LA 70112, USA 

Background: A convenient and reliable assay 

system is needed for the measurement of hepatitis C 

virus replication and viral infectivity in a cell culture. 

Recently, a unique HCV clone derived from Japanese 

patients has been demonstrated to replicate efficiently 

in cell culture and produce infectious HCV particles. 

Aim: We sought to develop chimeric clones between 

green fluorescence protein (GFP) with full-length and 

sub-genomic JFH-1 clones such that HCV replication 

and infection can be examined directly using 

fluorescence microscopy. 

Methods: Chimeric full-length (JFH-1) and replicon 

(pSGR-JFH-1) clones were prepared by inserting 

the GFP coding sequences in frame with the coding 

sequences of HCV NS5A protein of JFH-1 clone. 

HCV RNA transcripts were prepared from each clone 

by T7 RNA polymerase and transfected to Huh-7 

cells by the electroporation method. The success 

of HCV transfection and replication was examined 

by developing stable replicon cell lines and in vitro 

infectivity of fluorescence virus particles using Huh- 

7.5 cells. 

Results: Both the replicon and full-length chimeric 

clone showed a fairly high-level expression of NS5A- 

GFP fusion protein that visualized by fluorescence 

microscopy and fusion protein can be detected by 

Western blot analysis. The replication of chimeric 

clones was confirmed by detecting positive strand 

HCV RNA by ribonuclease protein assay and the 

ability to form G-418 cell colonies. We now have 


established several stable cell lines that support 

high-level replication of GFP-tagged sub-genomic 

HCV RNA. The level of viral replication between the 

wild type and chimeric clone is comparable. Huh-7 

cells transfected with GFP tagged viral genomes 

produced infectious virus particles since expression 

of GFP was demonstrated in naïve Huh-7.5 cells after 

virus infection. As a practical validation, we showed 

that replication and infectivity totally abolished by 

treatment with alpha interferon. 

Conclusion: We established a HCV cell culture 

systems using GFP tagged viral genomic and subgenomic 

clones that allow direct visualization of 

infected cells by fluorescence microscopy. These 

systems provide a powerful tool for the understanding 

of host-virus interactions and may provide a 

reliable model system to test varieties of anti-HCV 

substances. 

Abstract 14 

Development of Mouse Model for 

Hepatitis C Virus Replication 

S Dash, S Hazari, K Zackson, RF Garry, M Burrow, and 

T Mandal 

Department of Pathology and Laboratory Medicine, 

Microbiology, Medicine, Tulane University Health 

Sciences Center, Pharmacy Department, Xavier University 

of Louisiana, New Orleans, LA, USA 

Background: We previously published that 

intracellular immunization with recombinant 

antibodies to viral helicase and small interfering RNA 

can effectively eliminate hepatitis C virus replication 

and expression in vitro cell culture models. We realized 

that a reliable small animal model is needed to test 

the success of the intracellular immunization strategy 

against hepatitis C virus in vivo. 

Methods: We prepared interferon sensitive Huh-7 

cell line replicating green fluorescence (GFP)-tagged 

HCV sub-genomic RNA. Mouse adapted GFPtagged 

HCV replicon cell clone was isolated from 

subcutaneous tumors and serially passaged in SCID/ 

bg mice followed by selection with G-418. Replicon 

cells (S-3/GFP) were implanted subcutaneously into 

gamma irradiated SCID mice to form a subcutaneous 

tumor. This model was validated by subcutaneous 

injection of interferon alpha (15,000 IU) daily up to 

2 weeks. 

Results: Highly tumorgenic sub clone of 

original interferon sensitive replicon (S-3/GFP) 

was isolated that express high levels of GFP after 

four passages in SCID mice. These replicon cells 

developed subcutaneous tumors in gamma irradiated 

SCID/bg mice in approximately two weeks. High 

levels of HCV replication in the Xenograft tumors 

were confirmed by detection of HCV positive 

strand RNA by ribonuclease protection assay (RPA) 

and GFP expression by fluorescence microscopy. 

Interferon alpha treatment completely cleared HCV 

RNA replication and expression in the subcutaneous 

tumors within two weeks. 

Conclusions: We prepared a mouse adapted GFPlabeled 

replicon cell line that formed a subcutaneous 

tumor and supported high levels of HCV replication 

in SCID mice. HCV replication in the subcutaneous 

tumors can be effectively inhibited by interferon 

alpha suggesting that this mouse model can be used 

to test novel therapeutics against HCV. 

Aim: To develop a mouse model in which HCV 

replication can be studied for a short-term in 

Xenograft tumor. 


Abstract 15 

A Cell-Based, Miniaturized 

Infection System to Identify 

Bioactive Molecules Against 


P Gastaminza 1 , A Montero 2 , S Pitram 2 , R Ghadiri 2 , 

V Fokin 2 , B Sharpless 2 , and FV Chisari 1 

1 Department of Molecular and Experimental Medicine; 

2 Department of Chemistry, The Scripps Research 

Institute, 10550 N. Torrey Pines, La Jolla, CA 92037, USA 

BACKGROUND: In the past, the lack of a cell culture 

and small animal infection models has limited the 

discovery of new drugs in the field. Although the 

establishment of surrogate models (e.g. replicons, 

pseudoparticles) has greatly contributed to the 

discovery of potential new drugs, only limited 

aspects of the virus replication cycle can be targeted 

with those systems. The recent development of a 

robust model of HCV infection in cell culture allowed 

rescuing a recombinant genotype 2a virus from a 

consensus cDNA cloned from a japanese patient with 

fulminant hepatitis (JFH-1) and reproduction of the 

entire replication cycle of HCV in vitro. 

METHODS: Taking advantage of this new system 

we designed a simple yet sensitive and efficient 

colorimetric screening assay that allows evaluation 

of viral spread in a 96-well format. This technology 

permits the screening of large libraries to identify 

compounds that can target any aspect of the viral 

replication cycle, thus greatly increasing the potential 

yield of candidate antiviral molecules for further 

development. 

RESULTS: Applying this methodology, the screening 

of various chemical libraries including peptides and 

small molecules allowed the discovery of several 

new classes of molecules with antiviral activity in 

the submicromolar to low-micromolar range. The 

significance of the various hits obtained in the 

primary screening was evaluated by determining 

the potency and toxicity of the candidate molecules. 

Time-of-addition experiments as well as specific 

studies performed in surrogate models of HCV 

infection completed the characterization of the 

identified molecules and provided insights on the 

mode of action of the new antiviral compounds. 

CONCLUSION: We have developed a miniaturized 

HCV infection assay that allows screening chemical 

libraries for compounds with antiviral capacity. 

By reproducing the entire HCV replication cycle 

in vitro, this assay creates the unique opportunity 

to identify novel cellular and viral targets and 

therefore novel inhibitors of HCV infection since 

aspects of the viral replication cycle that could not 

be examined in previous screening assays (e.g. viral 

assembly, trafficking and secretion) can be efficiently 

interrogated in this system. Using this technology a 

new class of inhibitory peptides and three classes of 

novel small molecules where shown to significantly 

inhibit HCV infection. 

Abstract 16 

Generation of HCV E2 Specific 

Neutralizing Monoclonal Antibody 

SJ Park 1 , YJ Lee 1 , ET Park 1 , SH Lee 1 , SY Seol 1 , 

HJ Won 2 , YJ Jeong 2 , SG Park 2 , IH Choi 2 , JW Shin 3 , 

and NH Park 3 

1 Department of Internal Medicine; 2 Department of 

Microbiology, College of Medicine, Inje University, Busan; 

3 Department of Internal Medicine, University of Ulsan 

College of Medicine, Ulsan University Hospital, Ulsan, 

Korea 

Objectives: This study aimed to develop a human 

monoclonal antibody neutralizing hepatitis C virus 

infection from naïve human antibody library with 

phage display techniques. 

Methods: For selection of antibody clones reacted 

to HCV E2 protein, panning and screening was 

carried out with E2 protein immobilized CM5 chip in 

BIAcore 2000 from naïve antibody library sized 1 x 


10 10 cfu. Selected antibody clones were analyzed by 

nucleotides sequencing and affinity measurements. 

Neutralizing activity was determined with HCVpp 

infection test into Huh7 cells. 

Results: Four clones (A4, F4, G4, and H6) were 

selected after panning and screening. A4 clones 

showed the highest affinity (9.60 x 10 -8 M -1 ). In the 

neutralizing assay, 10 µg/ml of A4 scFv was inhibited 

30% of HCVpp infection. 

Conclusions: This study generates human 

monoclonal antibody fragment, A4, neutralizing 

HCV E2 proteins. It might be useful in the passive 

immunotherapy for hepatitis C. 

Abstract 17 

A Mutation within the Hepatitis 

C Virus NS3 Helicase Domain 

Promotes Virion Assembly in 

Cultured Hepatoma Cells 

M Yi, Y Ma, J Yates, and SM Lemon 

The Center for Hepatitis Research, Institute for 

Human Infections and Immunity and Department of 

Microbiology and Immunology, University of Texas 

Medical Branch, Galveston, TX, USA 

BACKGROUND: A unique substitution in the amino 

acid sequence of the hepatitis C virus (HCV) NS3 

protein (Q1251L, Yi et al., J. Virol 81:629-38, 2007) 

rescues a defect in production of infectious virus in 

cells transfected with a chimeric RNA (H-NS2/NS3-J, 

designated “HJ3”) comprising the genotype 1a core- 

NS2 sequence placed within the background of the 

genotype 2a JFH-1 sequence. This is surprising as 

NS3, which possesses protease, helicase and NTPase 

activities, has not been suggested previously to play 

a role in virus assembly or release. We have studied 

how the Q1251L mutation contributes to infectious 

virus production by this chimera in order to better 

understand the role of NS3 in this process. 

METHODS: We assessed the effect of the 

Q1251L mutation on RNA replication by two 

different methods; quantitative Taqman RT-PCR 

measurements of genomic RNA, and a reporter assay 

using subgenomic replicon constructs. We measured 

the titer of intracellular and extracellular infectious 

virus produced from HJ3 RNA with or without the QL 

mutation to determine whether this mutation affects 

virus assembly and/or release. Similar experiments 

were performed using JFH1 and a second H77-JFH1 

chimera (H-(NS2)-J, designated HJ2). To further 

understand the NS3-mediated assembly process, 

we analyzed cell lysates by centrifugation through 

equilibrium density gradients and rate-zonal velocity 

gradients, determining the quantity of core protein 

and infectious virus in each fraction. 

RESULTS: Although Q1251 is highly conserved 

across HCV genotypes, the Q1251L substitution 

had no effect on replication of subgenomic replicon 

RNA, and did not impair NS3-associated enzymatic 

activities including processing at the NS2-NS3 

junction. However, it profoundly influenced assembly 

of the chimeric virus. While transfection of unmodified 

HJ3 RNA failed to produce either extracellular or 

intracellular infectious virus, transfection of HJ3 

RNA containing the Q1251L substitution (HJ3/QL) 

resulted in rapid and efficient release of infectious 

virus, which was also detected in cell lysates. 

Quantitative assessment of core protein abundance 

in fractions of isopycnic gradients loaded with lysates 

of transfected cells confirmed that core expressed 

by HJ3 did not assemble into rapidly sedimenting 

particles in the absence of the Q1251L substitution, 

suggesting a role for NS3 early in particle assembly. 

While the introduction of the Q1251L substitution 

into the parental JFH-1 sequence enhanced 

production of JFH-1 virus, it had no significant effect 

on assembly or release of a second chimeric virus, 

H/J2. This differs from H/J3 only in the C-terminal 

19 amino acid residues of NS2, providing genetic 

evidence for an interaction between NS3 and NS2 

during virus assembly. 

CONCLUSION: These results indicate a previously 

unsuspected role for NS3 in early virion morphogenesis 

and provide a new perspective on assembly of the 

infectious HCV particle. 


Diagnosis and New Treatments 

for HBV and HCV 


Abstract 18 

HCV Replicons vs. Infectious Virus 

Systems in Drug Discovery 

SM Lemon 

Center for Hepatitis Research, University of Texas Medical 

Branch, Galveston, TX 77551-1073, USA 

The development of subgenomic and subsequently 

genome-length HCV replicons provided highly 

needed tools for antiviral drug discovery efforts. 

These autonomously replicating RNAs allowed 

early validation of the antiviral activity of NS3/4A 

protease and NS5B polymerase inhibitors that were 

active in cell-free enzyme assays, and in screening 

assays have led to the identification of compounds 

that hit novel targets within the HCV replicase, such 

as NS5A, for which there are no cell-free, enzymatic 

assays. However, while replicon models recapitulate 

most if not all aspects of HCV RNA replication, 

they are typically di-cistronic and place translation 

of the nonstructural proteins under control of a 

heterologous internal ribosome entry site. Moreover, 

they are selected for amplification capacity and 

typically contain one or more cell culture-adaptive 

mutations that generally limit replication of virus 

in the chimpanzee. While such mutations may have 

relatively little relevance for protease and polymerase 

inhibitors, their presence is a concern in evaluating 

compounds that target other nonstructural proteins 

with less well-defined roles in the virus replication 

cycle. In addition, replicons are of no use in efforts to 

target virus attachment and entry, or later steps in the 

assembly and egress of virus from infected cells. These 

steps in the virus replication cycle can be targeted 

using more recently available cell culture-infectious 

virus systems, but not without significant limitations. 

The genotype 2a JFH-1 virus has a robust replication 

phenotype, and viable chimeric viruses have now 

been constructed that express the structural proteins 

of the more prevalent and relatively interferonresistant 

genotype 1a and 1b viruses from within 

the JFH-1 background. These new viruses represent 

valuable tools for the characterization of virus entry 

and the development of entry inhibitors, yet the 

buoyant density of these infectious virus particles 

is significantly less than that of virus produced 

within the liver in situ, indicating a need for caution 

in extrapolating from results obtained with cell 

culture-derived virus. The genotype 1a H77S virus 

has a substantially less robust replication phenotype 

than JFH-1 and its related inter-genotypic chimeras. 

However, in its most recent stage of development, 

cells transfected with H77S.2 RNA release ~10 3 FFU/ 

ml of infectious virus into supernatant fluids, and 

this virus is capable of limited serial cell-free passage. 

While not suitable for high throughput screening, 

H77S.2 is useful in validation experiments as it is 

comprised of entirely genotype 1a sequence with 

defined cell-culture adaptive mutations. 

Abstract 19 

The Size of the Viral Inoculum 

Determines the Kinetics, 

Magnitude and Outcome of 

Hepatitis B Virus Infection 

S Asabe 1 , SF Wieland 1 , R Engle 2 , RH Purcell 2 , and 

FV Chisari 1 

1 Scripps Research Institute, La Jolla, CA, USA; 

2 National Institutes of Health, Bethesda, MD, USA 

The factors that determine the outcome of hepatitis 

B virus (HBV) infection include the age of onset of 

the infection and the immune responsiveness of the 

host. Little is known about the impact of virological 

determinants in this regard. Based on precedent in 

other viral systems, it is assumed that high antigen 

burden will exhaust or otherwise suppress the 

immune response and, thus, favor viral persistence. 

To test that hypothesis we examined the impact of 

inoculum size on the outcome of HBV infection 

in experimentally infected chimpanzees. Animals 

were injected intravenously with 10 10 , 10 7 , 10 4 , 10 1 

or 10 0 genome equivalents (GE) of a monoclonal 


HBV inoculum and monitored for 1 year thereafter. 

Animals receiving 10 10 , 10 7 , and 10 4 GE of HBV 

developed self-limited infections characterized by 

dose-related onset kinetics and magnitude of infection 

that resolved within 12-16 weeks of inoculation. 

Resolution of infection in these animals was preceded 

by an early HBcAg-specific CD4 T cell response and 

coincided with a highly synchronized, early interferon 

gamma-producing T cell response and sharply elevated 

serum alanine aminotransferase (ALT) activity. 

Unexpectedly, virus spread to 100% of the hepatocytes 

in all animals that were inoculated with 10 1 or 10 0 

GE of HBV, and they developed either chronic (>52 

weeks) infections with the histological characteristics 

of chronic active hepatitis, or greatly prolonged (36- 

42 weeks) infections that ultimately resolved in the 

setting of an ALT flare. The immunological correlates 

of these unexpected outcomes were the absence of 

early CD4 T cell responses to HBcAg, the delayed 

onset of poorly synchronized, interferon gammanonproducing 

peripheral CD8 T cell responses, and 

the induction of delayed but vigorous interferon 

gamma-producing intrahepatic T cell responses that 

were functionally unable to terminate the infection. 

Interestingly, serum IL10 and intrahepatic PD1 mRNA 

levels were higher and sustained in the persistent/ 

prolonged low dose infections than in the rapidly 

controlled high dose infections. These results suggest 

that subviral antigens in the infectious inoculum may 

prime antiviral T cell responses that precede viral 

spread through the liver and prepares the host for an 

anamnestic response that controls the infection when 

viral antigens are subsequently produced by infected 

cells. In contrast, failure to prime the T cell response 

early in HBV infection results in uncontrolled viral 

spread in the liver where intrahepatic T cell priming 

and persistent high level antigen production induce 

negative immunological regulators (e.g. IL10 and 

PD1) that further impair CD8 T cell function and lead 

to prolonged or persistent infection. 

Abstract 20 

Population Genetics of HBV and 

HCV Quasispecies: Implications 

for Drug Development and 

Drug-Resistance Testing 

RW Shafer 

Departments of Medicine and Pathology, Stanford 

University, Stanford, CA, USA 

BACKGROUND: HBV and HCV, like HIV, share the 

following characteristics: (i) they each cause a chronic 

life-threatening disease, (ii) they are each treatable 

with small molecular weight compounds, and (iii) 

they each have highly error-prone mechanisms 

of replication causing them to exist in vivo as a 

quasispecies – or mixture of innumerable related 

sequence variants. 

METHODS: My laboratory has been using ultra-deep 

pyrosequencing (UDPS; 454 Life Sciences, Bradford 

CT), to characterize the extent of genetic variability in 

the molecular targets of HIV, HBV, and HCV therapy 

in treatment-naïve and treatment-experienced 

individuals. My presentation will focus on our labs 

results using UDPS for sequencing HBV and HIV and 

on our plans for using UDPS for sequencing HCV. I 

will also review the main UDPS experimental design 

choices including (i) approaches to maximize the 

number of sequencable virus templates, (ii) the choice 

of sequencing strategy: “shotgun” vs tailed primers, 

(iii) the relative importance of PCR enzyme fidelity, 

and (iv) statistical analyses for handling random 

errors and G to A hypermutation. 

RESULTS: We have sequenced the RT and protease 

quasispecies in 48 clinical plasma HIV-1 isolates and 

a 1.2 bp pol region in 32 clinical plasma HBV isolates. 

Our first 32 HIV-1 isolates were sequenced using the 

first generation, GS20 sequencing platform. Each of 

the HBV isolates and our most recent HIV-1 isolates 

were sequenced using he second generation, FLX 

sequencing platform. In each experiment, we have 


characterized the UDPS error rate using plasmid 

clones and have developed a robust statistical 

approach to distinguishing authentic minor 

variants from possible sequencing errors. For most 

experiments, the sensitivity for detecting minor 

variants which ranged from 0.2% to 2.0%, was 

primarily limited by the number of templates that 

could be introduced into the sequencing reaction 

(rather than by the sequencing technology itself). HIV 

and HBV infected persons receiving antiviral therapy, 

were consistently found by UDPS to have clinically 

relevant drug-resistant mutations detected at levels 

ranging from 1% to 20% that were rarely detected 

by standard population-based sequencing. Extensive 

molecular and limiting dilution clonal sequencing, 

furthermore, confirmed that the vast majority, if not 

all of these minor variants, were authentic. 

CONCLUSIONS: In theory, UDPS is an eminently 

logical approach to characterizing genetic variability 

and detecting drug-resistance mutations at the 

earliest stages of their development. In practice, 

however, both the upstream processing of samples 

prior to UDPS and the downstream analysis of UDPS 

sequence data are uncharted areas that require 

considerable amounts of optimization before this 

technology can achieve the reliability that currently 

exists for standard dideoxynucleoside sequencing. 

Abstract 21 

Nitazoxanide: A Potent Antiviral 

Agent Against HCV and HBV 

BE Korba 1 , AB Montero 1 , JS Glenn 2 , MS Ayers 3 , and 

J-F Rossignol 3 

1 Dept. of Microbiology & Immunology, Georgetown 

Univ. Med. Ctr., Washington, DC, USA; 2 Division of 

Gastroenterology & Hepatology, Dept. of Med., Stanford 

Univ. Sch. of Med., Stanford, CA, USA; 3 The Romark 

Institute for Medical Research, Tampa, FL , USA 

Nitazoxanide (NTZ), a thiazolide, is a broad spectrum 

anti-infective drug marketed in the United States 

(Alinia ® ) for treating gastroenteritis caused by 

Cryptosporidium parvum and Giardia lamblia, and is in 

clinical development for treating Clostridium difficile 

and rotavirus-associated diseases. NTZ and its active 

circulating metabolite, tizoxanide (TIZ), exhibited 

potent and selective antiviral effects in cell culture 

models of HCV and HBV replication (Korba, et al., 

2007, Antivir. Res., in press). Both NTZ and TIZ were 

effective against HCV replication in both genotype 

1b (CON1) and 1a (H77) replicon cell lines. Moderate 

synergistic interactions against HCV were observed 

between NTZ and either interferon alpha 2b (IFN), or 

2’C-methyl cytidine in combination treatments. NTZ 

was equally effective against NS5B S282T, and NS3 

A156S/V/T drug-resistant mutants. Pretreatment of 

cultures with NTZ monotherapy further enhanced 

the potency of NTZ + IFN (but not NTZ + 2’CmeC) 

combinations. Although it was possible to select 

NTZ and TIZ resistant HCV replicon cell lines, the 

resistance phenotype was not transferred to naïve 

cells by transfection of HCV RNA from these cell 

lines. Both compounds also inhibited intracellular 

HBV replication and virion production in 2.2.15 

cells. NTZ exhibited moderately synergistic anti- 

HBV interactions with either of two licensed anti- 

HBV agents, lamivudine (LMV) or adefovir dipovoxil 

(ADV), and was equally effective in inhibiting the 

replication of several LMV and ADV-resistant 

mutants. Most notably, NTZ induced a reduction 

in the production of several HBV proteins (HBsAg, 

HBeAg, HBcAg) without a corresponding reduction 

in HBV RNA, indicating a post-transcriptional/ 

translation mechanism. 

Clinical trials against chronic HCV infection are 

in progress in the USA and Egypt. Interim reports 

on clinical efficacy (STEALTH C-1 trial) have been 

recently presented (Rossignol, et al., 2007, Hepatol. 

46(Suppl. 1):316A). In naïve patients, combination 

therapy consisting of 12 weeks pretreatment with 

NTZ followed by 36 weeks of NTZ in combination 

with PegIFNα-2a+ribavirin was significantly more 

effective than 48 weeks of PegIFNα-2a+ribavirin 

(standard of care) (SVR12, 79% vs. 45%, p=0.007). 

Detailed studies of mechanisms against both HBV and 

HCV are currently under investigation. Preliminary 


data indicate that host cell processes are targeted, 

most likely components of the unfolded protein 

and/or ER stress response pathways. Current data 

indicate that the intracellular environment induced 

by each virus may influence the consequences of druginduced 

changes in cellular responses. Nitazoxanide 

is a promising new antiviral agent that, due to its 

probable novel mechanism of action, has substantial 

potential for use as an adjunct to current and future 

therapies to enhance sustained response rates against 

chronic hepatitis virus infection and disease. 

Abstract 22 

Clinical Implications of Combined 

Intra-Cellular and Cellular 

Evolution of HCV Resistance 

during Direct Anti-HCV 

Treatment 

AU Neumann and J Guedj 

Bar-Ilan University, Ramat-Gan, Israel 

Background and goal: The current paradigm for 

modeling development of viral resistance, based on 

the HIV experience, considers evolution of resistance 

only on the cellular infection level. While this is correct 

for HIV, a retrovirus RNA virus for which mutation 

occurs mainly at the RT step, it is known that for 

HCV all processes of resistance evolution – mutation, 

selection and amplification – can occur on a faster 

time-scale of RNA synthesis at the intra-cellular level. 

Here we explore, with a novel mathematical model 

that considers both intra-cellular level evolution and 

cellular infection level viral dynamics, the clinical 

implication of intra-cellular resistance evolution for 

direct anti-HCV drugs. 

Methods: In the model, intra-cellular RNA (ICR) 

is used to form replication units (RU), which in turn 

synthesize more ICR that is partially packaged and 

secreted as virions. Direct anti-HCV drugs can have 

an anti-viral effect through blocking of RU formation, 

ICR synthesis and/or virion export. The development 

of resistance is modeled in the intra-cellular level by 

the evolution and dynamics of different strains of RU 

and ICR with different drug-sensitivity and different 

relative-fitness. Resistance evolution also impacts the 

cellular infection level as result of the exported virus 

of different strains. 

Results: Evolution of resistant virus is faster when 

it occurs at the intra-cellular level as compared to 

occurring only at the cellular infection level, assuming 

similar rates of mutation, and similar distribution of 

sensitivity to drug and relative fitness of the different 

strains in both models. An analytical estimate - based 

on the relative-fitness, drug sensitivity and mutation 

rate parameters - was obtained for the time it takes 

a resistant strain to reach equal concentration as the 

wild-type, and for the resistance rebound slope. 

The combination of resistance evolution on the intracellular 

level with cellular infection viral dynamics 

level allows for more complex viral kinetic patterns 

than possible with resistance evolution on the cellular 

infection level only. In general, a complete rebound, a 

tri-phasic decline, a bi-phasic decline with a shoulder, 

a decline at the delta mode (with the rate of infected 

cell loss) or a decline at the gamma mode (with the 

faster rate of intra-cellular RU loss) are all possible 

as function of the mutation rate and the distribution 

of fitness and sensitivity. Of particular interest, we 

predict that it is possible for the total viral load to 

decline (in the delta mode, after a transient rebound) 

even after takeover by a virus strain fully resistant to 

a drug given as a mono-therapy. 

Conclusions: More rapid and more complex 

patterns of viral resistance evolution are predicted 

when considering HCV resistance evolution at the 

intra-cellular level together with cellular level viral 

dynamics. Some of the patterns predicted by the 

model were already observed in data publicly released 

for different direct anti-HCV drugs. The model 

makes several predictions with important clinical 

implications, such as the possibility for decline 

with fully resistant virus during monotherapy, and 

analytical estimates allow long-term prediction based 

on early kinetic information. 


Abstract 23 

Phase 2 Studies with 

Albinterferon alfa-2b (alb-IFN) 

Dosed q4wk Provide Insights into 

Dose Selection for Future Studies 

I Jacobson 1 and D Nelson 2 

1 Weill Medical College of Cornell University, New York, 

USA; 2 University of Florida at Gainesville, Florida, USA 

BACKGROUND: alb-IFN is a novel, long-acting 

interferon (IFN) with a half-life of ~150 h, achieving 

substantial drug exposure levels, and the capability 

of maintaining antiviral activity detected over a 28-d 

dosing interval. Phase 2 studies have been conducted 

in IFN-naïve patients with chronic hepatitis C (CHC) 

to assess the efficacy and safety of q4wk dosing. 

METHODS: Study 1 (genotype [Gt] 1, CHC): alb-IFN 

1200 μg q4wk (n = 116) was compared with alb-IFN 

900 and 1200 μg q2wk (n = 118 and 110, respectively), 

and peginterferon alfa-2a (PEG-IFNα-2a) 180 μg 

qwk (n = 114), plus ribavirin 1000-1200 mg/d in all 

arms. Study 2 (Gt 2/3, CHC): alb-IFN 1500 μg q4wk 

(n = 22) was compared with alb-IFN 1500 μg q2wk 

(n = 21), plus ribavirin 800 mg/d. Primary efficacy 

endpoint was sustained virologic response (SVR) in 

both studies. 

RESULTS: In study 1, the SVR rates with alb-IFN 

1200 μg q4wk by intent-to-treat analysis vs alb-IFN 

900 μg q2wk and PEG-IFNα-2a were 51% vs 58% and 

58%, respectively (P = .28 vs PEG-IFNα-2a). Although 

rapid virologic response rate at wk 4 (hepatitis C virus 

[HCV] RNA < limit of quantitation [43 IU/mL]) was 

lower in patients with alb-IFN 1200 μg q4wk vs alb- 

IFN 900 μg q2wk and PEG-IFNα-2a (18% vs 25% 

and 26%, respectively), those patients had a 100% 

positive predictive value of achieving SVR. Viral 

breakthrough and relapse rates with alb-IFN 1200 μg 

q4wk were comparable to the q2wk and PEG-IFNα-2a 

arms. alb-IFN 1200 μg q4wk was associated with less 

hematologic toxicity, which was reflected in the need 

for fewer dose reductions. In study 2, alb-IFN 1500 

μg q4wk resulted in an SVR rate of 77% vs 62% for the 

q2wk regimen. Early response rates (HCV RNA

of at least 4 distinct non-nucleoside inhibitor (NNI)- 

binding sites on the HCV polymerase. We previously 

reported a series of indole-based HCV polymerase 

NNIs that bind the enzyme at an allosteric site 

found at the junction of the thumb domain and the 

N-terminal finger loop (“finger-loop” NNIs). Although 

inhibitors of this class have shown to be highly active 

in biochemical and cell-culture assays, evidence of in 

vivo antiviral activity associated with this mechanism 

of action was still missing. 

Methods: A finger-loop NNI of HCV genotype (gt) 

1 and gt 3 NS5B polymerases, with an IC 50 

values of 

30 nM against the genotype 1b replicon, was dosed 

for 5 days at 10 mg/kg bid orally to one chimpanzee 

infected with gt 1a HCV, and at 2 and 10 mg/kg to a 

second chimpanzee infected with gt 1b HCV. 

Results: We observed a 1.1 and 3.8 log 10 

viral load 

reduction in the gt 1b HCV-infected animal at doses 

of 2 and 10 mg/kg, respectively. Viremia rebounded 

after treatment ended. In the gt 1a HCV-infected 

chimp, a dose of 10 mg/kg elicited a 1.4 log 10 

drop 

in viral load in 48 h. Cloning and sequencing of the 

NS5B polymerase gene from the viral isolates present 

in the two different chimps did not reveal any obvious 

genetic reason for the difference in antiviral response, 

and only small differences in susceptibility to this 

class of inhibitors were observed with the polymerase 

genes from the two viral isolates when assayed 

in the replicon system. Moreover, no resistance 

mutations or treatment related sequence changes 

were identified. When antiviral response (log 10 

drop 

in viremia) observed in the 3 dosing intervals of the 

two chimps was analyzed as a function of the ratio 

of trough plasma concentration and normalized for 

replicon EC 50 

, a very steep dose-response curve was 

obtained. Thus, small differences in exposure/potency 

produced large effects on viremia. 

Conclusions: This study demonstrated that 

finger-loop NNIs are efficacious at inhibiting viral 

replication in the HCV infected chimpanzee model 

and suggests that the chimpanzee model could prove 

useful for determining the PK/PD relationship for 

experimental compounds with novel mechanism of 

action. 

Abstract 25 

Preclinical Development of the 

Amphipathic DNA Polymer REP 

9AC for the Treatment of HBV 

Infection 

F Noordeen 1 , A Vaillant 2 , JM Juteau 2 , and A Jilbert 1,3 

1 School of Molecular and Biomedical Science, The 

University of Adelaide, Australia; 2 REPLICor Inc, Laval, 

Quebec, Canada; 3 Infectious Diseases Laboratories, 

Institute of Medical and Veterinary Science, Adelaide, 

Australia 

Background: Amphipathic DNA polymers (APs) 

are a novel class of nucleic acid medicine based 

on phosphorothioate modified DNA which have a 

broad spectrum activity against enveloped viruses. 

In type 1 fusion viruses, APs interact with conserved 

amphipathic alpha helical domains on surface 

glycoproteins blocking their entry or release activities. 

APs are also effective against non-type 1 enveloped 

viruses such as HCV. 

Methods: The antiviral efficacy of the lead AP 

candidate REP 9AC, a 40mer poly AC phosphorothioate 

oligonucleotide, against another non-type 1 enveloped 

virus, the human hepatitis B virus (HBV), was studied 

using the duck hepatitis B virus (DHBV) model both 

in vitro in primary duck hepatocytes (PDH) and in 

vivo in the Pekin duck. 

Results: REP 9AC demonstrated potent antiviral 

activity against DHBV infection of PDH suggesting 

that a large amphipathic domain related to those 

found in type 1 fusion glycoproteins is involved in 

the entry or release of DHBV. In vivo, REP 9AC was 

assessed for its ability to block virus entry and cellto-cell 

spread of DHBV infection. Fourteen-day-old 

ducks were infected with a defined dose of DHBV 

and treated intraperitoneally with REP 9AC (10mg/ 

kg/day) or orally with the Bristol-Myers Squibb 

nucleoside analogue, Entecavir (ETV; 1 mg/kg/day) 

for 14 days. REP 9AC was well tolerated by the ducks 

with no detectable side effects. Biopsy liver tissue 


collected on day 4 post-inoculation (pi) showed a 

small percentage of DHBV-infected hepatocytes in 

all REP 9AC and control ducks. However, by day 14 

pi 100% of ducks treated with REP 9AC showed no 

evidence of continuing DHBV infection in the liver 

using immunohistochemistry for DHBV surface 

antigen, or Southern blot detection of DHBV DNA. 

As expected from previous studies treatment of ducks 

with ETV inhibited DHBV replication and prevented 

the cell-to-cell spread of DHBV infection, while in all 

saline treated ducks DHBV infection spread to infect 

>95% of hepatocytes (Foster et al., J Virol 2005). In 

a subsequent in vivo dose response experiment, REP 

9AC was similarly effective at doses as low as 1mg/ 

kg/day. 

Conclusions: AP chemistry (phosphorothioated 

DNA) has an established record of being well 

tolerated in human patients and has a liver ½ life in 

excess of one month in human patients, suggesting 

that REP 9AC could be an effective treatment for 

HBV infection either as mono or combination 

therapy, which can likely be given once a week. More 

importantly, the novel mode of action of APs strongly 

suggest that treatment with REP 9AC will not result 

in the development of antiviral drug resistance. The 

compounds may also address the still uncertain 

role of virus spread in maintenance of chronic HBV 

infections, as well as preventing the spread of antiviral 

drug resistant HBV mutants in treated patients. 

Abstract 26 

Inhibition of Hepatitis C 

Virus Replication by 

Octadecyloxypropyl -(S)-HPMPA 


KY Hostetler, DL Wyles, KA Kaihara, JR Beadle and 

RT Schooley 

Division of Infectious Diseases, University of California, 

San Diego, La Jolla, CA, USA 

BACKGROUND: Alkoxyalkyl esters of (S)-9-[3- 

hydroxy-2-(phosphonomethoxy)-propyl]-adenine 

((S)-HPMPA) show broad spectrum antiviral activity 

against double stranded DNA viruses as well as HIV-1 

and hepatitis B viruses, and are orally active in animal 

models of poxvirus, cytomegalovirus and hepatitis 

B (HBV). The NS5B RNA polymerase of hepatitis 

C virus (HCV) is required for viral replication and 

several inhibitors are under evaluation. Inhibitors 

of this polymerase would be particularly attractive 

since the active site is highly conserved among HCV 

genotypes. We synthesized octadecyloxypropyl-(S)- 

HPMPA (ODE-(S)-HPMPA) and tested it against 1B 

and 2A replicons. 

METHODS: Genotype 1B and 2A replicons 

containing the firefly luciferase gene were transfected 

into Huh 7.5.1 cells. The cells were incubated with or 

without (S)-HPMPA or ODE-(S)-HPMPA for 48 to 72 

hours. Viral replication was assessed by measuring 

luciferase activity and cytotoxicity was measured with 

MultiTox-Fluor. The dose response data was analyzed 

and the EC 50 

and CC 50 

values were obtained by fitting 

a sigmoidal dose-response curve to the luciferase data 

using Prism software (GraphPad, v4). The selectivity 

indexes were calculated (CC 50 

/EC 50 

). 

RESULTS: Unmodified (S)-HPMPA was inactive 

in both 1B and 2A luciferase replicon systems 

(EC 50 

>100 µM). However, ODE-(S)-HMPA exhibited 

substantial antiviral activity in both 1B and 2A 

replicons with EC 50 

values in the 0.5 to 1.5 µM range. 


Selectivity indexes ranged from 25 to 50. We studied 

the cellular penetration and conversion of 14 C-labeled 

(S)-HPMPA and ODE-(S)-HPMPA to HPMPA-diphosphate 

in HepG2 cells in vitro. The absence of antiviral 

activity observed with (S)-HPMPA appears to be due 

to minimal cellular uptake and conversion of (S)- 

HPMPA to HPMPA-diphosphate; ODE-(S)-HPMPA 

uptake and conversion to HPMPA-diphosphate is 

many-fold greater than that of (S)-HPMPA itself. 

The mechanism of inhibition of viral replication is 

not known at the present time. 

CONCLUSIONS: The octadecyloxypropyl prodrug 

of (S)-HPMPA is a potent and selective inhibitor 

of HCV genotype 1B and 2A replicons. This is 

surprising since (S)-HPMPA is an analog of 

deoxyadenosine monophosphate and is known to 

inhibit double stranded DNA viruses. Its structure 

differs substantially from other nucleoside analog 

lead compounds identified to date which tend to 

be analogs of ribonucleotides. ODE-(S)-HPMPA is 

worthy of further evaluation since it has already been 

shown to be effective orally in animal models of HBV 

and would be expected to have HCV activity in vivo. 

Abstract 27 

FKBP8 Plays a Crucial Role in the 

Replication of Hepatitis C Virus 

T Okamoto, K Moriishi, and Y Matsuura 

Department of Molecular Virology, Research Institute for 

Microbial Diseases, Osaka University, Osaka, Japan 

BACKGROUND: Hepatitis C virus (HCV) nonstructural 

protein 5A (NS5A) is a component of 

the viral replication complex and is well known to 

modulate the functions of several host proteins. 

Although NS5A could be a candidate as the drug 

target for HCV, host factors that associate with NS5A 

for HCV replication have not been clarified yet. 

METHODS: To gain further insight into the functional 

role of NS5A in HCV replication, we screened human 

libraries by a yeast two-hybrid system using NS5A 

as bait and identified FKBP8, a member of the 

FK506-binding protein family, as an NS5A-binding 

protein. We further examined the host proteins 

interact with FKBP8 by immunoprecipitation and 

LC-MS/MS analyses. We investigated the biochemical 

characteristics and the intracellular localization of 

NS5A and FKBP8 by immunoprecipitation, surface 

plasmon resonance analysis, and fluorescence and 

electron microscopy. 

RESULTS: The siRNA-mediated knockdown of FKBP8 

in HCV RNA replicon cells suppressed RNA replication, 

and this reduction was reversed by the expression of 

an siRNA-resistant FKBP8 mutant. Furthermore, the 

knockdown of FKBP8 could also impair production of 

infectious virus in HCV cell culture system. Analysis 

of the cellular proteins interacting with FKBP8 by 

LC-MS/MS revealed that Hsp90 is an FKBP8-binding 

protein and forms a complex with FKBP8 and NS5A. 

The complex was shown to be critical for HCV 

replication, as based on the finding that treatment 

of the HCV replicon cells with geldanamycin, an 

inhibitor of Hsp90, suppressed RNA replication in a 

dose-dependent manner. Surface plasmon resonance 

analysis revealed that the dissociation constant of the 

interaction between the purified FKBP8 and NS5A 

expressed in bacteria was 82 nM. Mutational analyses 

of FKBP8 and NS5A indicated that three sets of 

tetratricopeptide repeats in FKBP8 are responsible for 

interactions with NS5A and that a single amino acid 

residue of Val or Ile at 121 position of NS5A is critical 

for the specific interaction with FKBP8. Substitution 

of the Val 121 to Ala drastically impaired the replication 

of HCV replicon cells, and the drug-resistant replicon 

cells emerging after drug selection were shown to have 

reverted to the original arrangement by replacing 

Ala 121 with Val. Examination of individual fields of 

the replicon cells by both fluorescent microscopy 

and electron microscopy (the correlative fluorescent 

microscopy-electron microscopy technique) revealed 

that FKBP8 is partially co-localized with NS5A in the 

cytoplasmic structure known as the membranous 

web. 

CONCLUSION: These results indicate that NS5A 

directly binds to FKBP8 through the Val 121 and FKBP8 


further recruits Hsp90 to the replication complex. 

Recently, geldanamycin was shown to have a drastic 

impairment of the replication of poliovirus without any 

emergence of escape mutants. Therefore, disruption 

of the specific interaction of NS5A with FKBP8 might 

be an ideal target for a novel therapeutic of chronic 

hepatitis C with a low frequency of emergence of 

drug-resistant breakthrough viruses. 

Abstract 28 

Initial Recommendations for HCV 

Drug Resistance Analysis: 

A Consensus Statement from the 

HCV Drug Resistance Advisory 

Group 

K Lin 1 , D Hazuda 2 , M Otto 3 , C Boucher 4 , C Schooley 5 , 

J O’Rear 6 , D Kempf 7 , A Kwong 8 , and N Parkin 9 for the 

HCV Drug Resistance Advisory Group 

1 Novartis, Cambridge, MA, USA; 2 Merck & Co., West 

Point, PA, USA; 3 Pharmasset, Princeton, NJ, USA; 

4 Universitair Medical Center, Utrecht, Netherlands; 

5 University of California, San Diego, San Diego, CA, USA; 

6 FDA/CDER/OND/OAP/ Division of Antiviral Products, 

Silver Spring, MD, USA; 7 Abbott, Abbott Park, IL, USA; 

8 Vertex Pharmaceuticals, Inc., Cambridge, MA, USA; 

9 Monogram Biosciences, South San Francisco, CA, USA 

Background: As new anti-HCV drugs, especially 

protease and polymerase inhibitors, are evaluated in 

clinical trials, interest in assays for monitoring the 

development of resistance to these agents and for 

avoiding cross-resistance has increased. However, 

there is little guidance about the nature (phenotypic 

or genotypic) of the resistance assays, when to use 

them, or desired performance characteristics. The 

HCV Drug Resistance Advisory Group (DRAG) was 

formed in late 2006 to formulate recommendations 

for methods to measure resistance and how such 

methods should be integrated into clinical trials, and 

potentially following drug approval. 

Methods: Knowledge about mutations associated 

with resistance to investigational protease inhibitors 

(PIs), nucleoside polymerase inhibitors (NIs), and 

non-nucleoside polymerase inhibitors (NNIs) 

was gathered from the literature and conference 

presentations. Recommendations for the design and 

use of HCV resistance assays were based on diagnostic 

assay validation standards, previous experience in 

the HIV drug resistance field, and consultation with 

regulatory agencies, hepatologists and infectious 

disease experts. 

Results: Based on exposure of HCV genotype 1 to 

PIs both in vitro and in vivo, positions in NS3 associated 

with resistance include 36, 54, 155, 156, 168 and 

170. NIs (4’-Azido cytidine and 2’-C-Me nucleosides) 

select for mutations in NS5B at positions 96 and 282 

(respectively). NNIs belong to one of 4 classes, based 

on chemical structure and binding site, and select for a 

multitude of mutations in the thumb or palm regions 

of NS5B, including 201, 316, 411, 414, 419, 423, 

448, 451, 495, 496, and 499. Mutations conferring 

resistance to inhibitors may pre-exist in viruses from 

naïve patients as polymorphisms. Issues surrounding 

the desired performance characteristics, validation 

criteria, and implementation during clinical trials of 

sequence analysis and phenotypic assays have been 

discussed in the DRAG and draft recommendations 

have been formulated. Sequence analysis can be 

performed on a population or clonal basis, depending 

on the question being addressed (dominant mutations 

or minority species, pre-existing resistant variants, 

and linkage of multiple mutations). Amplification 

sensitivity targets of approximately 1,000 RNA 

copies/ml are desired, and sensitivity for detection 

of minority species must take into account the viral 

load and number of amplifiable genome templates in 

the plasma sample. Phenotypic susceptibility assays 

are crucial for the understanding of resistance and 

interpretation of genotypic data; prototype assays 

based on genotype 1 replicons have been described 

and are currently a popular approach, complemented 

by enzymatic assays using protein expressed in 

recombinant or cell-based systems. 

Conclusion: The recommendations made by the 

HCV DRAG are a starting point for future discussions 

about the role of resistance testing for HCV and for 

the establishment of standardized validation methods 

and criteria. 


Abstract 29 

Early Biochemical and Virological 

Response of Clevudine Therapy 

in Patients with HBV Associated 

Liver Cirrhosis 

KW Chung 1 , CY Ha 1 , SJ Baik 1 , CH Lee 2 , JS Lee 3 , 

HJ Lee 4 , BH Han 5 , WC Choi 6 , SW Paik 7 , KC Koh 7 , 

JH Lee 7 , WY Tak 8 , YJ Lee 9 , and K Yoo 1 

1 Ewha Womans University, Seoul, South Korea; 

2 Konkuk University Hospital, Seoul, South Korea; 3 Inje 

University Ilsan Paik Hospital, Gyeonggi-do, South Korea; 

4 Youngnam University Medical Center, Daegu, South 

Korea; 5 Kosin Medical School, Busan, South Korea; 6 Inje 

University Sanggye Paik Hospital, Seoul, South Korea; 

7 Sungkyunkwan University Samsung Medical Center, 

Seoul, South Korea; 8 Kyungpook National University 

Hospital, Daegu, South Korea; 9 Inje University Busan 

Paik Hospital, Busan, South Korea 

BACKGROUND: In the pivotal phase III clinical 

trials, clevudine 30 mg for 6 months showed 

potent antiviral activity and significant biochemical 

improvement along with a marked post-treatment 

antiviral effect. This analysis was performed to 

evaluate the early biochemical and virological 

response of clevudine in chronic hepatitis B patients 

with cirrhosis. 

METHODS: The data of 13 patients who were 

diagnosed chronic hepatitis B patients with cirrhosis 

were collected from 1 hospital. Preliminary results who 

have been treated for at least 1 month are presented 

here. HBV DNA was quantified by bDNA assay with a 

lower limit of detection of 141,500 copies/mL. 

RESULTS: Median HBV DNA levels before therapy 

was 7.3 log 10 

copies/mL and the median changes in 

HBV DNA levels from baseline was –1.5 log 10 

copies/ 

mL after 1 month of therapy (n=13) and –2.7 log 10 

copies/mL after 3 month of therapy (n=4). Serum 

HBV DNA levels were below 141,500 copies/mL in 

46% (6/13) at 1 month and 75% (3/4) after 3 months 

of therapy. 

At baseline, overall median ALT was 51 IU/L and 3 

patients had normal ALT. Forty-six percent (46%, 

6/13) at month 1 and 100% (4/4) of patients at 

month 3 had normal ALT. 

CONCLUSION: Clevudine 30 mg once daily therapy 

demonstrated early viral suppression and significant 

biochemical improvement in patients with liver 

cirrhosis. 

Abstract 30 

Clevudine was Superior to 

Lamivudine in the Patients with 

HBeAg(+) Chronic Hepatitis B 

GK Lau 1 , N Leung 2 , CK Hui 1 , A Kwok 2 , A Wong 2 , 

R Chan 2 , SG Hwang 3 , and HS Lee 4 

1 The University of Hong Kong, Hong Kong, PRC; 2 Alice 

Ho Miu Ling Nethersole Hospital, Hong Kong, PRC; 

3 Bukwang Pharmaceutical Co., Ltd., Seoul, Korea; 4 Seoul 

National University Hospital, Seoul, Korea 

BACKGROUND: Clevudine is a pyrimidine analogue 

with potent anti-HBV activity in vitro. In the 

pivotal phase III clinical trials, clevudine 30 mg for 

24 weeks showed profound viral suppression with 

normalization of serum ALT levels. 

METHODS: The aim of this study is to compare the 

efficacy and safety of clevudine versus lamivudine for 

48 weeks in chronic hepatitis B (CHB) patients in a 

randomized and blinded way. The study is ongoing 

at two sites in Hong Kong. Eligible patients were 

treatment-naïve HBeAg(+) CHB patients with HBV 

DNA levels ≥ 3 × 10 6 copies/mL and 1.0 ≤ALT

RESULTS: The clevudine group demonstrated greater 

viral suppression at Week 48 when compared with 

the Lamivudine group [median reduction (range) 

4.9 (3.3-6.2) vs. 3.2 (0.8-5.6) log 10 

copies/ml at Week 

48 respectively, p < 0.05]. Serum HBV DNA levels 

were below 300 copies/mL in 73% and 27% at week 

32 and in 82% and 36% at week 48 in the clevudine 

and lamivudine groups, respectively. Nine patients 

of clevudine group and 10 patients of lamivudine 

group had normal ALT at week 48 (p=0.82). In the 

lamivudine group, viral breakthrough occurred in 3 

patients during week 32-48, but no patient had viral 

breakthrough for 48 weeks in the clevudine group. 

Lamivudine-resistant mutations (rtL180M and 

rtM204V) were detected in all of the three patients 

with viral breakthrough in the lamivudine group. 

Clevudine was well tolerated with the incidence of 

adverse events comparable to lamivudine. 

CONCLUSIONS: A 48-week dosing with clevudine 

30mg showed superior viral suppression to 

lamivudine 100mg without the emergence of viral 

breakthrough in HBeAg(+) CHB patients, while 3 

in the 11 patients (27.3%) in the lamivudine group 

had viral breakthrough during 48 weeks which were 

associated with lamivudine-resistant mutations. 

Abstract 31 

Clevudine Monotherapy Showed 

Rapid Viral and Biochemical 

Response in Chronic Hepatitis B 

Patients with Cirrhosis 

CH Lee 1 , KW Chung 2 , JS Lee 3 , HJ Lee 4 , BH Han 5 , 

WC Choi 6 , SW Paik 7 , KC Koh 7 , JH Lee 7 , WY Tak 8 , 

YJ Lee 9 , and HY Lee 10 

1 Konkuk University Hospital, Seoul, South Korea; 

2 Ewha Womans University, Seoul, South Korea; 3 Inje 

University Ilsan Paik Hospital, Gyeonggi-do, South Korea; 

4 Youngnam University Medical Center, Daegu, South 

Korea; 5 Kosin Medical School, Busan, South Korea; 6 Inje 

University Sanggye Paik Hospital, Seoul, South Korea; 

7 Sungkyunkwan University Samsung Medical Center, 

Seoul, South Korea; 8 Kyungpook National University 

Hospital, Daegu, South Korea; 9 Inje University Busan 

Paik Hospital, Busan, South Korea; 10 Chungnam 

National University Hospital, Daejeon, South Korea 

BACKGROUND: In the pivotal phase III clinical trials, 

clevudine 30 mg for 6 months showed potent antiviral 

activity and significant biochemical improvement 

along with a marked post-treatment antiviral effect. 

This analysis was performed to evaluate the efficacy 

of clevudine in chronic hepatitis B patients with 

cirrhosis. 

METHODS: The data of 23 patients were collected 

from the hospitals. The patients were diagnosed 

chronic hepatitis B patients with hepatitis B-related 

cirrhosis. Among them, 5 patients were diagnosed with 

decompensated liver cirrhosis or HCC. Preliminary 

results from the 23 patients who have been treated 

for at least 12 weeks are presented here. 

RESULTS: Median HBV DNA levels before therapy 

was 6.03 log 10 

copies/mL (n=23). The median changes 

in HBV DNA levels from baseline was –3.43 log 10 

copies/mL (n=18) after 12-16 weeks of therapy and 

–3.50 log 10 

copies/mL (n=12) after 20-24 weeks of 

therapy. Serum HBV DNA levels were below 300 

copies/mL in 39 % (n=18) after 12-16 weeks of 

therapy and 75 % (n=12) after 20-24 weeks of therapy. 


From the 23 patients, 20 patients (87%) had HBV 

DNA

polymerase. The molecular mechanism of action of 

clevudine inhibition is not completely understood. In 

this study, we have examined the unique mechanism 

of action of clevudine. 

METHODS: An endogenous HBV polymerase assay 

was employed to investigate the mechanism of action 

of CLV-TP. The reaction was followed by measuring the 

incorporation of 32 P-labeled nucleoside triphosphates 

into HBV DNA using HBV core particles. In order 

to determine the mode of inhibition, reactions were 

followed in the presence of varying concentrations 

of CLV-TP and a natural nucleotide substrate. A 

control experiment was performed using a known 

competitive inhibitor, 3TC-TP. 

RESULTS: Since clevudine is a thymidine analog, 

inhibition of HBV DNA polymerase by CLV-TP 

was examined by varying both CLV-TP and TTP 

concentrations. The data were fit to both competitive 

and non-competitive equations. The plots clearly fit 

the non-competitive equation with the inhibition 

constant (K i 

) of 0.67 µM. The non-competitive 

inhibition of CLV-TP was confirmed by performing the 

same experiment in the presence fixed concentration 

of TTP and varying dCTP which should not compete 

with CLV-TP. A similar inhibition profile was observed 

with the K i 

of 1.15 µM. A control experiment 

was performed with 3TC-TP and as expected, it 

competitively inhibited HBV DNA polymerase with 

the K i 

of 0.0075 µM which was comparable to the 

published data. 

CONCLUSIONS: Using an endogenous HBV 

polymerase assay, the mode of inhibition for CLV-TP 

was determined to be non-competitive. Since several 

active site mutations are known to confer resistance 

to Clevudine in vitro, CLV-TP may be binding at or near 

the active site of the HBV polymerase without being 

utilized as a substrate. To the best of our knowledge 

this is the first example of a nucleoside analog 

triphosphate showing non-competitive inhibition. 

Abstract 34 

Mechanistic Characterization 

of Potent Small Molecule HCV 

Inhibitors that Target NS5A 

A Sandrasagra, AMI Lam, R Fathi, Z Yang, J Cao, 

Y Liu, G Li, Y Liao, Q Zhu, K Nawoschik, H-J Cho, 

R Wu, G Westby, and CR Wobbe 

XTL Biopharmaceuticals Inc, Valley Cottage, New York, 

USA 

Background: Target based screening has led to the 

discovery of a number of small molecule therapeutics 

that inhibit HCV by acting at enzymes critical for 

viral replication. Complimentary phenotypic replicon 

based screening approaches, which are un-biased 

toward a specific molecular target, can enable the 

identification of otherwise inaccessible and novel 

targets or pathways. We have employed replicon 

based screening to identify a novel series of small 

molecule HCV replication inhibitors that affect NS5A 

dependent functions. 

Methods and Results: We have generated potent 

HCV replicon inhibitors that have no toxicity against 

a range of cultured cell lines, and that induce a rapid 

multiple-log reduction of HCV RNA with no rebound 

upon inhibitor withdrawal. Our leads do not inhibit 

in vitro assays for HCV protease, helicase, polymerase 

and IRES. Consistent with these findings, replicons 

with resistance mutations to NS3 protease and NS5B 

polymerase inhibitors are susceptible to our leads. 

XTL leads were also found to be inactive vs. a number 

of cellular metabolic pathways and targets including 

nucleotide pools, caspases, a panel of 50 in vitro protein 

kinases assays, and a panel of 60 receptor binding 

assays. Combination studies with other known HCV 

replication inhibitors demonstrated that our leads 

are either additive or synergistic. Analysis of replicon 

mutations conferring resistance to our leads indicated 

that resistance could arise by the alteration of specific 

viral sequences outside of the NS3 protease or NS5B 

polymerase regions. Furthermore, a single mutation 


(Y93H) within domain 1 of NS5A is sufficient to confer 

6 to 100 fold resistance vs. a panel of representative 

leads. Data from molecular genetic studies and in vitro 

binding assays suggest that XTL leads interact with 

NS5A. Affinity models constructed using the NS5A 

domain 1 crystal structure predict that our leads bind 

at or near the dimer interface and make contacts that 

are distinct from other compounds reported to target 

NS5A. 

Conclusion: Using a phenotypic replicon based 

screening approach in combination with Diversity 

Oriented Synthesis chemistries, we have generated 

potent HCV replication inhibitors with nanomolar 

EC 50 

s. Resistance selection, molecular genetic, in vitro 

binding, and molecular modeling studies suggest that 

our leads that act by interacting with NS5A. 

Abstract 35 

GSK949614; a Novel and Potent 

Thumb Site Inhibitor of the HCV 

NS5B Polymerase 

PA Thommes 1 , NR Parry 1 , EM Amphlett 1 , G Bravi 2 , 

H Bright 1 , AG Cheasty 1 , MA Convery 2 , JA Corfield 1 , 

R Fenwick 1 , DF Gray 1 , RM Grimes 1 , D Harrison 1 , 

CD Hartley 4 , RL Jarvest 4 , KJ Medhurst 4 , ML Meeson 4 , 

D Mesogiti 1 , F Mirzai 1 , JE Mordaunt 4 , NA Roughley 3 , 

P Shah 4 , MJ Slater 4 , JH Thorpe 2 , CS Wilkinson 1 , 

and E Williams 1 

1 Infectious Diseases CEDD, Stevenage, 2 Computational 

& Structural Chemistry, GSK Medicines Research 

Centre, Gunnels Wood Road, Stevenage SG1 2NY, United 

Kingdom 

Background: The HCV NS5B gene encodes the 

viral RNA-dependent RNA polymerase which is 

essential for HCV replication and an attractive 

target for antiviral therapy. In common with other 

polymerases, the HCV polymerase has a structure 

similar to a right hand, with distinguishable ‘finger’, 

‘palm’ and ‘thumb’ domains. Several classes of 

inhibitors directed against HCV NS5B have been 

described, which inhibit both enzymatic activity and 

replication. These compounds bind to distinctive 

sites on the polymerase molecule and thus have been 

termed ‘palm’, ‘thumb’, ‘finger-loop’ binders, etc. 

We have previously reported the identification of 

two distinct series with different binding modes in 

the palm site of the polymerase; thiadiazines and 

acylpyrrolidines. Here we describe a pyrazole based 

inhibitor series which binds at the thumb site of the 

enzyme. 

Methods: The development of the pyrazole series 

was driven by structure-based design with parallel 

use of NS5B enzyme and HCV genotype 1a and 1b 

replicon assays and by monitoring PK parameters of 

key compounds in the rat. Resistant mutants were 

selected by growing replicon cells in the presence 

of compound and individual mutations assessed in 

enzyme and transient replicon assays. Selectivity 

was measured against the polymerases of other 

members of the Flaviviridae and human cellular DNA 

polymerases. Cytotoxicity was determined in Vero 

cells. 

Results: Using structure activity relationship driven 

by potency in in vitro assays the pyrazole series was 

optimised to provide GSK949614 as a lead molecule. 

This compound was shown to be a selective inhibitor 

of the HCV polymerase with sub-micromolar potency 

against HCV genotypes 1a and 1b in both enzyme 

and replicon assays. Its activity is synergistic with 

that of interferon alpha 2a in replicon assays. 

GSK949614 has little cytotoxicity and no significant 

activity against either human DNA polymerases or 

the polymerases of related viruses. Replicon cells 

grown in the presence of GSK949614 gave rise to 

mutations at amino acids 423 and 419, located in the 

thumb region of the enzyme. Each of these mutations 

was shown to be critical for the sensitivity of the 

enzyme to the inhibitor. The compound retains full 

activity against a panel of enzymes resistant to drugs 

binding to other sites on the polymerase, including 

the palm site binders. GSK949614 has bioavailability 

in both rat and dog that will allow extended safety 

assessments. 


Conclusion: We have developed the pyrazole series 

binding in the thumb region of the NS5B molecule 

to yield GSK949614. Its in vitro properties, together 

with its PK profile, make it suitable for further study 

as a potential drug candidate. 

Abstract 36 

Pyrrolo[1,2-b]pyridazin-2-ones 

Show Promising In Vitro Antiviral 

Activity Against HCV NS5B 

Polymerase 

F Ruebsam 1 , SE Webber 1 , MT Tran 1 , CV Tran 1 , 

DE Murphy 1 , J Zhao 2 , PS Dragovich 1 , SH Kim 3 , 

L Li 4 , Y Zhou 1 , Q Zhao 1 , CR Kissinger 1 , RE Showalter 1 , 

M Lardy 1 , A Shah 5 , M Tsan 6 , R Patel 1 , L LeBrun 1 , 

R Kamran 7 , MV Sergeeva 1 , and L Kirkovsky 1 

1 Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, 

San Diego, CA 92121, USA; 2 Celgene Corporation, 4550 

Towne Centre Court, San Diego, CA 92121, USA; 3 Arena 

Pharmaceuticals, Inc., 6166 Nancy Ridge Drive, San 

Diego, CA 92121, USA; 4 Intellikine, Inc., 10931 North 

Torrey Pines Road, Suite 103, La Jolla, CA 92037, USA; 

5 SGX Pharmaceuticals, Inc., 10505 Roselle Street, San 

Diego, CA 92121, USA; 6 Illumina, Inc., 9885 Towne 

Centre Drive, San Diego, CA 92121, USA; 7 Takeda San 

Diego, 10410 Science Center Drive, San Diego, CA 92121, 

USA 

Background: Hepatitis C virus (HCV) is a major 

cause of acute hepatitis and chronic liver disease, 

including cirrhosis and liver cancer. Low response 

rates to the current standard of care, in particular 

for patients infected with genotype 1 HCV, along 

with significant side-effects of current HCV therapy 

result in a continuing medical need for improved 

treatments. 

Methods: As part of our efforts to identify novel 

small molecule, non-nucleoside inhibitors of the HCV 

NS5B protein using a structure based design approach, 

we investigated a series of pyrrolo[1,2-b]pyridazin-2- 

ones (Figure 1). We systematically explored variation 

of the substituents at the 1-, 6- and 7’-positions of 

the pyrrolo[1,2-b]pyridazin-2-ones. We also assessed 

the biological properties of the resulting molecules 

including inhibition of the 1b NS5B enzyme, activity 

in the HCV genotype 1b subgenomic replicon in tissue 

culture, cytotoxicity, and stability toward human liver 

microsomes (HLM). 

Results: Described here are the structure-activity 

relationships observed by varying the 1-, 6- and 7’- 

substituents of the pyrrolo[1,2-b]pyridazin-2-one 

NS5B inhibitors under study. We observed that while 

certain alkyl groups in R 2 , such as isoamyl or tertbutyl 

ethyl fragments, led to compounds with low 

nanomolar potencies in biochemical and cell-based 

assays, the introduction of a 4-fluorobenzyl moiety 

provided the best overall balance between potency 

and metabolic stability. We also noted that the R 3 

position was very sensitive to structural changes and 

that a sulfonamide moiety was critical for activity. 

The combination of optimal substituents at positions 

1, 6 and 7’ resulted in a compound that demonstrated 

potent inhibition against the 1b NS5B enzyme (IC 50 

Abstract 37 

4-(1,1-Dioxo-1,4-dihydro-1λ 6 - 

benzo[1,4]thiazin-3-yl)-5- 

hydroxy-2H-pyridazin-3-ones are 

a Novel Series of Molecules which 

Inhibit the HCV NS5B Polymerase 

CV Tran 1 , DA Ellis 2 , J Blazel 3 , PS Dragovich 1 , Z Sun 4 , 

F Ruebsam 1 , HM McGuire 5 , AX Xiang 1 , J Zhao 6 , 

L Li 7 , Y Zhou 1 , SE Webber 1 , Q Han 1 , CR Kissinger 1 , 

RE Showalter 1 , M Lardy 1 , A Shah 2 , M Tsan 8 , R Patel 1 , 

L LeBrun 1 , R Kamran 9 , MV Sergeeva 1 , and L Kirkovsky 1 

1 Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, 

San Diego, CA 92121, USA; 2 SGX Pharmaceuticals, Inc., 

10505 Roselle Street, San Diego, CA 92121, USA; 

3 Ardea Biosciences, Inc., 3300 Hyland Avenue, Costa 

Mesa, CA 92626, USA; 4 Merck & Co., Inc., 126 E. 

Lincoln Avenue, Rahway, NJ 07065, USA; 5 AstraZeneca 

Pharmaceuticals LP, 35 Gatehouse Drive, Waltham, MA 

02451, USA; 6 Celgene Corporation, 4550 Towne Centre 

Court, San Diego, CA 92121, USA; 7 Intellikine, Inc., 

10931 North Torrey Pines Road, Suite 103, La Jolla, CA 

92037, USA; 8 Illumina, Inc., 9885 Towne Centre Drive, 

San Diego, CA 92121, USA; 9 Takeda San Diego, 10410 

Science Center Drive, San Diego, CA 92121, USA 

Background: Hepatitis C virus (HCV) is a major 

cause of acute hepatitis and chronic liver disease, 

including cirrhosis and liver cancer. Globally, an 

estimated 170 million individuals, 3% of the world’s 

population, are chronically infected with HCV and 

3 to 4 million people are newly infected each year. 

Currently, there is no vaccine available to prevent 

hepatitis C, nor a HCV-specific antiviral agent 

approved for treatment of chronic hepatitis C. The 

current standard of care is a combination of pegylated 

interferon (IFN) with ribavirin. However, response 

rates in patients infected with genotype 1 HCV, 

along with adverse side-effects, result in a continuing 

medical need for improved therapy. 

determined using the NS5B 1b enzyme. EC 50 

and 

cytotoxicity (CC 50 

) values were generated using a Huh-7 

HCV replicon cell line. Metabolic stability studies 

were conducted using human liver microsomes. 

Results: The newly discovered class of molecules, 

4-(1,1-dioxo-1,4-dihydro-1λ 6 -benzo[1,4]thiazin-3- 

yl)-5-hydroxy-2H-pyridazin-3-ones, showed excellent 

in vitro characteristics. The majority of compounds 

in this series were generally not cytotoxic (CC 50 

), 

had moderate to high stability towards human liver 

microsomes (HLM) and were potent inhibitors of 

both the NS5B polymerase enzyme (IC 50 

< 18µM) 

and the HCV replicon (EC 50 

< 1µM). One optimized 

compound exhibited an IC 50 

< 10 nM against the 1b 

NS5B enzyme and an EC 50 

of 1.1 nM when tested 

against the 1b HCV replicon in cell culture. This 

compound was also moderately stable in the presence 

of HLM with a half-life of 32 minutes. 

Conclusion: The novel series of 4-(1,1-dioxo-1,4- 

dihydro-1λ 6 -benzo[1,4]thiazin-3-yl)-5-hydroxy-2Hpyridazin-3-ones 

(shown in Figure 1) displayed an 

excellent in vitro profile against HCV NS5B polymerase. 

The inhibitors had low nanomolar activity against 

the NS5B enzyme as well as HCV replicon, and 

were reasonably stable in the presence of HLM. The 

encouraging in vitro data support further preclinical 

evaluation of this series in order to confirm its utility 

for the treatment of chronic HCV infection. 

O O 

S 

OH 

R 1 

N 

H 

N N O 

R 2 

Figure 1 

R 3 

Methods: Using a structure based design approach, 

we identified and optimized a novel series of 

compounds that inhibit HCV NS5B polymerase 

with excellent in vitro properties. IC 50 

values were 


Abstract 38 

Interference of Hsp90 Activity 

by Hsp90 Inhibitor Suppresses 

Hepatitis C Virus (HCV) 

Replication 

S Ujino 1 , S Yamaguchi 1 , K Shimotohono 3 , and 

H Takaku 1,2 

1 Department of Life and Environmental Sciences; 2 High 

Technology Research Center; 3 Research Institute, Chiba 

Institute of Technology, Chiba, Japan 

its inhibitory effects were caused by NS3 degradation. 

Moreover, as a result of immunoprecipitation, 

interaction between Hsp90 and NS3 was detected. 

CONCLUSIONS: Here, we present a new and potent 

strategy for therapy of hepatitis C virus (HCV) 

infection with Hsp90 inhibitor. We found that Hsp90 

inhibitor has an anti HCV activity. Furthermore, 

Hsp90 inhibitor was elucidated as non-escape drug by 

long-term treatment. HCV NS3 was found to mediate 

HCV replication after interaction that it is interacted 

with Hsp90. These results illustrate implications 

for the HCV replication cycle and novel antiviral 

strategies. 

BACKGROUND: The Hsps induced by cells stress are 

expressed at high levels in a wide range of tumors 

and are closely associated with a poor prognosis and 

resistant therapy. The principal folding protein belong 

to the Hsp70 and Hsp90, which bind to unfolded 

sequences in polypeptide substrates and shown for 

hydrophobic regions. As numerous oncoproteins 

have been shown to be Hsp90 client proteins, Hsp90 

inhibitors have become a arrest the growth of tumor 

cells. Previously we demonstrated the inhibition of 

HCV RNA replication in the HCV replicon with Hsp90 

inhibitor. 

METHODS: We used two types of HCV replicon cells. 

One is HCV full genome replicon(NNC) and the other 

is HCV subgenomic reilicon. HCV inhibiting effect was 

measured by Real-Time PCR. Western blot analysis of 

core and NS protein expression in NNC cells treated 

with 17-AAG. Lysates were immunoblotted with core 

and NS specific antibodies. Immunoprecipitation 

of Hsp90 and NS3 in cultured cells. NS3 was 

immunoprecipitated from cell lysates with anti-FLAG 

or anti-Hsp90 antibodies and immunoblotted by 

using respective antibodies as indicated. 

RESULTS: Replication of HCV was inhibited when 

treated with Hsp90-inhibitor(0.25~250nM). Hsp90 

inhibitor showed neither cytotoxicity nor virus escape 

drug in long-term culture assay. We also found that 

Hsp90 inhibited the formation of the NS3–Hsp90 

chaperone complex during HCV replication, and that 


Hepatitis B Foundation Symposium: 

Early Detection and 

Management of Cirrhosis and 

Hepatocellular Carcinoma 


Abstract 39 

Hepatocellular Carcinoma: Why 

Early Diagnosis is Needed 

AM Di Bisceglie 

Saint Louis University Liver Center, Saint Louis, MO, USA 

Hepatocellular carcinoma (HCC) is one of the frequent 

solid malignancies world-wide and is increasing in 

incidence in the United States and developed western 

world. The occurrence of HCC is very closely linked 

to the presence of underlying chronic liver disease, 

including chronic viral hepatitis and cirrhosis. In the 

United States, the frequency of various underlying 

diseases varies regionally but overall, chronic viral 

hepatitis accounts for 50 to 60% of all cases, while 

cirrhosis due to alcohol and other conditions accounts 

for another substantial proportion. 

Although it has been shown that early diagnosis of 

HCC is possible, most patients seem to present when 

this tumor is at an advanced stage. Typical symptoms 

that lead to the diagnosis include abdominal pain, 

weight loss and further hepatic decompensation in 

someone known to have cirrhosis. Unfortunately, 

when such symptoms are present, the tumor is often 

very large and may even be metastatic, either within 

or beyond the liver. At this stage, the tumor is rarely, 

if ever, respectable and there are limited potentially 

curative therapeutic options available for the patient. 

Liver transplantation is associated with optimal 

results when the tumor is within the so-called “Milan 

criteria” – that is, a single tumor 3cm in diameter. Local ablative treatments such 

as ethanol injection or radio-frequency ablation are 

similarly most effective with tumor diameters

Serological tests have the ability to correctly define 

fibrosis stage as either mild (F0/F1) or significant 

(F2-4) in up to 80% of patients with some indices 

better at predicting advanced disease and others at 

excluding advanced disease. 

Serological tests are accurate enough to abrogate the 

need for liver biopsy in 40% of patients. 

References: 

Bedossa P, et al, Hepatology 2003;38:1449-57. 

Cales P, et al, Hepatology 2005;42:1373-81. 

Imbert-Bismut F, et all, Lancet 2001;357:1069-75. 

Adams LA, et al, Clin Chem 2005;51:1867-73. 

Patel K, et al, J Hepatol 2004;41:935-42. 

Ziol M, et al, Hepatology 2005;41:48-54. 

Castera L, et al, Gastroenterology 2005;128:343-50. 

Tests of liver stiffness with elastography are able 

to predict cirrhosis in 90% of patients and can be 

complimentary to blood tests. 

Algorithms of combination of biopsy, serological 

tests and elastography may be the optimal way to 

stage and monitor fibrosis. 

Table: Serological Tests for Liver Fibrosis 

Patients 

Name (Serum Markers) 

AUROC 

(95% CI) 

Sens Spec PPV NPV 

Wai et al (2003) 192 

APRI 

(AST, platelets) 

0.88 

(.80-.96) 

41% 95% 88% 64% 

Rosenberg et al (2004) 1021 

ELF 

(Propeptide III collagen, TIMP 1, 

HA) 

0.80 

(.76-.85) 

90.5% 41% 99% 92% 

Ziol et al 

(2005) 

327 

Fibroscan 

(hepatic elastography) 

0.79 

(.73-.84) 

56 % 91% 88% 56% 

Imbert-Bismut et al 

(2001) 

339 

Fibrotest 

(α 2 

macroglobulin, α 2 

globulin, γ 

globulin, apolipoprotein A 1 

, γGT 

and total bilirubin) 

0.87 

(SD 

0.34) 

87 % 59% 63% 85% 

Castera et al (2005) 183 

Combined Fibroscan and 

Fibrotest 

0.88 

(.82-.92) 

NA NA NA NA 

Patel et al 

(2004) 

402 

Fibrospect 

hyaluronic acid, tissue inhibitor of 

metalloproteinase 1 (TIMP-1) and 

α 2 

macroglobulin 

0.831 77% 73% 74% 76% 

Adams et al. (2005) 221 

Hepascore 

Bilirubin, γGT, hyaluronic acid, α 2 

macroglobulin, age and sex 

0.82 

0.90 

0.89 

63% 

88% 

71% 

89% 

74% 

89% 

88% 

88% 

95% 

98% 

98% 


Abstract 41 

Screening Fibrosis: “La Révolution 

Française” 

T Poynard 


Non-invasive tests, biomarkers and liver stiffness 

measurements (LSM) have advantages over other 

proposed strategies for liver injury assessment. Several 

patented biomarkers are already on the market, the 

most validated being FibroTest (FT). FT has similar 

diagnostic value in the most frequent chronic liver 

diseases, and at least similar false positive or false 

negative rates than routine biopsy (Poynard, BMC 

Gastroenterology 2007). 

Practices are evolving rapidly and in France 81% of 

hepatologists used FT and 32% used LSM (Castera, J 

Hepatol 2007). 

French health authorities concluded that FT was 

a valid alternative to biopsy in chronic hepatitis C 

and the reimbursement is pending (http://www.hassante. 

fr/portail/display.jsp?id=c_476486). 

We conducted three fibrosis screening studies. The 

first study (FibroGras) analyzed retrospectively 

the frozen sera of a consecutive cohort of 1,909 

hyperlipidaemic patients prospectively in followed 

a lipid centre. A total of 925 blood donors have 

been taken as controls (Ctl). Advanced fibrosis was 

presumed by FT in 2.8% hyperlipidaemic patients 

vs. 0% in Ctl (P < 0.0001); advanced steatosis was 

presumed by SteatoTest in 30.1% vs. 4.9% Ctl (P < 

0.0001) and NASH was presumed by NashTest in 

7% vs. 0%, respectively (P < 0.0001). There was a 

highly significant and linear association between 

the number of metabolic syndrome factors and liver 

disease prevalence presumed with biomarkers – the 

highest being for type 2 diabetics: advanced steatosis 

66%, NASH 24% and advanced fibrosis 6% (Ratziu, 

Aliment Pharmacol Ther 2007). 

The second study (FibroSucre) analyzed prospectively 

1,131 consecutive patients, without a history of 

liver disease, followed in a diabetes center. The 

biomarker predicted advanced fibrosis in 5.6%. A 

total of 45 patients with presumed advanced fibrosis 

were re-investigated using LSM and standard tests, 

and advanced fibrosis was confirmed in 32 patients 

(2.8%), with 5 cases of cirrhosis including 4 cases with 

of hepatocellular carcinoma. Most of liver diseases 

previously unknown were NASH but interestingly in 

2 patients a chronic hepatitis C has been discovered. 

In the population with type 2 diabetes, 45 years or 

older, the prevalence of confirmed advanced fibrosis 

was 4.3% and hepatocellular carcinoma was 5.7/1,000 

(Jacqueminet, J Hepatol 2006;44:A260). 

The third study (FibroCPAM) is on going in a general 

population. Consecutive apparently healthy subjects 

>40 years had free health check-up offered by social 

security centres. Subjects with presumed advanced 

fibrosis at FT, were re-investigated by a hepatologist 

using LSM, and if necessary, ultrasonography, 

endoscopy or liver biopsy. A scheduled intermediate 

analysis was performed in the first 5,013 subjects. 

FT predicted advanced fibrosis in 3%, including 0.4% 

cirrhosis. At the time of abstract submission 40% 

of subjects with presumed advanced fibrosis have 

been fully reinvestigated. Fibrosis was confirmed in 

50%, mostly with LSM >8.8kPa including 8 cases of 

cirrhosis previously unknown. As expected in France, 

most of liver diseases discovered were NASH and 

alcoholic liver diseases, but 5 previously unknown 

hepatitis C and one hepatitis B were also identified. 

Finally in a world without perfect Gold Standard, 

we developed in 2,004 patients a new non-invasive 

methodology for improving accuracy of fibrosis 

markers using the strength of concordance between 

FT and LSM (Poynard, Hepatology 2007;46:1071). 

According to the respective risk of false negative/ 

positive, the present results strongly suggest for 

fibrosis screening, to use FT at first line and LSM at 

second line. Patients not concordant at second line 

must be re-investigated by repeated biomarkers or 

liver biopsy. 


In conclusion, waiting for an ideal non-invasive 

biomarker is a dream, and it is time to screen for 

advanced fibrosis. 

Abstract 42 

Treatment of HCC Over the Past 

Decade: The Experience of Over 

Four Thousand Patients in Japan 

M Omata 

Department of Gastroenterology, University of Tokyo 

Etiology: Approximately 34,000 patients died 

of hepatocellular carcinoma last year in Japan. Of 

these, 11% and 83% of the patients with the cancer 

were positive for HBV and for HCV, respectively. This 

indicates that 94% of our patients with hepatocellular 

carcinoma are currently infected with either of two 

hepatitis viruses. In contrast, only 3% (1.5% for HBV 

and 1.5% for HCV) of general population are infected 

with the viruses. 

Natural Course: Our follow-up study indicates 

that there is difference between B-viral and C-viral 

disease to develop into hepatocellular carcinoma, e.g., 

C-viral hepatocellular carcinoma often develop with 

the background of advanced fibrosis and/or cirrhosis, 

whereas B-viral HCC sometimes without. Therefore, 

surgical resection or complete ablation of nodules not 

necessary leads to complete cure of hepatocellular 

carcinoma, especially in C-viral HCCs. 

Our Treatment Strategy: In fact, our 

experience of more than 5000 patients treated by PEIT 

(Percutaneous Ethanol Injection Therapy), PMCT 

(Percutaneous Microwave Coagulation Therapy) and 

recent RFA (Radio Frequency Ablation), indicates 

frequent recurrence. It is clear that you need the 

treatment both for backgrounds and tumor nodules. 

Otherwise, the recurrence of cancer from background 

(cirrhotic nodules) could reach 80% within 5 years. 

Strategy of ours is to treat cancer nodules by 

PEIT, PMCT and RFA. Approximately 85% of our 

patients who are admitted to our Department of 

Gastroenterology, University of Tokyo, were treated 

with one of the above percutaneous methods. 

Recently, we have completed a prospective 

controlled study of PEIT and RFA (Gastroenterology 

2005;129:122-130). In that study, the RFA treated 

patients’ survival were significantly better than 

those of PEIT. Thus, majority of the patients are now 

treated by RFA , resulting in 3000 cases so far and 

only exceptionally cases are treated by PEIT. 

Recurrence: However, there are several problems. 

The biggest is the recurrence from cirrhotic background. 

Treatment for the backgrounds is basically to 

cure cirrhosis or advanced fibrosis. We have indicated 

that eradication of HCV by interferon eventually 

induces the resolution of fibrosis (Ann Intern Med 

2000;132:517-524) . In fact, this reduction of the 

fibrosis due to the interferon treatment were related 

to decrease of incidence of hepatocellular carcinoma 

(Ann Intern Med 1999;131:174-181). We initiated a 

prospective controlled study for the patients who had 

hepatocellular carcinoma, treated by percutaneous 

injection therapy and interferon. The result indicates 

that 21 patients treated by ablation and interferon 

which induced good response, 5-year survival were 

83%, compatible with liver transplantation (Ann 

Intern Med 2003;138:299-306). 

Advanced HCC: We still have many patients 

suffering from advanced hepatocellular carcinoma, 

especially with portal vein tumor invasion (PVI) 

who usually live only for 6 months. The combination 

of 5-FU and Interferon was given to more than 300 

patients with 17% of CR (Complete Response) (Cancer 

2006;106:1990-1997). Furthermore, the significance 

and the limitation of molecular targeting drugs on 

advanced cases could be shown in our patients. 


Abstract 43 

Use of Pre-emptive Nucleoside 

Analogue Therapy for Hepatitis B 

Reactivation after Chemotherapy 

GKK Lau 

Queen Mary Hospital, The University of Hong Kong, 

Hong Kong SAR, China 

In areasc where hepatitis B infection is endemic, 

hepatitis due to hepatitis B virus reactivation after 

chemotherapy or immunosuppressive therapy, 

is a serious cause of liver-related morbidity 

and mortality. With the characterization of the 

underlying pathogenesis, much progress in the 

management of this important clinical problem has 

been made in the past 2 decades. By year 2007, it is 

mandatory to screen for hepatitis B surface antigen 

status before initiating intensive chemotherapy 

or immunosuppressive therapy. All those who are 

hepatitis B surface antigen positive should be started 

pre-emptive nucleos(t)ide analogues. However, there 

remains important issues, such as the type and length 

of nucleos(t)ide analogues therapy. As not all hepatitis 

B surface antigen positive patients will suffer from 

HBV reactivation, it will be useful to identify risk 

factors related to HBV reactivation so that patients 

will not be treated unnecessary with nucleos(t)ide 

analogues. In addition, there is an increase awareness 

of reactivation of occult hepatitis B virus, especially 

in hepatitis B virus endemic area, such as Asiapacific 

region. Careful epidemiological study will be 

needed to clarify the impact of occult hepatitis B 

infection in patients treated with chemotherapy or 

immunosuppressive therapy. 


Immunology & Pathogenesis 


Abstract 44 

Cellular Immune Responses 

Against Hepatitis C in Acute and 

Chronic Infection 

MJ Koziel 

Beth Israel Deaconess Medical Center, Harvard Medical 

School, Boston MA, USA 

Hepatitis C virus (HCV) induces persistent infection 

and causes chronic liver diseases in most infected 

patients; however, the mechanisms whereby HCV 

evades an effective immune response and the role 

of immune responses in liver damage once chronic 

infection is established are unknown. Vigorous 

HCV-specific CD4+ and CD8+ T cell (CTL) responses 

against HCV multiple epitopes are necessary for 

spontaneous viral clearance during the acute phase, 

but the virus appears to have multiple strategies to 

evade these defenses. These include interference with 

the endogenous interferon response, attenuation and 

alteration of cellular function, and mutation to avoid 

immune recognition. Based on prospective treatment 

studies, it appears that the likelihood of establishing 

chronic infection is set as early as 12 weeks into 

infection. T cell responses may be associated with the 

likelihood of sustained virologic response in interferon 

and ribaviirn based regimens, but there is a relative 

paucity of data whether they are required for SVR. 

Despite an abundance of studies determining the 

correlates of protective immunity, there are relatively 

few studies on the role of immune responses during 

the chronic phase of infection. HCV-specific immune 

responses measured in the peripheral blood are weak 

and often barely detectable, whereas they can be 

found in liver tissue. CD4+ T cell responses appear 

to protect against liver injury and may be important 

to clearance during interferon and ribavirin based 

therapy. The classic understanding of CD8+ cells is 

that they are primarily involved in tissue injury, but 

there may be subpopulations of T cells that protect 

against liver inflammation. 

Abstract 45 

Broadly Neutralizing Antibodies 

to HCV: Definition of Epitopes and 

Antiviral Activity In Vitro and 

In Vivo 

M Law 1 , T Maruyama 1 , J Lewis 2 , E Giang 1 , AW Tarr 3 , 

Z Stamataki 4 , P Gastaminza 1 , FV Chisari 1 , IM Jones 5 , 

RI Fox 6 , JK Ball 3 , JA McKeating 4 , NM Kneteman 2 , and 

DR Burton 1 

1 The Scripps Research Institute, California, USA; 

2 University of Alberta, Alberta, Canada; 3 The University 

of Nottingham, Nottingham, UK; 4 University of 

Birmingham, Birmingham, UK; 5 University of Reading, 

Reading, UK; 6 Scripps Memorial Hospital, California, 

USA 

BACKGROUND: A major problem facing HCV vaccine 

design and attempts to prevent HCV transmission by 

passive antibody therapy, e.g. in the setting of liver 

transplantation, is the extreme variability of the virus. 

Indeed, several antibody preparations, including 

hepatitis C immune globulin (HCIG) prepared from 

human plasma pools, have been shown unable to 

protect against challenge with HCV quasispecies 

in animal models, or to prevent recurrence of HCV 

following transplantation. Key questions are whether 

neutralizing antibodies of sufficient breadth can be 

generated to protect against quasispecies challenge 

and whether the corresponding epitopes can be 

defined to facilitate vaccine design. 

METHODS: To investigate these questions, a 

phage-display antibody (Fab fragment) library was 

constructed using bone marrow RNA from a donor 

with chronic HCV infection and panned against 

HCV E1E2 envelope glycoprotein antigens to isolate 

specific human monoclonal antibodies (MAbs). 

Isolated Fabs with unique binding properties were 

converted into full length IgG1 molecules and their 

activity against a panel of diverse HCV isolates was 

examined to identify broadly neutralizing MAbs. The 

conserved neutralizing epitopes were mapped by 

competition with well-defined MAbs and by alanine 


mutagenesis scanning. The correlation between in 

vitro neutralization and in vivo protection of the 

broadly neutralizing MAbs was investigated in passive 

antibody transfer experiments using a human liverchimeric 

mouse model. 

RESULTS: A panel of 36 distinct human Fabs specific 

for the HCV E2 envelope protein was isolated to define 

three antigenic regions (ARs) in E2. Seven distinct 

Fabs were converted to full length IgG1s to facilitate 

the characterization of the ARs; a series of in vitro 

assays showed that AR1 and AR3 partially overlap 

with the putative CD81 binding site but only AR3 is 

a target for multiple cross-neutralizing antibodies. 

Alanine mutagenesis scanning of regions Q412-S424, 

R483-P491 and G523-F550 of E2 identified shared 

and distinct residues used by CD81 and the MAbs, 

providing an explanation for such overlaps. Two 

AR3-antibodies (AR3A and AR3B), when given at a 

high dose (200 mg/kg animal), protected the human 

liver-chimeric mice against intravenous challenge 

with a heterologous HCV-infected human serum 

(2 x 10 5 IU, genotype 1a). While all 4 control mice were 

infected; AR3A antibody delayed virus replication and 

protected 2 of 5 mice while AR3B antibody protected 

3 of 4 mice. 

CONCLUSIONS: From a large panel of phage-display 

recombinant antibodies to HCV, we identified human 

MAbs that recognize a conserved antigenic region 

on the virus envelope glycoprotein E2, neutralize 

genetically diverse HCV isolates and protect against 

heterologous HCV quasispecies challenge in vivo in 

a human liver-chimeric mouse model. The results 

provide evidence that broadly neutralizing human 

antibodies to HCV protect against viral infection and 

suggest that a prophylactic vaccine against HCV may 

be achievable. 

Abstract 46 

Innate Immune Studies in 

Hepatitis B: Novel Studies in 

Pathogenesis and Treatment 

K Visvanathan 

Monash Institute of Medical Research, Australia 

Background/Aims: Toll-like receptors (TLR’s) 

are critical receptors that promote innate immune 

responses to pathogen-associated molecular 

patterns. Activation of TLR’s leads to production of 

cytokines such as TNF and IL-6. We have previously 

demonstrated that patients with HBeAg+ve CHB have 

suppressed TLR2 expression on peripheral monocytes 

and hepatocytes associated with decreased cytokine 

expression. 

Methods and Patients: Three different cohorts 

of patients were used for these studies. For the LMV 

studies, PBMCs was measured from 21 patients with 

untreated HBeAg-positive and HBeAg-ve CHB and 10 

uninfected control patients. Individual patient blood 

was stimulated with specific TLR ligands and the 

resultant supernatant was assayed for TNF and IL-6. 

In addition serum HBeAg was measured by enzyme 

immunoassay. For the IFN studies 15 HBeAg+ve 

patients all treated with IFN were used. Similar 

experiments to those for the LMV study were done 

for this study. 

Using a third cohort and using novel flow cytometric 

techniques we have further investigated the ability of 

hepatocytes and Kupffer cells from HBV patients to 

respond to culture with specific TLR stimuli. Primary 

human liver cells were cultured either unstimulated or 

with specific TLR ligands. After culture the cells were 

stained for flow cytometric analysis with intracellular 

anti-cytokine antibodies. 

Results: TLR2 levels increased within four weeks of 

effective therapy in HBeAg-positive patients (P

level of TLR4 expression did not differ significantly 

between the groups. The functional relevance of these 

findings was established by the demonstration in 

HBeAg positive patients of initial reduced cytokine 

production (TNF and IL-6) and phospho-p38 

kinase production after stimulation of monocytes 

with specific TLR ligands. This immunosuppresion 

improved rapidly during treatment (P

Abstract 48 

Therapeutic Vaccination Against 

Chronic Hepatitis C Virus 

Infection: Towards a Proof-ofconcept 

in Man? 

CS Klade 1 , H Wedemeyer 2 , C Sarrazin 3 , E Schuller 1 , 

K Lingnau 1 , MP Manns 2 , and E Tauber 1 

1 Intercell AG, Campus Vienna Biocenter, 1030 Vienna, 

Austria; 2 Hannover Medical School, Center for Internal 

Medicine, Carl-Neuberg-Str. 1, 30625 Hannover, 

Germany; 3 Saarland University Hospital, Internal 

Medicine II, Kirrberger Straße, 66424 Homburg/Saar, 

Germany 

Novel approaches for treatment of chronic HCV 

infection including therapeutic vaccination are 

urgently needed. We have comprehensively identified 

disease relevant epitopes applying PBMC from 

therapy responders and spontaneous resolvers. IC41 

is a prototypic peptide vaccine containing CD8 and 

CD4 T cell epitopes and poly-L-arginine as synthetic 

adjuvant. Its safety, immunogenicity and occasional 

RNA responses in patients refractory to standard 

therapy (ST) have been reported. Correlation of 

immune and RNA response showed that CD4 

proliferation or IFN-gamma ELIspot were not 

sufficient for RNA response, but a prerequisite for 

IFN-gamma CD8 T cells. Tetramer specific CD8 T cells 

showed a partial shift from CCR7+ central memory 

to CCR7- effector memory phenotype, also not 

associated with RNA decline. The single parameter 

correlating was IFN-gamma CD8 CTL above a critical 

threshold. Responses were dominated by NS3-1073, 

and in one patient an amino acid exchange resulting 

in reduced epitope recognition emerged prior RNA 

rebound. This evidence for mutational T cell epitope 

escape corroborates a causal relationship of T cell 

induction and HCV RNA decline. Next, IC41 was 

investigated as late add-on to ST: 35 genotype 1 

patients, RNA-negative (

Abstract 49 

Amelioration of Metabolic 

Disturbances and Oxidative Stress 

in Hepatitis C Viral Infection by 

FK506 (Tacrolimus) 

K Moriya 1 , H Miyoshi 1 , S Shinzawa 1 , T Tsutsumi 1 , 

H Fujie 1 , Y Shintani 1 , H Yotsuyanagi 1 , T Suzuki 2 , 

T Miyamura 2 , Y. Matsuura 3 , and K Koike 1 

1 University of Tokyo, Tokyo, Japan; 2 National Institute 

of Infectious Diseases, Tokyo, Japan; 3 Osaka University, 

Osaka, Japan 

BACKGROUND: Oxidative stress is assumed to play 

a pivotal role in the pathogenesis of liver diseases 

in hepatitis C virus (HCV) infection, and the 

mitochondria dysfunction may be responsible for 

it. FK506 (Tacrolimus) is supposed to protect the 

mitochondrial respiratory function. We determined 

whether or not FK506 protects the function of 

mitochondria and contributes to improvement of 

HCV-associated liver disease. 

METHODS: FK506 was administered to HepG2 

cells expressing HCV core protein or HCV core gene 

transgenic mice, which develop hepatic steatosis, 

insulin resistance and liver cancer. 

RESULTS: FK506 reduced the production of oxidative 

stress, dose-dependently and significantly, in coregene-expressing 

cells compared to the control cells. 

There was a significant reduction in lipid content and 

the concentration of carbon 18 monounsaturated 

fatty acids by FK506 in core-gene-expressing cells. 

In addition, administration of FK506 thrice a week 

for three months to HCV core gene transgenic mice 

resulted in a significant improvement of hepatic 

steatosis. Furthermore, FK506 treatment significantly 

lowered the serum insulin level that is provoked by the 

core protein, in addition to the reduction of oxidative 

stress and DNA damage in core gene transgenic mice. 

Taken together, our results indicate the antioxidant 

nature of FK506, which blocks oxidative stress 

production in hepatocytes expressing the core protein 

both in vitro and in vivo. 

CONCLUSION: FK506 reversed the effect of the core 

protein in the pathogenesis of HCV infection. This 

result may provide a new therapeutic aid for chronic 

hepatitis C, in which oxidative stress and metabolic 

abnormalities in lipid and glucose contribute to liver 

pathogenesis. 

Abstract 50 

Poor Costimulatory Effects 

of CD 137 in Patients with 

Hepatocellular Carcinoma 

JW Shin 1 , SJ Park 2 , NH Park 1 , and YJ Lee 2 

1 Department of Internal Medicine, University of Ulsan 

College of Medicine, Ulsan University Hospital, Ulsan, 

Korea; 2 Department of Internal Medicine, College of 

Medicine, Inje University, Busan Paik Hospital, Busan, 

Korea 

Background/Aims: The 4-1BB, a member of TNF 

receptor superfamily, is expressed on activated T 

cells and antigen presenting cells. 4-1BB generates 

costimulatory signals which involved T cell activation 

and proliferation, and tumor suppression. s4- 

1BB which is generated by proteolytic cleavage or 

alternative splicing inhibits biological activities of 

4-1BB. High circulating levels of s4-1BB have been 

suggested to suppress immune response and this 

finding was observed in hematologic malignancy. 

In this study, we investigated expression of 4-1BB 

on peripheral blood mononuclear cell (PBMC) and 

serum concentration of s4-1BB in patients with 

Hepatocellular carcinoma (HCC) and healthy control. 

We also analyzed correlations between s4-1BB 

concentrations and clinical characteristics of HCC 

patients. 

Methods: 4-1BB expressions on PBMC from twenty 

three HCC patients and twenty four healthy controls 

were analyzed by flow cytometry after stimulation 


with CD3. Serum levels of s4-1BB were measure by 

an enzyme linked immunosorbent assay. 

Results: The expression of 4-1BB was significantly 

lower on PBMC of HCC patients than that of healthy 

control (10.35 ± 5.3 % vs 23.08 ± 6.73%; p

is currently under phase III clinical trial in a larger 

number of chronic hepatitis B patients. 

Abstract 52 

HBsAg-HBIg Complex Modulates 

Antigen Presentation of Dendritic 

Cells from Chronic Hepatitis B 

Patients 

highest in DCs stimulated with YIC. When T cells 

from patients were incubated with YIC-loaded DCs, 

higher numbers of cells produced IFN-γ, IL-2 and L-5, 

IL-10 were observed. 

CONCLUSIONS: YIC surpasses HBsAg or anti-HBs 

in modulating DC functions from chronic hepatitis B 

patients in vitro. 

B Zheng 1 , X Yao 2 , J Zhou 1 , and Y-M Wen 2 

1 Department of Microbiology, the Hong Kong University, 

Hong Kong SAR, China; 2 Key Laboratory of Medical 

Molecular Virology, Shanghai Medical College, Fudan 

University, Shanghai, China 

BACKGROUND: YIC (HBsAg complexed with anti- 

HBs) has shown to revert immune tolerance in a 

transgenic mouse model. In phase I and II clinical 

trials, YIC induced high titer of anti-HBs in healthy 

adults, and serum IFN-γ and IL-2 in chronic hepatitis 

B patients. The highest rates of HBeAg loss (23.1%), 

HBeAg sero-conversion (21.8%), and higher than 

2 log 10 

decrease in serum HBV DNA (31.2%) were 

observed in the 60 µg YIC immunized group of chronic 

hepatitis B patients. The therapeutic mechanism of 

YIC was predicted to modulate antigen presenting cells 

by the Fc fragments of anti-HBs in YIC. This study is 

thus to investigate how YIC modulates dendritic cell 

(DCs) functions from chronic hepatitis B patients. 

METHODS: After DCs from chronic hepatitis B 

patients were separately incubated with yeastderived 

HBsAg, HBsIg or YIC, HLA II, CD80, CD86, 

CD40 molecules on DCs were monitored by FACS, 

and IL-12 levels were monitored using ELISA kits. 

T cells from the same patient were incubated with 

their DCs previously loaded with HBsAg, HBIg or 

YIC and numbers of Th cells produced cytokines were 

determined by ELISPOT. 

RESULTS: The mean HLA II, CD 86 and CD40 

molecules per dendritic cell were the highest in 

samples incubated with YIC. IL-12 was also the 


New Therapeutic 

Approaches 


ABSTRACT 53 

HCV Entry Pathways and 

Implications for the Development 

of Entry Inhibitors 

MJ Evans, T von Hahn, AJ Syder, A Ploss, 

DM Tscherne, and CM Rice 

Center for the Study of Hepatitis C, The Rockefeller 

University, New York, USA 

The hepatitis C virus (HCV) is a serious global 

public health problem, yet many aspects of the viral 

replication cycle remain mysterious. In particular, 

only basic steps required for viral entry into a host cell 

have been defined. Historically, technical limitations 

have made this a difficult step in the HCV replication 

cycle to study. However, recently developed systems 

including retroviral pseudotyped particles harboring 

functional HCV glycoproteins (HCVpp) and cell culture 

produced infectious particles (HCVcc) now enable 

the study of HCV infection of cultured cells. While 

HCVcc provides a means to study the entry processes 

of authentic HCV virions, HCVpp appear to mimic all 

early entry processes of HCV. Furthermore, HCVpp 

can infect more divergent cell types that may lack 

the factors necessary for efficient RNA replication of 

HCVcc. Through the use of these systems, HCV entry 

has been defined as a temperature and pH-dependent, 

clathrin-mediated process requiring at least several 

cellular molecules, including the tetraspanin CD81, 

scavenger receptor class B type I (SR-BI), and perhaps 

glycosaminoglycans (GAGs). We have recently 

identified claudin-1 (CLDN1), a component of tight 

junction complexes, as an essential HCV entry 

factor. Even this list of HCV entry factors appears 

incomplete, as numerous cell lines express all of them 

yet remain uninfectable. 

Despite great progress on developing specific HCV 

antivirals, it is clear that the emergence of viral 

resistance will be a problem facing future HCV 

treatment options. Thus it is a priority for HCV 

research to develop a spectrum of inhibitors targeting 

diverse steps in the virus lifecycle. HCV cell entry may 

represent one such unique stage of the HCV replication 

cycle that can be targeted. Without a complete picture 

of the dynamics of HCV infection within the host, it 

is difficult to speculate how effective such inhibitors 

would be in a therapeutic setting. HCV cell entry 

inhibitors may be exceptionally useful post-liver 

transplantation, where universal graft reinfection 

frequently results in rapid fibrosis progression and 

subsequent graft failure. A greater understanding of 

the HCV entry process, including the definition of the 

complete set of entry factors and their specific roles, 

may be required for the development of therapies 

targeting HCV entry. In addition, the identification 

of the tight junction protein CLDN1 as an essential 

HCV entry factor suggests the importance of 

understanding HCV entry into polarized cells, which 

may greatly impact the HCV entry process. Such 

studies may lead to an understanding of why HCV 

selectively infects human hepatocytes with obvious 

implications for developing transgenic mouse models 

that support productive HCV infection. 

Abstract 54 

The Role of CD81 and Scavenger 

Receptor Class B Member I in HCV 

Entry 

J Timpe, HJ Harris, J Grove, C Mee, P Balfe, and 

JA McKeating 

Hepatitis C Research Group, Division of Immunity and 

Infection, University of Birmingham, Vincent Drive, 

Birmingham, West Midlands, UK, B15 2TT 

The selective association of a virus with a target 

cell is initially defined by interactions between the 

viral encoded glycoproteins and specific cell surface 

molecules or viral receptors. The observation that 

HCVpp show a restricted tropism for human liver 

cells suggests that liver-specific receptors may help 

define HCV tropism. Recent evidence suggests the 

involvement of at least three host cell molecules in 

HCV entry: the tetraspanin CD81, scavenger receptor 

class B member I (SR-BI) and the tight junction 


protein Claudin-1 (CLDN1). Other factors, such as 

glycosaminoglycans and low density lipoprotein 

receptor, have also been implicated in HCV entry, 

although their role is less well established. 

Since the primary reports describing soluble 

truncated HCV E2 interacting with CD81 and SR-BI, 

investigators have sought to investigate the role of 

these molecules in the viral entry process. Antibodies 

specific for CD81 and recombinant forms of the 

second extracellular loop of CD81 (sCD81) inhibit 

the infectivity of cell bound particles, suggesting that 

CD81 does not confer viral attachment and acts as a coreceptor 

mediating virus internalization. In contrast, 

SR-BI appears to define the primary attachment of 

HCV to target cells. Over-expression of SR-BI in Huh- 

7.5 hepatoma cells promotes HCV infection, leading 

to an increase in focal size and suggesting that SR-BI 

facilitates cell-cell transmission of infectivity. 

To quantify cell-cell transfer of HCV infectivity we 

developed an infectious center assay, where infected 

cells are co-cultured with fluorescently labelled naïve 

target cells in the presence or absence of neutralizing 

antibodies to inhibit the infectivity of cell-free 

virus. Enumeration by flow cytometry and indirect 

immunofluorescence demonstrated efficient cell-cell 

transmission of HCV infectivity. sCD81 and anti- 

CD81 inhibit cell-free particle infection of Huh-7.5 

and partially reduce cell-cell transmission. CD81 

negative HepG2 hepatoma cells, which are resistant 

to cell-free virus infection, became infected after coculturing 

with JFH-1 infected cells, confirming that 

CD81 independent routes of cell-cell transmission 

exist. Target cell expression of SR-BI and CLDN1 

promotes cell-cell transfer of infectivity. These data 

demonstrate that HCV can transmit in vitro by cellfree 

virus infection and by direct transfer between 

cells and these different routes of transmission may 

utilize different receptors. 

Imaging techniques that take advantage of fluorescence 

resonance energy transfer between fluorescent 

tagged receptors allow us to study localization 

and protein associations in non-polarized and 

polarized cell systems. These data allow us to model 

the stoichiometry of the receptor molecules and 

provide information on novel targets for antiviral 

therapy. 

Abstract 55 

Lipid Droplet is an Important 

Organelle for Production of 

Infectious Hepatitis C Virus 

K Shimotohno 

Center for Integrated Medical Research, Keio University, 

Tokyo, Japan 

BACKGROUND: Hepatitis C virus (HCV) is a 

causative agent of chronic hepatitis, cirrhosis and 

hepatocellular carcinoma. Patients infected with HCV 

often associate with diseases such as diabetes as well as 

steatosis, which are believed to be promoting factors 

to the development of hepatocellular carcinoma. 

Animal model experiments showed that HCV capsid 

protein (Core) was involved in the development of 

these diseases. HCV Core regulates metabolism of 

lipid synthesis, which may result in accumulation of 

lipid in cells. Moreover, HCV Core often associates 

with the lipid droplet (LD) in cells expressing Core 

solitary. However, the role of lipid accumulation as 

well as Core-association to the LD on HCV replication 

remains elusive. 

METHOD: Wild and mutated HCV replicons derived 

from JFH1, an infectious molecular clone of HCV- 

2a, were introduced into HuH7 cells and subcellular 

localization of HCV proteins was analyzed in 

connection with production of infectious virus into 

culture medium. 

RESULT: HCV Core protein associated with the LD, 

which not only confirmed the previous reports from 

other group but also showed the association of Core 

even when expressed with whole HCV proteins. In 

addition, we observed close association of other virus 

proteins with the LD in Core-dependent manner. This 

observation was further confirmed by the following 


evidence; (1) Core which lacks association with the 

LD failed to recruit other HCV proteins to the LD, (2) 

No association of other HCV proteins with the LD was 

observed in HuH7 cells bearing HCV replicon lacking 

Core production. Infectious HCV was produced only 

in those cells expressing HCV proteins that associate 

with the LD. Importanly, non-infectious HCV was 

released from cells irrespective to association of HCV 

proteins with the LD. 

DISCUSSION: Lipid droplet plays important roles 

in production of infectious HCV particle. Our result 

suggests that Core plays major roles to recruit other 

HCV proteins around the LD, which may generate the 

environment in where production of infectious virion 

proceeds. Accumulation of lipid in cells upon HCV 

infection may be requisite for efficient production of 

HCV progeny and also may play as a trigger to develop 

steatosis. 

Abstract 56 

The Role of Cyclophilins and 

Cyclophilin Inhibitors in the 

Replication of HCV 

R Crabbé 

Debiopharm, Switzerland 

Cyclophilins are ubiquitously present proteins with 

peptidyl-prolyl cis-trans isomerase activity that 

play an important role in protein folding and in 

isomerization of native proteins in several cellular 

systems. Cyclophilin B (CypB) is targeted to the 

secretory pathway via an endoplasmic reticulum 

signal sequence. It is involved in the regulation of 

inflammatory processes, mainly through interaction 

with CD147 and is also a potent chemotactic agent. It 

has recently been suggested that it plays a role in HCV 

replication. Growing evidence indicates that CypB is a 

positive modulator of the HCV RNA dependent RNA 

polymerase in the replication complex. CypB may act 

as a functional regulator of NS5B RNA-dependent 

RNA polymerase and cyclophilin inhibitors seem 

to interfere with this interaction. Consistent with 

this finding, a model for the role of CypB in HCV 

replication has been proposed. CypB interaction 

with NS5B enhances RNA binding and promotes 

RNA replication. Cyclosporin A and other cyclophilin 

inhibitors would therefore block the ability of CypB to 

interact with NS5B, giving rise to weaker RNA binding 

and the inability to form a functional RNA replication 

complex. A reduced HCV replication of replicon cell 

lines by shRNA after knockdown of CypA, CypB, or 

CypC has also been reported. Therefore, the precise 

mechanism of interaction of CypB and/or other Cyps 

with the RNA-dependent RNA polymerase NS5B or 

other HCV proteins still needs to be elucidated. It 

remains unknown if all HCV genotypes exploit Cyp(s) 

in the same manner. 

Recently, CsA derivatives lacking immunosuppressive 

action have been synthesised. These drugs (NIM811, 

Debio 025 and SCY-635) showed potent anti-HCV 

activity in the replicon system with IC 50 

values of 0.07 

μM to 0.22 μM for Debio 025 and from 0.35 to 0.66 μM 

for NIM811. Debio 025, alone or in combination with 

other anti-HCV drugs, was also particularly efficient 

in curing replicon containing cells from their replicon, 

while Cyclosporin A or a HCV protease inhibitor 

did not result in clearance of the HCV replicon. The 

only cyclophilin inhibitor for which patient data are 

available is Debio 025. In HIV-1 and HCV co-infected 

subjects with compensated liver disease, a dose of 

1200 mg BID of Debio 025 for 14 days resulted in a 

3.6 log 10 

decrease of viral load. 

The present data indicate that cyclophilins seem to play 

an important role in the replication of the hepatitis C 

virus. Although the exact mechanism of action at the 

molecular level is not yet fully elucidated, Cyclophilin 

inhibition seems to represent a new approach to anti- 

HCV treatment mainly by interfering at the level of 

the host-viral interaction. 


Abstract 57 

HCV Infection Increases Claudin-1 

and Claudin-7 Expression by 

Inducing Cirrhosis 

A Kiss 1 , A Holczbauer 1 , E Batmunkh 1 , G Lotz 1 , 

P Kupcsulik 2 , and Z Schaff 1 

1 Semmelweis Medical University, 2nd Inst. of Pathology, 

Budapest, Hungary; 2 Semmelweis Medical University, 1st 

Inst. of Surgery, Budapest, Hungary 

BACKGROUND: Claudins (CLDNs) (1-24) have been 

recently identified as integral proteins of tight junction 

strands. They have been reported to be differentially 

regulated in malignancies and implicated in the 

process of carcinogenesis and tumor progression. 

Changes in claudin-1 and claudin-10 expression have 

been recently found in the development of primary 

hepatocellular carcinoma (HCC). Three host cell 

molecules have been reported as significant entry 

factors for hepatitis C virus (HCV): beside CD81 and 

scavenger receptor B I. claudin-1 has been recently 

described as co-factor in the entry of HCV into 

hepatocytes. Further, claudin-6 and claudin-9 may 

function as additional co-receptors for HCV as well. 

The objective was to characterize the mRNA and 

protein expression of claudin-1, 2, 3, 4 and 7 in HCCs, 

surrounding and normal livers with respect to HCV 

infection and the presence of cirrhosis. 

between the presence of cirrhosis and elevated 

levels of CLDN-1 and -7 protein expression while 

no correlation was revealed between HCV infection 

and claudin expression. Claudin-1 and 7 protein 

expression was indeed significantly elevated in 

cirrhosis (2.86-9.25 fold) compared with normal liver 

and non-cirrhotic surrounding liver. HCC developed 

in cirrhotic livers showed even higher expression of 

claudin-1 contrary to decreased CLDN-7 expression 

in HCC when compared to cirrhosis. Western blot 

analysis confirmed immunohistochemical data. 

mRNA expression of claudin-1 and -7 regarding to 

the presence of cirrhosis showed similar differences 

as found in protein expression. 

CONCLUSIONS: Our data indicate that cirrhosis 

finally increases the expression of claudin-1 and -7 

in the surrounding liver and in the developing HCCs 

as well. Since HCV infection alone does not alter 

claudin expression dramatically our data suggest 

that the elevated claudin-1 and -7 expression is the 

result of an indirect effect of HCV infection achieved 

by the induction of cirrhosis. This would be a new 

mechanism how HCV virus infection and following 

hepatitis and cirrhosis could enhance the effectivity 

of viral entrance and therefore boost the chronicity 

of the viral infection. The project was supported 

by grants: OTKA-T049559, ETT-049/2006, NKFP 

1A/002, ETT-156/2006, NKFP 0056/2004 

METHODS: 25 surgically resected HCCs with surrounding 

tissues and ten normal livers were examined 

by real-time RT-PCR and immunohistochemistry 

and Western blot analysis. Immunoreactivity of 

CLDNs were quantified by morphometry. 

RESULTS: Claudin-1 and -7 protein expression was 

significantly elevated in HCV infected surrounding 

livers and HCCs when compared to normal liver 

samples. On the other hand HCCs or surrounding 

livers of HCV infected samples did not show significant 

alteration in claudin 1, 2, 3, 4 and 7 mRNA or protein 

expression compared to HCV negative specimens. 

However, Spearman analysis indicated correlation 


Abstract 58 

Development of Novel 

Hyperglycosylated Type 1 

Interferons: A Strategy to Improve 

PK Performance Without Loss of 

Biological Potency 

LM Blatt 

Alios BioPharma Inc., South San Francisco, California, 

USA 

Interferons are cytokines that are induced as a 

consequence of viral and microbial infections that 

have pleiotropic biological effects that play a role 

in modulating innate and adaptive immunity. Over 

the past decade, the use of type 1 interferon to treat 

chronic hepatitis C has led to sustained virologic 

response in approximately 50% of patients. Although 

the use of interferons has become widespread, 

several challenges still exist with respect to treatment 

optimization and at least half of the patients cannot 

obtain a sustained response to existing therapies. 

These challenges include poor PK profiles, limited 

biological activity, and suboptimal therapeutic 

indices. The addition of polyethylene glycol to type 

1 interferon has been shown to dramatically increase 

plasma exposure following dosing and has led to 

increased response rates in the treatment of chronic 

hepatitis C. It is important to note that the addition 

of polyethylene glycol to recombinant proteins has 

the effect of reducing the specific activity of the 

molecule in vitro. This is due to the fact that the large 

polyethylene glycol molecule that is necessary to 

block renal filtration can sterically hinder the active 

binding of the interferon to the cell surface receptor. 

were identified that were quantitatively modified by 

addition of carbohydrate moieties. A single variant 

carrying both sites was quantitatively modified at 

both sites. Glycosylation substantially increased 

molecular weight with no resultant loss of biological 

potency. In cell culture models, consensus interferon 

demonstrates increased potency when compared to 

naturally occurring type 1 interferons. Comparisons 

of the antiviral potency of glycol-variants with the 

parent non-glycosylated consensus interferon and 

PEG-IFN alfa 2a using the VSV on A549 cells are 

shown in the table below: 

IFN 

EC 50 

(pg/mL) 

Consensus IFN 1.83 ± 0.41 

Single Glyco-A 2.53 ± 0.53 

Single Glyco-B 3.98 ± 0.85 

Double Glyco-A+B 2.27± 0.41 

PEG-IFN alfa 2a 1200± 600 

Statistical analysis of the EC 50 

values obtained 

demonstrated that all glycol-variants retain similar 

biological potency when compared to the consensus 

interferon molecule and all are approximately 1000X 

more potent when compared to PEG-IFN alfa 2a 

(P

Abstract 59 

Bioinformatics Resources 

Supporting the Analysis of 


EJ Lefkowitz 1 , C Kuiken 2 , B Peters 3 , A Sette 3 , and 

C Upton 4 

1 University of Alabama at Birmingham, Birmingham, AL, 

USA; 2 Los Alamos National Laboratory, Los Alamos, NM, 

USA; 3 The La Jolla Institute for Allergy and Immunology, 

La Jolla, CA, USA; 4 University of Victoria, Victoria, BC, 

Canada 

Several groups that provide bioinformatics resources 

to the scientific community to aid in the study 

of Hepatitis C virus (HCV) have recently begun 

collaborating to expand the available informational 

and analytical tools supporting HCV research. The 

Viral Bioinformatics Resource Center (VBRC), the 

Immune Epitope Database and Analysis Resource 

(IEDB), and the Hepatitis C virus resource at the 

Los Alamos National Laboratories (HCV-LANL) are 

coordinating the acquisition of new and existing data, 

the annotation of that data, and the development 

of new analytical tools, to maximize efficiency and 

provide cross-connected web sites to access to all of 

these resources. 

The VBRC (www.vbrc.org) is one of eight NIHsponsored 

Bioinformatics Resource Centers established 

to make available informational and 

analytical resources to the scientific community. The 

goal of these Centers is to aid research directed at 

providing a better understanding of microorganisms 

included on the NIH list of priority pathogens. 

The VBRC was specifically directed to study 

viruses belonging to the Arenaviridae, Bunyaviridae, 

Filoviridae, Flaviviridae, Paramyxoviridae, Poxviridae, 

and Togaviridae families, and includes HCV. In addition 

to sequence data, the VBRC provides curation for 

each of the viral genomes and gene records, resulting 

in a searchable, comprehensive mini-review of gene 

function relating genotype to biological phenotypewith 

special emphasis on pathogenesis. The VBRC 

also provides a variety of analytical tools on its web 

site to aid in the understanding of the available data, 

including tools for genome annotation, comparative 

analysis, whole genome alignments, and phylogenetic 

analysis. 

The IEDB (www.immuneepitope.org) project catalogs 

and organizes information regarding antibody and T 

cell epitopes from infectious pathogens, experimental 

antigens, and self-antigens, and now includes HCV 

epitopes. The IEDB contains information on epitopes 

curated manually from the scientific literature. Both 

intrinsic structural and phylogenetic features, as well 

as information relating to the interactions of the 

epitopes with the host’s immune system are stored, 

and a variety of tools for querying the database and 

analyzing epitope information are provided. 

The HCV-LANL resource (hcv.lanl.gov) provides 

access to a database of annotated HCV sequences 

that includes detailed isolate and host (patient) 

information. The sequence diversity of HCV requires 

methods to track variants and assess their association 

with individual infections and with epidemiologically 

related outbreaks. This interactive resource provides 

flexible retrieval tools for sequences, clinical 

information, and meta-data, as well as utilities for 

scientific data analysis, including tools to assist in the 

analysis of sequence variation. 

By distributing responsibilities, sharing data, and 

interacting with the scientific community, these three 

groups will expand existing resources to establish an 

even more comprehensive set of easy-to-use data 

query and analytical tools that provide a useful and 

used platform supporting HCV research. 


Abstract 60 

A Combination of Direct Antiviral 

Compounds Can Achieve 

Sustained Viral Response in 

Hepatitis C Virus-infected 

Chimpanzees 

DB Olsen 1 , L Handt 1 , K Koeplinger 1 , S Ludmerer 1 , 

D Graham 1 , M MacCoss 2 , NJ Liverton 1 , JP Vacca 1 , 

JA McCauley 1 , D Hazuda 1 , and SS Carroll 1 

1 

Merck Research Laboratories, West Point, PA, 19486, 

USA; 2 Merck Research Laboratories, Rahway, NJ, 07065, 

USA 

BACKGROUND: Current pegylated interferon-α and 

ribavirin combination therapies to treat infection by 

hepatitis C virus (HCV) have significant side effects 

and show limited efficacy in patients infected with 

HCV genotype 1. Efforts to develop novel therapies 

that enhance both efficacy and tolerability have 

focused on direct antiviral agents targeting the virally 

encoded RNA polymerase, NS5B, and protease, 

NS3/4A. 

METHODS: To explore the potential to achieve 

sustained virologic response by administration 

of a combination of polymerase and protease 

inhibitors, HCV-infected chimpanzees were dosed 

with MK-0608, a nucleoside analog inhibitor of 

HCV RNA polymerase, or with a novel, macrocyclic 

inhibitor of NS3/4A protease, or a combination of 

both compounds. Plasma samples were collected 

at time points before, during and after the dosing 

period and plasma viral loads determined using the 

Taqman assay (limit of quantitation, LOQ, 20 IU/ 

mL). Population sequencing and a novel allele specific 

Taqman assay (AUGER) were used to assess levels of 

viral variants known to be resistant to inhibition by 

the two compounds. 

RESULTS: Administration of potent inhibitors of 

either NS5B or NS3/4A to HCV-infected chimpanzees 

results in profound suppression of viral loads in 

plasma. All of the chimpanzees receiving combination 

dosing experienced decreases in plasma viral titer 

to below the LOQ. The viral titer of one of three 

chimpanzees receiving the longest duration of 

combination dosing remained below the LOQ 26 

weeks after the end of dosing. Viral variants resistant 

to inhibition by the NS3/4A inhibitor were detected 

in rebounding viral populations in the other animals. 

CONCLUSIONS: The results demonstrate that 

profound long-term suppression of viral titer can 

be achieved by administration of a combination of 

direct antiviral agents in the chimpanzee model of 

HCV infection. The results of these studies regarding 

the efficacy, duration of response, and potential for 

development of antiviral resistance have implications 

for designing strategies to achieve SVR with direct 

antiviral therapies in HCV infected patients. 

Abstract 61 

Potent Antiviral Activity of the 

Nucleoside HCV Inhibitor, R7128, 

in Prior IFN Non-responders 

JG McHutchison 1 , R Reddy 2 , M Rodriguez-Torres 3 , 

E Gane 4 , R Robson 5 , J Lalezari 6 , GT Everson 7 , 

E DeJesus 8 , HE Vargas 9 , A Beard 10 , GZ Hill 11 , M Otto 10 , 

W Symonds 10 , and M Berrey 10 

1 Duke Clinical Research Institute, Durham, NC, USA; 

2 University of Pennsylvania, Philadelphia, PA, USA; 

3 Fundacion de Investigacion d Diego, Santurce, PR, 

USA; 4 Auckland Clinical Studies Limited, Auckland, New 

Zealand; 5 CCST, Christchurch, New Zealand; 6 Quest 

Clinical Research, San Francisco, CA, USA; 7 University 

of Colorado, Aurora, CA, USA; 8 Orlando Immunology 

Center, Orlando, FL, USA; 9 Mayo Clinic, Phoenix, AZ, 

USA; 10 Pharmasset, Inc., Durham, NC, USA; 11 Roche, 

Palo Alto, CA, USA 

Background: R7128 is a pro-drug of PSI-6130, a 

cytidine nucleoside analog polymerase inhibitor, for 

treatment of HCV. Safety and PK have been evaluated 

following single oral doses in healthy volunteers; 

preliminary antiviral activity was assessed following 

14d R7128 in subjects with HCV genotype 1 

infection. 


Methods: Single doses of R7128 were administered 

to 46 healthy subjects. Six active and two placebo 

subjects per group were enrolled in 5 sequential dose 

groups (500 mg, 1500 mg, 4500 mg, 6000 mg, and 

9000 mg) and a food effect group (1500 mg). In Part 

2, multiple oral doses of R7128 were administered 

for 14 days to 40 HCV-infected patients (8 active & 

2 placebo per cohort) at doses of 750mg QD, 1500mg 

QD, 750mg BID & 1500mg BID. 

Results: Following single doses, nineteen (19) 

adverse events were reported; all were mild to 

moderate, none were dose-dependent, and no 

gastrointestinal AEs were observed. In the multiple 

dose study, all subjects had HCV genotype 1 (30 – 1a; 

10- 1b), had previously failed alpha-interferon and 

were non-cirrhotic. There were no SAEs reported 

and no AEs required dose modification. No clinically 

significant changes in vital signs, ECGs, hematology, 

renal or other laboratory parameters occurred. 

Preliminary data on AEs reported during treatment 

in subjects receiving R7128 include a total of 51 

events in 18 of 32 subjects, most of mild intensity. 

The most frequently reported AEs for patients 

receiving R7128 were headache (13) and dry mouth 

(3). 34 adverse events occured in 7 subjects receiving 

placebo, with headache (4) and diarrhea (4) most 

commonly reported. After both single and multiple 

doses, plasma exposure to the prodrug, R7128, was 

negligible, while concentrations of the active moiety, 

PSI-6130 increased less than proportionally with 

dose. PSI-6130 C max 

occured 2-3 hours after dosing. 

The terminal half-life was ~5h for PSI-6130. Mean 

plasma HCV RNA in all 4 dose groups decreased 

in a dose-dependent manner with placebo values 

remaining at baseline. The mean reduction in HCV 

RNA with the 1500 mg BID dose was -2.7 log 10 

IU/mL 

and ranged from -1.2 to -4.2 log 10 

(below the limit of 

detection) at Day 15. 

Conclusions: This study demonstrated that 

R7128, a direct antiviral, can deliver sufficient 

antiviral potency via monotherapy to suppress HCV 

below the level of detection (

METHODS: 104 patients were randomized to: Dual 

1500: R1626 1500 mg bid + PEG-IFNα-2a (n=21); 

Dual 3000: 3000 mg bid + PEG-IFNα-2a (n=32); 

Triple 1500: 1500 mg bid + PEG-IFNα-2a + RBV 

(n=31); SOC (standard of care): PEG-IFNα-2a + RBV 

(n=20). 

RESULTS: At week 4 HCV RNA was undetectable (

Methods: These double-blind studies randomized 

mono-infected subjects with HBeAg+ or HBeAg- 

CHB 2:1 to TDF or ADV. Entry criteria included 18- 

69 years of age, compensated liver disease, a Knodell 

necroinflammatory score≥3, ALT >2xULN (HBeAg+) 

or >ULN (HBeAg-), HBV DNA>10 6 c/mL (HBeAg+) or 

> 10 5 c/mL (HBeAg-). Biopsies were performed pretreatment 

and between Weeks 44 and 48. HBV DNA 

was measured using the Roche COBAS TaqMan assay 

(LLQ=169c/mL). 

Results: HBeAg+: 266 nucleos(t)ide naïve subjects 

(176 TDF:90 ADV) were randomized and treated; 

mean baseline HBV DNA was 8.72 log 10 

c/mL and ALT 

was 147 IU/mL. HBeAg-: 375 subjects (250 TDF:125 

ADV) both naïve and lamivudine experienced (18%) 

were randomized and treated for 48 weeks; mean 

baseline HBV DNA was 6.9 log 10 

c/mL and ALT was 

140 IU/mL. More than 90% of the subjects completed 

primary endpoint assessments and overall 1% TDFtreated 

subjects discontinued due to an adverse 

event. A statistically significantly greater response 

was observed for TDF-treated subjects: HBV DNA 

99% homology to the 5’- 

NCR. (ii) BLAST alignments show no assignment to 

any known HCV geno-/subtype. (iii) HCV sequence 

stretches (about 82 bp) are contained within repeated 


sections containing several annealing sites for the 

primers. (iv) Amplified DNA fragments up to 1288 

bp with a 23 bp sequence in between show high 

homology to a center sequence stretch of the 5’-NCR 

(nt 89 to nt 170) of HCV, i.e., with three substitutions 

and a gap. 

We further could identify CD34(+) cells, enriched 

with these DNA sequence section. In particular, this 

marker is expressed on hematopoietic progenitor 

cells. 

Conclusions: Our working hypothesis: These 

particular DNA sequences are part of longer ‘extra 

chromosomal’ DNA molecules. 

This kind of ‘extra chromosomal’ DNA of various 

length could have been generated by incremental 

acquisition from these numerous short sequence 

stretches in the human genome through the activity 

of mobile genetic elements, a common feature of 

them is to generate repeats in the target sequences. 

This may point at different stages during their 

evolution. 

The findings point at three directions at least: (i) 

large parts of the HCV’s 5’-NCR have reached a DNA 

level with yet unknown functioning independently of 

those (ii) being part of an infectious RNA unit named 

HCV. (iii) These HCV homologous sequences could be 

part of non-coding RNA families. 

Abstract 65 

A Novel Anti-viral Compound, 

BIT225, Inhibits Bovine Diarrhea 

Virus (BVDV) In Vitro, and has 

Synergistic Antiviral Activity in 

Combination with Recombinant 

Interferon Alpha-2b (rIFNα−2b) 

and Ribavirin 

CA Luscombe 1 , Z Huang 2 , M Murray 2 , M Miller 1 and 

G Ewart 1 

1 Biotron Limited, Canberra, ACT, Australia; 2 Hepatitis 

Research Program, Southern Research Institute, 

Frederick, MD, USA 

Background: The hepatitis C virus (HCV) p7 

protein is a “viroporin” (virus encoded ion channel), 

potentially involved in virus entry and/or assembly 

of progeny virions: It is required for HCV infection 

in chimpanzees, validating it as a target for antiviral 

chemotherapy. Biotron Limited has developed a 

library of over 300 potential antiviral compounds, 

specifically designed to target viroporins, and has 

identified the compound BIT225 as an inhibitor of p7 

ion channel activity. BIT225, therefore, has potential 

for development as an anti-HCV therapeutic, 

particularly as this compound has already successfully 

completed phase 1 studies in healthy volunteers with 

no significant adverse events reported. 

Bovine viral diarrhea virus (BVDV) is commonly 

studied as a model system for HCV. BVDV encodes a 

homologous p7, ion channel forming protein that is 

essential for virus replication. 

Methods: A cytoprotection assay was used for 

evaluation of compounds against BVDV replication in 

Madin-Darby bovine kidney cells in vitro. BIT225 was 

tested either alone or in combination with rIFNα- 

2b and/or ribavirin. For triple drug combination 

experiments, two fixed, sub EC 50 

concentrations of 

rIFNα-2b (5 and 10 IU/ml) were tested against 8 twofold 

dilutions of BIT225 (from 4µM) and 5 two-fold 


dilutions of ribavirin (from 20µg/ml). Effects of the 

drug combinations were analysed using MacSynergy 

software to determine synergy, additivity or 

antagonism. 

Results: BIT225 alone inhibits BVDV replication 

with an EC 50 

value of 314 nM. The EC 50 

for rIFNα-2b 

alone was 21.7 IU/ml, while ribavirin, which is well 

known to enhance the activity of interferons, had 

minimal antiviral effect up to 20 µg/ml when tested 

alone. In combination with BIT225, ribavirin slightly 

antagonized the strong antiviral activity of BIT225, 

but only at the high concentrations of both drugs. In 

contrast, the combination of rIFNα-2b and BIT225 

showed slight to high synergism against BVDV. As an 

example, in the presence of 10 IU/ml rIFNα-2b, the 

EC 50 

value for BIT225 dose response was reduced to 

138 nM. The triple drug combinations showed even 

higher synergism: Complete virus inhibition was 

seen with 31nM BIT225 in the presence of 10 IU/ml 

rIFNα-2b and 2.5 µg/ml ribabivirin. 

Conclusions: These studies show strong synergism 

between BIT225 and interferon. While the 

combination of BIT225 and ribavirin is not beneficial 

in the absence of interferon, the addition of ribavirin 

in a triple compound combination further boosts 

antiviral efficacy, allowing all three compounds to 

be used at lower concentrations. BIT225 may act by 

inhibiting an additional step in the virus replication 

cycle (p7 activity) and/or by enhancing the effect 

of interferon on the cell. A Phase Ib evaluation of 

BIT225 in chronic HCV subjects is planned for the 

last quarter of 2007. 

Abstract 66 

Identification of Therapeutic 

Targets in Hepatitis B Virus 

(HBV) Associated Hepatocellular 

Carcinoma (HCC) 

MA Feitelson 1 , Z Lian 1 , J Liu 2 , HY Wang 3 , WC Wu 3 , 

P Arbuthnot 4 , MC Kew 4 , and MM Clayton 1 

1 Department of Biology, College of Science and 

Technology, Temple University, Philadelphia, PA, USA; 

2 Department of Digestive Diseases, Fourth Military 

Medical University, Xi’an, P.R. China; 3 Shanghai 

Eastern Hospital & Institute of Hepatobiliary Surgery, 

Second Military Medical University, Shanghai, P.R. 

China; 4 Department of Medicine, University of the 

Witwatersrand, Johannesburg, South Africa 

BACKGROUND: Intrahepatic expression of hepatitis 

B x antigen (HBxAg) is associated with the 

development of HCC, perhaps through up-regulated 

expression of selected cellular genes. 

METHODS: HepG2 cells were stably transduced 

with recombinant retrovirus making HBxAg or the 

bacterial chloramphenicol acetyltransferase (CAT) 

product. RNA isolated from HepG2X and HepG2CAT 

cells was subjected to PCR select cDNA subtraction. 

Differentially expressed genes from these cultured 

cells were validated in clinical samples by in situ 

hybridization, northern and western blotting, and 

by immunohistochemistry. Selected up-regulated 

proteins were then functionally characterized by 

individually over-expressing them in liver cell cultures 

and then measuring their impact upon hepatocellular 

growth, survival and tumor formation. Some of 

the underlying signaling molecules regulating cell 

growth/survival were also identified. 

RESULTS: When expression patterns of cellular 

genes were examined in HBxAg positive compared 

to negative HepG2 cells, expression of the unique 

cellular proteins, up-regulated genes 7 and 11 (URG7 

and URG11), were also strongly up-regulated in 

liver surrounding HCC and in some tumor nodules. 


URG7 over-expression promoted resistance of liver 

cells to Fas and TNFα mediated killing. URG11 

over-expression in liver cells strongly promoted 

growth in soft agar and accelerated tumorigenesis 

in transplantable HepG2 cells. Further examination 

showed that over-expression of URG7 and URG11 

were associated with up-regulated expression of 

wild type β-catenin in liver cell culture, in infected 

liver, and in some HBV infected tumors. Extensive 

co-staining between HBxAg, URG7, URG11 and 

β-catenin was observed in infected liver, suggesting 

a close relationship in vivo. Up-regulated expression 

of β-catenin correlated with HBxAg trans-activation 

function. Transient transfection assays with a 

fragment of the β-catenin promoter showed that it was 

activated by HBxAg, URG7 and URG11, suggesting 

transcriptional activation of the β-catenin gene in 

resistance to apoptosis and tumor development. 

URG11 specific siRNA partially inhibited the ability 

of URG7 to protect against TNFα killing and inhibited 

the growth of HCC cells in serum free medium. This 

correlated with depressed levels of β-catenin. 

CONCLUSIONS: HBxAg, URG7, URG11, and 

β-catenin are important therapeutic targets in 

hepatocarcinogenesis for the application of existing 

drugs and for the development of novel ones. 

Abstract 67 

A Phase 1, Randomized, Blinded, 

Placebo-controlled, Single-dose, 

Dose-escalation Study of PEG- 

Interferon lambda (PEG-rIL-29) in 

Healthy Subjects 

DF Hausman 1 , JA Freeman 1 , SM Souza 1 , IA Nestorov 1 , 

and T Zhang 1 

1 ZymoGenetics, Inc., Seattle, WA, USA 

BACKGROUND: IL-29 is a Type III interferon (IFN) 

induced in response to viral infection. IL-29 binds to 

a receptor with a more restricted expression pattern 

than the IFN-α receptor. PEGylated interferon 

lambda or PEGylated recombinant IL-29 (PEG‐rIL- 

29) is under development as a potential treatment 

for hepatitis C virus (HCV) infection that may be 

associated with better tolerability than current IFNs. 

PEG-rIL-29 inhibits HCV replication in vitro, and 

administration of PEG-rIL-29 to cynomolgus monkeys 

results in increased serum beta‐2‐microglobulin 

(B2M) and hepatic 2’5’ oligoadenylate synthetase 

(OAS). The purpose of this Phase 1 study was to 

evaluate the safety, tolerability, pharmacodynamic 

and pharmacokinetic profile of a single subcutaneous 

dose of PEG-rIL-29 in healthy human subjects. 

METHODS: Subjects were randomized 5:1 to receive 

PEG‐rIL-29 or placebo on Day 1, with continuous 

monitoring for 48 hours post-dose and followup 

on Days 4, 8, 15, 29, and 59. Assessments included 

standard safety measures, electrocardiogram, 

echocardiogram, serum levels of PEG-rIL-29, anti- 

PEG‐rIL-29 antibodies, and markers of biological 

activity including B2M and OAS. 

RESULTS: Seventeen subjects were treated with PEGrIL-29 

(5 each at 0.5, 1.5, and 5 μg/kg, and 2 at 7.5 

μg/kg) and 3 with placebo. Dose-dependent increases 

in B2M were noted starting at 1.5 µg/kg, and in 

serum OAS starting at 5 µg/kg. Pharmacokinetics 

were dose‐dependent, with the half life estimated 

to be 56 hours. PEG-rIL-29 was well tolerated at 

doses up to 5µg/kg and was not associated with 

fever or significant hematologic or cardiac effects 

at any dose level. Dose limiting toxicity, consisting 

of reversible Grade 3 alanine aminotransferase 

(ALT) elevation associated with Grade 2 increase 

in aspartate aminotransferase (AST), occurred in 1 

of 2 subjects treated at 7.5 µg/kg. All other adverse 

events (AEs) were Grade 1 or 2. The most frequently 

reported AEs were pharyngolaryngeal pain (n=3), 

increased transaminases/ALT (n=3), leukocytosis 

(n=2), contact dermatitis (n=2), and cough (n=2). 

Of these, only the transaminase increases and one 

event of pharyngolaryngeal pain were considered 

related to study drug. Laboratory abnormalities 

included dose-dependent Grade 1 decreases in 

fibrinogen and transient increases in prothrombin 


time not associated with bleeding or decreases in 

platelets. Reversible Grade 1 to 2 elevations in ALT 

and AST without associated increases in bilirubin 

also occurred in 3/5 subjects treated at 5 µg/kg. No 

significant changes were seen in serum chemistries, 

renal or hematologic parameters at any dose level. No 

subjects developed specific antibodies to PEG-rIL-29. 

CONCLUSIONS: Administration of single doses of 

PEG-rIL-29 was well tolerated at doses up to 5 µg/kg 

without flu-like symptoms or changes in hematologic 

parameters and was associated with evidence of 

biologic activity. Additional studies are being planned 

to further evaluate the safety and potential antiviral 

activity of PEG-rIL-29 in subjects with chronic HCV 

infection. 

Abstract 68 

Influence of Laboratory 

Parameters on Appearance and 

Course of Bleeding in Patients 

with Portal Hypertension and 

Liver Cirrhosis 

L Husova, J Lata, M Senkyrik, and M Dastych 

University of Brno, Brno, Czech Republic 

BACKGROUND: Acute variceal bleeding is the main 

reason of mortality in patients with liver cirrhosis. 

The development of esophageal varices as well as 

their rupture depends on the level of portal pressure, 

however, a number of other factors may play a negative 

role in the rise of bleeding and its prognosis. 

METHODS: 115 patients with liver cirrhosis were 

enrolled (53 with esophageal variceal bleeding, 62 

non-bleeding cirrhotic patients. There was a difference 

found in Child-Pugh classification (the Childs C 

patients were significantly more frequent in the 

bleeding group). Immediately after hospitalization; a 

panel of hematological (prothrombin time, leukocytes, 

hematocrite, thrombocytes) and biochemical tests 

(urea, creatinine, bilirubin, natrium, C-reactive 

protein, procalcitonin, total blood protein, albumin) 

was obtained. 

RESULTS: In bleeding patients we found significantly 

lower hematocrite (p

(on borderline of statistical significance) in surviving 

patients. Higher levels of serum urea and creatinine 

were found in patients who died so it probably has an 

influence on mortality of bleeding patients. 

Abstract 69 

Structural Studies of the Hepatitis 

C Virus NS5A Protein 

R Love, O Brodsky, M Hickey, P Wells, A Zou, W Hao, 

R Duggal, and C Cronin 

Pfizer Global Research and Development, San Diego, CA, 

USA 

Background: Non-structural protein NS5A 

is a critical component of HCV replication and 

is involved in several cellular processes such as 

interferon resistance and apoptotic regulation. It is a 

phosphoprotein of 447 residues with 3 domains, and 

an amphipathic N-terminal helix which promotes 

membrane association. Domain I consists of a novel 

zinc-binding motif and two subdomains, as shown 

by a crystal structure reported in 2005 (Nature, 

435:374). That structure revealed a dimer, which in 

turn suggested a potential RNA binding cleft. We 

have investigated new methods of preparation of 

domain 1, with subsequent determination of a novel 

crystal structure for the dimer of this protein. 

Methods: A novel pET/T7-based HCV NS5A 

protein expression vector, utilizing a TEV-cleavable 

N-terminal polyhistidine purification tag, was used 

to obtain multimilligram quantities of recombinant 

NS5A domain I protein (amino acids 33-202). Protein 

expression was carried out using Ultra-Yield Flask 

technology in Terrific Broth. Soluble NS5A domain I 

protein was isolated by passage over Probond IMAC 

resin followed by Q-Sepharose chromatography. 

The polyhistidine tag was subsequently removed by 

cleavage with TEV protease and the protein further 

purified by passage over a second ProBond column 

followed by size exclusion chromatography. After 

crystallization screening to find initial conditions, 

Hampton detergent-additives were employed to 

optimize crystals of domain 1. 

Results: The structure of NS5A domain 1 was 

determined to 1.9Å resolution by molecular 

replacement, and found to exist as a dimer. The 

polypeptide fold within each monomer and the 

zinc site are very similar to that reported in 2005, 

however, the dimer interface between monomers is 

significantly different. In our dimer the long axes of 

the monomers are parallel, and related by approximate 

2-fold symmetry. The two N-termini are located 

on the same end of the dimer, which theoretically 

permits membrane association of this end via two 

amphipathic N-terminal helices (not present in this 

structure). Our dimer interface shows extensive 

buried surface area and involves interactions between 

a number of conserved residues. Residues defining the 

previously proposed RNA-binding cleft now lie fully 

exposed and on the side of each monomer farthest 

from the dimer interface. 

Conclusions: The crystal structure of NS5A 

domain 1 that we have determined reveals a mode 

of dimerization different from that originally 

reported, yet is nevertheless consistent with a 

membrane association mechanism. It therefore 

offers an alternative possibility for the physiological 

configuration of NS5A. 

Abstract 70 

Dose Selection of Albinterferon 

Alfa-2b (alb-IFN) for a Phase 3 

Clinical Program 


Duke Clinical Research Institute and Division of 

Gastroenterology, North Carolina, USA 

BACKGROUND: alb-IFN, a novel, long-acting 

interferon (IFN) for treatment of chronic hepatitis C 

(CHC), has demonstrated promising antiviral activity 


and tolerability in preclinical and early clinical studies. 

The results of a phase 2b clinical study of alb-IFN were 

used to determine dosage regimens for evaluation in 

ongoing phase 3 studies. 

METHODS: 458 IFN treatment-naïve patients with 

genotype 1 CHC were randomized to 4 treatment 

arms (peginterferon alfa-2a [PEG-IFNα-2a] 180 μg 

qwk or one of 3 alb-IFN arms [900 or 1200 μg q2wk, 

or 1200 μg q4wk], all in combination with weightbased 

oral ribavirin 1000–1200 mg/d) in an active 

controlled, open-label, 48-wk, dose-ranging study. 

RESULTS: For alb-IFN 900 μg q2wk (n = 118), the 

sustained virologic response (SVR) rate by intent-totreat 

analysis was comparable to PEG-IFNα-2a (n = 

114): 59% vs 58% (P = NS). The wk-4 and 12 response 

rates (defined as hepatitis C virus RNA < limit of 

detection, ie,

experiment, the same treatment schedule was use 

to determine that the minimal effective CLDC dose 

was between 0.5 to 0.05 µg/mouse. CLDC treatment 

increased the expression of inflammatory cytokines 

IL-1α, MCP-1, RANTES and the T H 

1 cytokine IL-12 

were statistically increased in the liver the day after 

the last treatment. IL-12, MCP-1 and RANTES were 

also statistically increased in the serum. To better 

understand the temporal expression of the cytokines 

in response to CLDC, the serum of C57BL/6 mice at 

1, 3, 8 or 24 hr after a single IV injection of CLDC 

(5 µg/mouse) was assayed for IFN-α, IFN-γ, IL-6, and 

IL-10 (Figure 4). All cytokines were increased with a 

peak at 3 hr after CLDC administration, except for IL- 

10, which was not affected. The levels of IFN-α, IFN-γ 

and IL-6 nearly reached baseline levels by 24 hr. 

CONCLUSION: CLDC were effective in reducing liver 

HBV DNA probably by non-cytolytic blockage of HBV 

DNA replication mediated by IFN-α/β or IFN-γ. 

FUNDING: HHSN266200500036C, Enteric and Hepatic 

Diseases, NIAID, NIH (JDM) 

Abstract 72 

Role of Stomatin in the Assembly 

of Hepatitis C Virus RNA Replicase 

Complex 

J-H Kim, DK Ahn, S-B Shim, and J-W Oh 

Department of Biotechnology, Yonsei University, 134 

Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea 

BACKGROUND: Hepatitis C virus (HCV) replication 

occurs on a lipid raft or detergent-resistant membrane 

(DRM) and is mediated by RNA replicase 

complex (RC) consisting of viral nonstructural 

proteins including RNA-dependent RNA polymerase 

(RdRp), NS5B protein, and cellular proteins. The 

DRM structure containing viral RCs can provide a 

microenvironment for efficient RNA replication by 

concentrating and compartmentalizing the RCs. 

Recently, various host proteins interacting with 

HCV NS5B have been identified. However, cellular 

proteins playing a structural and organizational role 

in the viral replication complex formation have not 

been yet identified and characterized. Here, we took 

a proteomic approach to identify cellular proteins 

that might participate in HCV RNA replication 

steps including the formation of RCs at cellular viral 

replication sites. 

METHOD: Cellular proteins interacting with the 

NS5B protein were pulled down, subjected to 

SDS-PAGE, and identified by mass spectrometry. 

Interaction with the NS5B protein was confirmed by 

co-immunoprecipitation and immunofluorescence 

studies. Cellular localizations of the identified protein 

and NS5B protein were assessed by subcellular 

fractionation and membrane flotation assays using 

the hepatoma stable cell line Huh7 harboring an 

HCV subgenomic replicon RNA. Knock-down of 

the expression of an NS5B-interacting protein was 

carried out using a small hairpin interfering RNA 

and an antisense peptide nucleic acid (PNA) specific 

to stomatin. Levels of HCV RNA and proteins were 

measured by quantitative real-time PCR and Western 

blot analysis, respectively. 

RESULTS: We identified stomatin as the most 

prominent cellular protein interacting with HCV 

NS5B. Stomatin has been known to participate 

in the formation of lipid rafts, which are 

membrane microdomains associated with protein 

complexes, cholesterol, and sphingolipids. Coimmunoprecipitation 

and co-localization studies 

confirmed the in vivo interaction between stomatin 

and HCV NS5B, both of which also co-fractionated 

with the mitochondria. Membrane flotation assays 

showed that these proteins are associated with 

the lipid-raft-like domain of the mitochondria. To 

characterize the potential role of stomatin in HCV RNA 

replication, stoamtin expression was knock-downed 

in HCV subgenomic replicon cells using a stomatinspecific 

siRNA and PNA. The results indicate that a 

decrease of stomatin expression suppresses HCV 

RNA replication, which is accompanied with release 

of NS5B protein from the DRM to the detergentsensitive 

membrane fraction. 


CONCLUSIONS: Our results identify stomatin as 

a cellular protein involved in HCV RNA complex 

formation on a mitochondria-associated detergent 

resistant membrane structure. 

Abstract 73 

Antiviral Activity of Amino Acid 

Derivatives of Monascus Pigment 

in Hepatitis C Virus Replicating 

Cells 

S-J Kim, J-H Kim, H Jung, J Jeun, CS Shin, and 

J-W Oh 

Department of Biotechnology, Yonsei University, 134 

Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea 

BACKGROUND: Hepatitis C virus (HCV) infection 

leads to a serious liver disease and the best present 

therapy for chronic hepatitis C is a combination of 

pegylated interferon with ribavirin. This therapy 

is only effective in half of the patients. Traditional 

antiviral drugs designed to target viral enzymes 

often generate resistant mutants rapidly. Therefore, 

the search for alternative, specific therapies for 

HCV chronic infection continues for more options. 

Several lines of evidence suggest that cellular lipid 

and cholesterol metabolism plays a role directly or 

indirectly in the HCV replication cycle. Particularly, 

cholesterol biosynthesis has been proposed as an 

integral part for HCV replication on lipid rafts and 

various inhibitors of 3-hydroxy-3-methylglutaryl 

coenzyme A (HMG-CoA) reductase, the key enzyme in 

cholesterol biosynthesis, have been shown to suppress 

HCV replication. We recently have produced various 

amino acid derivatives (AADs) of Monascus pigments 

and showed their inhibitory activity against HMG- 

CoA reductase. Here, we investigated the potency of 

Monascus pigment AADs in HCV replication. 

HCV-infected cells were treated with various 

concentrations of each AAD alone or in combination 

with interferon-alpha (IFN-α). Western blot analyses 

and quantitative real-time RT-PCR were carried out to 

analyze the levels of HCV nonstructural proteins and 

viral RNA in the treated cells. Cytotoxicity of AADs 

was evaluated by the MTT assay. Inhibitory activity 

of monascus AADs against HMG-CoA reductase was 

determined by an enzyme assay measuring NADPH 

oxidation level. Cholesterol and lipid levels in mice 

fed a high-cholesterol diet without or with AADs were 

measured using a commercial enzyme kit. Effect of 

AADs on HCV RNA polymerase activity was assessed 

by an in vitro polymerase assay with purified HCV 

RNA polymerase. 

RESULTS: Monascus pigment and some of its AADs 

significantly lowered total cholesterol and LDLcholesterol 

levels in mice fed a high-cholesterol 

diet. The pigments also showed various degrees of 

inhibitory effect on HMG-CoA reducatse. Treatment 

of HCV subgenomic replicon cells with AADs reduced 

the expression levels of HCV nonstructural proteins 

and resulted in suppression of HCV RNA replication. 

Furthermore, combination of AADs with IFN-α 

potentiated the anti-HCV activity of IFN-α with no 

significant increase in cytotoxicity. Similar antiviral 

activity was also observed in HCV genotype 2a 

JFH1-infected Huh7 cells. Finally, we observed no 

strict correlation between the inhibitory activity of 

AADs against HMG-CoA reducatse and their anti- 

HCV activity, raising a possibility that alternative 

mechanisms could explain the antiviral activity of 

AADs. 

CONCLUSIONS: Our results identify Monascus 

pigment AADs as a potential inhibitor of HCV 

replication and suggest that combination with INF-α 

or possibly with other anti-HCV drugs might offer 

an alternative strategy with which to control HCV 

replication. 

METHODS: The hepatoma stable cell line supporting 

autonomous replication of a genotype 1b HCV 

subgenomic replicon RNA and the genotype 2a 


Abstract 74 

Silymarin Displays Anti-Viral 

Anti-Inflammatory, and 

Immunomodulatory Effects 

Towards Hepatitis C Virus 

J Wagoner 1 , C Morishima 1 , O Kane 1 , D Lee 2 , and 

S Polyak 1 

1 University of Washington, Seattle, WA, USA; 2 Harvard 

University, Cambridge, MA, USA 

BACKGROUND: A striking feature of hepatitis C 

virus (HCV) infection is its propensity to establish a 

chronic disease state. Because state-of-the art antiviral 

treatments are costly, have serious side-effect 

profiles, and moderate probabilities for durable 

cures, many patients opt for complementary and 

alternative medicine (CAM)-based approaches to 

improve liver health. However, the mechanisms of 

hepatoprotection have not been characterized. 

METHODS: In the current study, we examined 

silymarin, derived from milk thistle, for anti-viral, 

anti-inflammatory and immunomodulatory effects 

towards hepatitis C virus. 

RESULTS: Silymarin inhibited expression of TNF-α 

in anti-CD3 stimulated human PBMC and NF-κB 

dependent transcription in T cells and in human 

hepatoma Huh7 cells. Silymarin also caused dosedependent 

inhibition of infection of Huh7 and 

Huh7.5.1 cells by JFH-1 virus, with both prophylactic 

and therapeutic effects. When combined with IFN-α, 

Silymarin inhibited HCV replication more than 

IFN-α alone. Anti-viral effects induced by Silymarin 

involved Jak-Stat dependent and independent 

signaling. These activities were observed with an 

independently standardized preparation as well as 4 

commercial sources of silymarin. HPLC fractionation 

of the botanical medicine permitted identification 

of the components eliciting anti-viral and antiinflammatory 

actions. 

CONCLUSIONS: The data demonstrate that standardized 

silymarin has anti-viral action against in 

vitro HCV infection. Furthermore, this botanical 

medicine also displays potent immunomodulatory 

and anti-inflammatory actions. Therefore, CAMbased 

approaches may assist in the management 

of patients with chronic hepatitis C. Identification 

of the bioactive molecules in Silymarin provides 

an opportunity for rationale drug design through 

medicinal chemistry approaches. 

Abstract 75 

Construction and Applications of 

a Liver-specific Lentivirus Vector 

with Host Range Determined by 

the Envelope Proteins of HBV 

N Chai, H Chang, E Nicolas, S Gudima, and J Taylor 

Fox Chase Cancer Center, Pennsylvania, USA 

BACKGROUND: There is a pressing need for liver 

specific vectors for treatment of genetic diseases 

affecting the liver and for therapy of chronic hepatic 

infections (e.g., by HCV). Construction of lentivirus 

vectors with a liver specific host range would be an 

important step towards these goals. HBV is highly 

liver specific, targeting hepatocytes and possibly 

bile duct epithelial cells. Therefore, if the envelope 

proteins of HBV could be presented on the surface of 

a lentivirus vector, liver-specific gene delivery should 

be achieved. We describe a vector that appears to 

fulfill this need, with an in vitro host range that is 

restricted to primary human hepatocyte cultures. 

METHODS: A combination of plasmids was used 

to transfect 293 cells, with the aim of making an 

HIV-1 pseudotype with the envelope proteins L and 

S of HBV. The pseudotyped HIV, HIV(LS), was tested 

for the ability to infect cultures of primary human 

hepatocytes (PHH) as well as a number of different 

cell lines. Infection was detected by the expression of 

a reporter gene, LacZ, either using X-gal staining or 


eal-time PCR for detection of LacZ mRNA. Vector 

uptake was compared to uptake of HBV and HDV 

using inhibitors previously reported to distinguish 

forms of receptor mediated endocytosis. 

RESULTS: HIV(LS) demonstrated the same in vitro 

host range as HBV and HDV, infecting PHH but not 

primary woodchuck hepatocyes, or established cell 

lines such as Huh7, HepG2, and 293 cells. Staining 

for LacZ expression revealed that ~10% of the cells 

in a typical preparation of PHH could be infected, 

similar to the infection efficiency of HDV as assessed 

by staining for delta antigen. However, the uptake 

of HIV(LS) appeared to involve a different pathway 

than either HBV or HDV. HBV and HDV behaved as 

if entry required an endosomal mechanism coupled 

with acidification. HIV(LS) appeared to enter cells 

by direct fusion of the virus particles at the plasma 

membrane. 

CONCLUSIONS: We have constructed a lentivirus 

vector that will specifically and efficiently infect 

human hepatocytes. It will find application in liverspecific 

gene therapies, including what might be an 

efficient one-shot vaccine against HBV. In addition, 

although the lentivirus uses the same envelope 

proteins as HBV and HDV, for attachment and entry, 

it uses them in a significantly different way. 

Abstract 76 

Designed Zinc Finger Proteins 

Bind Duck Hepatitis B Virus 

Enhancer DNA and Decrease 

Production of Viral RNA, Proteins 

and Progeny In Vitro 

KA Zimmerman, KP Fischer, MA Joyce, and 

DLJ Tyrrell 

University of Alberta, Edmonton AB, Canada 

BACKGROUND: Duck hepatitis B virus (DHBV) is 

a model virus for human hepatitis B virus (HBV), 

which persistently infects approximately 360 million 

individuals worldwide. Nucleoside analogs such as 

lamivudine, tenofovir and adefovir inhibit the viral 

polymerase and decrease virus production, however, 

due to persistence of the HBV episome, none 

completely clear the virus in most treated patients. 

HBV DNA is present in the nucleus as a closed 

covalently circular (cccDNA) form, where it drives 

viral transcription and virus production. cccDNA is 

the next target for therapeutics aiming to clear the 

viral infection entirely. 

METHODS: To specifically target cccDNA, we have 

designed zinc finger proteins (ZFPs) that bind 

to DNA in the DHBV enhancer region. We have 

designed and purified two ZFPs targeting 18bp 

sequences and two pairs of ZFPs each targeting 9bp 

sequences. Binding kinetics were assessed using 

surface plasmon resonance (SPR) and electrophoretic 

mobility shift assays (EMSA). In vitro pull-down 

assays were used to demonstrate the ability of ZFPs 

to bind authentic cccDNA. The ZFPs were cloned into 

a mammalian expression vector and co-transfected 

into LMH (chicken hepatoma) cells with the plasmid 

pDHBV1.3, which replicates the DHBV replication 

cycle. Transfected cells were analyzed by Western blot 

on whole cell lysates, Southern blot on intracellular 

viral (ICV) DNA and quantitative PCR on Trizol 

isolated RNA to investigate the effects of ZFPs on 

viral products. Cell viability after transfection was 

assessed using an MTT assay. 


RESULTS: Using SPR and EMSA, we determined the 

dissociation constants (Kd) to be in the nanomolar 

range (12.3-99nM) for four ZFPs, in the picomolar 

range (471pM) for one ZFP and the micromolar 

range (185uM) for the last ZFP. The in vitro pulldown 

assay demonstrated that the ZFPs could bind 

authentic cccDNA. Western blots for DHBV proteins 

on LMH cell lysates showed dramatic decreases in 

viral core and surface expression in the presence of 

all ZFPs compared to actin controls. Quantitative 

PCR for viral core, polymerase and surface RNA 

levels demonstrated significant decreases of 58-88%, 

compared to co-transfection with empty vector. In 

addition, ICV particle production was decreased in 

ZFP-transfected cells. 

CONCLUSION: Our designed ZFPs can bind target 

sequences with Kd’s in ranges adequate for drug 

development. They are able to bind authentic cccDNA 

in vitro and can decrease the production of viral 

products in the LMH tissue culture system. We suggest 

our designed ZFPs are binding the DHBV enhancer in 

vitro and preventing the transcriptional machinery 

from accessing it, resulting in decreased production 

of viral mRNA, proteins and genomic RNA. 


Resistance to 

Antiviral Agents 


Abstract 77 

Overcoming HCV Treatmentresistant 

Characteristics 

MW Fried 


The response to peginterferon and ribavirin, while 

effective for the majority of patients, is neither 

universal nor guaranteed and is highly related to 

multiple virologic and host factors 1-3 . Genotype 1 

and baseline levels of HCV RNA (above 400,000 IU/ 

mL) are the strongest virological factors associated 

with diminished response rates 1-3 . Among host 

characteristics, the presence of cirrhosis, African 

American race, obesity, insulin resistance and 

hepatic steatosis have also been demonstrated to 

decrease the rate of SVR 1, 2, 4-6 . Although it is expected 

that newer classes of drugs, such as protease and 

polymerase inhibitors, will improve sustained 

response rates for all individuals, these agents remain 

investigational. Furthermore, it has become apparent 

that peginterferon and ribavirin will serve as the 

backbone of triple drug regimens for the foreseeable 

future 7, 8 . Therefore, optimizing peginterferon and 

ribavirin remains an important goal. 

The mechanism by which increased body weight 

diminishes antiviral response, regardless of 

peginterferon preparation, is likely complex and 

multifactorial. Obesity, a surrogate marker for 

hepatic steatosis, has been associated with decreased 

antiviral efficacy as well as increased risk of fibrosis, 

while hepatic steatosis is also frequently associated 

with insulin resistance that impairs antiviral and 

immune-stimulating properties of peginterferon-alfa 

(5,10–12). 

Individual patients with chronic hepatitis C represent 

a blend of multiple attributes that can affect antiviral 

response. Thus, a patient with genotype 1 infection 

may also have high levels of viremia and be overweight, 

a combination of factors whose pretreatment 

probability of response has been shown to be lower 

than any single unfavorable predictive factor 9 . These 

poor prognostic factors tend to cluster together, 

defining a population of patients who are least likely 

to respond to conventional antiviral therapy with 

peginterferon and ribavirin 10 . 

Numerous strategies have been suggested to improve 

the SVR rates for patients predicted to be poorly 

responsive to interferon-based therapies. Increasing 

the dose of peginterferon, increasing the dose of 

ribavirin and prolonging the duration of therapy have 

all met with limited success 11-14 . The use of higher 

weight-based ribavirin doses in combination with 

peginterferon alfa-2b produced a significant, albeit 

modest, improvement in a large community-based 

study 15 . A recent study compared the impact on viral 

kinetics and SVR of intensified treatment regimens 

of peginterferon alfa-2a and ribavirin with standard 

regimens in patients with a cluster of poor prognostic 

factors (genotype 1, high baseline levels of HCV RNA 

and body weight >85 kg) 16 . The results demonstrated 

that it is possible to improve virologic response rates 

by increasing the intensity of treatment 16 . 

Peginterferon and ribavirin are important components 

of antiviral therapy for chronic hepatitis C. In 

combination with direct antivirals, fewer patients 

will demonstrate treatment-resistant characteristics 

although optimizing response to peginterferon and 

ribavirin will remain essential. 

References: 

1. Fried MW, Shiffman ML, Reddy KR, et al. 

Combination of peginterferon alfa-2a plus 

ribavirin in patients with chronic hepatitis C 

virus infection. New England Journal of Medicine 

2002;347:975-82. 

2. Manns MP, McHutchison JG, Gordon SC, et al. 

Peginterferon alfa-2b plus ribavirin compared 

with interferon alfa-2b plus ribavirin for initial 

treatment of chronic hepatitis C: a randomised 

trial. Lancet 2001;358:958-65. 

3. Hadziyannis SJ, Sette H, Jr., Morgan TR, et al. 

Peginterferon-alpha2a and ribavirin combination 

therapy in chronic hepatitis C: a randomized 

study of treatment duration and ribavirin dose. 

Ann Intern Med 2004;140:346-55. 


4. Conjeevaram HS, Fried MW, Jeffers LJ, et al. 

Peginterferon and ribavirin treatment in African 

American and Caucasian American patients 

with hepatitis C genotype 1. Gastroenterology 

2006;131:470-7. 

5. Zeuzem S, Hultcrantz R, Bourliere M, et al. 

Peginterferon alfa-2b plus ribavirin for treatment 

of chronic hepatitis C in previously untreated 

patients infected with HCV genotypes 2 or 3. J 

Hepatol 2004;40:993-9. 

6. Dev A, Patel K, McHutchison JG. Hepatitis C and 

steatosis. Clin Liver Dis 2004;8:881-92. 

7. Kieffer T, Sarrazin C, Miller J, al e. Combination 

of telaprevir and peg-IFN alfa suppresses both 

wild-type virus and resistant variants in HCV 

genotype-1 infected patients in a 14-day phase 

1b study. Hepatology 2006:Abstract 92. 

8. Tong X, Chase R, Skelton A, Chen T, Wright- 

Minogue J, Malcolm BA. Identification and 

analysis of fitness of resistance mutations against 

the HCV protease inhibitor SCH 503034. Antiviral 

Res 2006;70:28-38. 

9. Foster GR, Fried MW, Hadziyannis SJ, Messinger 

D, Freivogel K, Weiland O. Prediction of sustained 

virological response in chronic hepatitis C patients 

treated with peginterferon alfa-2a and ribavirin. 

Scandinavian Journal of Gastroenterology 

2007;42:247-55. 

10. Swain M, Foster GR, Hadziyannis SJ. Poor 

prognostic factors in heavier patients with chronic 

hepatitis C result in less favourable treatment 

outcomes. Journal of Hepatology 2006;44:227 

(abstract). 

11. Sanchez-Tapias JM, Diago M, Escartin P, et 

al. Peginterferon-alfa2a plus ribavirin for 48 

versus 72 weeks in patients with detectable 

hepatitis C virus RNA at week 4 of treatment. 

Gastroenterology 2006;131:451-60. 

12. Lodato F, Azzaroli F, Brillanti S, et al. Higher doses 

of peginterferon alpha-2b administered twice 

weekly improve sustained virological response in 

difficult-to-treat patients with chronic hepatitis 

C: results of a pilot randomized study. J Viral 

Hepat 2005;12:536-42. 

13. Bacon BR, et al. The DIRECT Trial (Daily-Dose 

Consensus Interferon and Ribavirin: Efficacy 

of Combined Therapy): Treatment of Non- 

Responders to Previous Pegylated Interferon plus 

Ribavirin: Sustained Virologic Response Data. 

Hepatology 2007:(abstract). 

14. Marcellin P, et al. Pegylated interferon alfa- 

2a (40KD) plus ribavirin (RBV) in prior nonresponders 

to pegylated interferon alfa-2b 

(12KD)/RBV: final efficacy and safety outcomes of 

the REPEAT study Hepatology 2007:(abstract). 

15. Jacobson IM, Brown RS, Freilich B, al 

e. Peginterferon alfa-2b and weight-based or flatdose 

ribavirin in chronic hepatitis C: a randomized 

trial. Hepatology 2007;46:971-81. 

16. Fried MW, Jensen D, Rodriguez-Torres M, et al. 

Improved outcomes in HCV patients with difficult 

to treat characteristics: randomized study of 

Intensified peginterferon α-2a and ribavirin. 

Hepatology (abstract) 2006. 

Abstract 78 

Genetic and Structural Variability 

of the Hepatitis C Viral 

Polymerase, NS5B: Implications 

for Resistance to Inhibitors 

PC Simister 1 , R Brillet 2 , V Lohmann 3 , J-M Pawlotsky 2 , 

and S Bressanelli 1 

1 

Laboratoire de Virologie Moléculaire et Structurale, 

CNRS, Gif-sur-Yvette, France; 2 Hôpital Henri Mondor, 

Créteil, France; 3 Department of Molecular Virology, 

University of Heidelberg, Heidelberg, Germany 

The HCV polymerase, NS5B, is an important target 

in the development of anti-HCV drugs. Since 1999, 

40 NS5B crystal structures have been solved and 

deposited in the Protein Data Bank (PDB). Of these, 

36 structures are based on genotype-1b sequences 

and 4 on genotype 2a. Furthermore, in our laboratory 

we have crystallised NS5B from the JFH1 strain 

(genotype 2a). The catalytic region of NS5B consists 

of three domains (thumb, palm and fingers), which 

are connected to a transmembrane helix at the 

C-terminus by a flexible linker. Significant domain 


movements are expected to occur during the different 

stages of polymerisation of the HCV RNA genome. 

However, all the structures published to date have 

been crystallised in very similar conformations. 

The available structures represent predominantly 

two truncated forms in which either the membrane 

anchor is deleted (delta-21) or both the anchor and 

the linker (delta-55). 

Part 1: After analysing the slight conformational 

differences between the available structures 

using computational methods, we found that the 

conformationally invariant regions are not necessarily 

identical between genotypes. Also, in the structures 

from genotype 1b, the more open conformation is 

seen only in the truncated delta-55 form. However, 

for genotype 2a both conformations are observed with 

the delta-21 truncation. Significantly, we discovered 

that global conformational changes appear not to 

occur upon inhibitor binding. Interestingly, a detailed 

comparison of models of the JFH1 and other 2a 

polymerases has provided structural insights helping 

to explain their different biological activities. 

Part 2: Consensus genotype-1b sequences were 

obtained from the HCV-genome database, euHCVdb 

(Combet et al., 2007). Additionally, we characterized 

the quasi-species in 2 untreated patients infected 

with HCV (genotype 1b) and obtained about 40 fulllength 

NS5b sequences. We analysed the location 

of polymorphisms in the consensus sequences and 

those from the clinical samples in order to understand 

how inherent mutability might affect the clinical 

application of anti-NS5B inhibitors. Many residues in 

the inhibitor-binding sites are polymorphic including 

those which correspond to known resistance 

mutations. Importantly, some polymorphisms in the 

quasi-species are not represented in the consensus 

sequences, implying that extra information is 

provided by the clinical samples. 

Thus, we confirm that the C-terminal region is 

essential for NS5B regulation. The structural 

differences observed might affect the efficacy of 

anti-NS5B inhibitors in a genotype-dependent 

manner. Furthermore, the presence of pre-existing 

polymorphisms associated with resistance in the 

quasi-species population may influence the speed of 

HCV rebound with important implications for the 

design of therapeutic strategies. 

Abstract 79 

Selection of Multidrug Resistant 

Hepatitis B Virus Following 

Sequential Monotherapy and 

Combination Therapy 

A Ayres 1 , A Bartholomeusz 1 , M Littlejohn 1 , D Colledge 1 , 

L Yuen 1 , P Angus 2 , A Thompson 1 , and S Locarnini 1 

1 Victorian Infectious Diseases Reference Laboratory, Nth 

Melbourne, Victoria, Australia; 2 Austin and Repatriation 

Medical Centre, Heidelberg, Victoria, Australia 

Background: Although more antiviral agents for 

treatment of hepatitis B virus (HBV) infection have 

become available in recent years, options remain 

limited. Currently they comprise conventional and 

pegylated interferons and four oral nucleos(t)ides 

analogues. The former show limited efficacy whilst 

long-term use of the latter may cause the emergence 

of drug resistant HBV; their sequential use may result 

in multidrug resistant HBV variants, which are posing 

an increasing clinical challenge. 

Aim: To perform clonal analyses of serial putative 

drug resistant HBV isolates in order to monitor the 

emergence of multidrug resistance in relationship to 

quasispecies diversity. 

Methods: The HBV polymerase gene was amplified, 

cloned and sequenced from HBV isolates obtained 

from four different patients. Patient A initially failed 

adefovir monotherapy and subsequently failed to 

respond to combination therapy with adefovir (ADV) 

and lamivudine (LMV). Patients B and C were already 

infected with LMV resistant mutants and also failed 

to respond to ADV and LMV in combination. HBV 

infection in patient D became sequentially refractory 

to monotherapy with LMV, entecavir (ETV) and 


ADV and subsequently became unresponsive to a 

combination of ADV and ETV. 

Results and Discussion: Direct sequencing 

and clonal analysis of HBV isolates from samples 

from all four patients revealed complex mutation 

profiles. Sequencing of HBV from Patient A revealed 

the presence of rtM250L + rtI233V substitutions 

in addition to those (rN236T + rtA181T) known to 

confer ADV resistance. Clonal analysis of HBV from 

patients B and C revealed the co-existence of two 

separate HBV populations, each of which encoded 

different primary resistance mutations. Lamivudine 

resistant mutants (encoding rtL180M+ rtM204V/I) 

comprised one population, whilst a second population 

harboured mutations encoding rN236T +/- rtA181V, 

which confer ADV resistance. Clones that encoded 

both rtN236T and rtM204I/V on the same genome 

were not detected. Isolates from patient D encoded 

a previously unreported variant (rtN236V) in 

association with changes that confer primary LMV 

resistance (rtL180M + rtM204V) and primary 

ETV resistance (rtL180M + rtT184G + rtM204V+ 

rtS202I). 

Conclusion: Multidrug resistant HBV mutants 

that encode complex changes in the viral polymerase 

are emerging in patients who have undergone 

sequential antiviral therapy. Resistance to more 

than one nucleoside inhibitor is a major concern and 

demonstrates the need for continuous surveillance. 

Monitoring the evolution of HBV mutants selected by 

during different treatment regimens will contribute 

to the development of optimal treatment regimes 

tailored to individual patients as well as contributing 

to our understanding of the factors that contribute to 

drug resistance. 

Abstract 80 

Multidrug Resistance and Crossresistance 

Pathways in HBV as a 

Consequence of Treatment Failure 

L Yuen 1,2 , A Ayres 1 , A Bartholomeusz 1 , M Littlejohn 1 , 

and S Locarnini 1 

1 VIDRL, North Melbourne, Australia; 2 Evivar Medical, 

East Melbourne, Australia 

Background/Aims: In recent years, the sequential 

use of anti-viral medications for the treatment of 

hepatitis B has lead to the emergence of complex 

multi drug resistant HBV. Primary drug resistance 

mutations result in reduced susceptibility to an 

antiviral agent, while secondary compensatory 

mutations restore replication defects associated with 

primary drug resistance, and may be associated with 

low-level reduced susceptibility. Several evolutionary 

pathways of drug resistance for HBV have been 

observed in patients treated with (nucleot(s)ides 

NAs. It is possible that the drug resistance mutations 

selected under one agent may affect the efficacy 

of subsequent agents. The aim of this study was 

to elucidate mutation pathways associated with 

resistance to NAs and to determine the potential 

cross-resistance profiles selected under a particular 

NA. 

Methods: The HBV reverse transcriptase (rt) gene 

was sequenced from patients pre-therapy and during 

virological breakthrough on antiviral therapy. A HBV 

sequence analysis program, SeqHepB, was used to 

analyse the treatment-associated mutations. The 

sequence data obtained from the most recent samples 

of 159 pre therapy patients and 215 patients during 

their virological breakthrough were compared while 

receiving lamivudine (LMV) and/or adefovir (ADV). 

Associations were evaluated using the Fisher’s Exact 

method and a pattern discovery program Magnum 

Opus. 

Results and Discussion: Treatment with LMV, 

L-nucleoside analogues (such as telbivudine (LdT)), 


and/or entecavir (ETV) can result in the selection of 

the mutation rtM204I/V. Statistical analysis identified 

the selection of some secondary compensatory 

mutations (rtL80I/V, rtV173L and rtL180M) during 

LMV monotherapy that can also affect response 

to ETV or LdT. In contrast, ADV treatment failure 

is typically associated with the selection of the 

mutations rtN236T and/or rtA181V/T. During ADV 

monotherapy the presence of the mutations rtA181T/V 

and/or rtN236T were statistically significant. The 

selection of rtA181V and/or rtN236T and loss of 

rtL180M and/or rtM204I/V was also statistically 

significant among LMV resistant patients who were 

subsequently switched to ADV monotherapy. The 

rtA181T mutation has been implicated with reduced 

sensitivity to LMV and ADV, thus it is common to 

both the “204 and 236 pathways”. 

Conclusion: Current emerging patterns of antiviral 

drug resistance in the HBV polymerase are 

complex and codon analysis has identified a number 

of amino acids which have occurred at statistically 

significant levels while under drug selection 

pressure. 

Three major HBV evolutionary NA-resistance 

pathways (rtM204I/V, rtN236T and rtA181T/V) 

have been characterised. The first two pathways are 

associated with clusters of secondary mutations that 

can affect subsequent treatment with NAs, whilst the 

third rtA181T/V is inherently a multi-drug resistance 

pathway. 

Abstract 81 

Antiviral Effect of Nucleoside 

Analogs and Interferon on a 

Novel Infectious Cloned Hepatitis 

C Virus Containing the S282T 

Mutation in the NS5B RNA 

Polymerase 

G Mateu 2 , L Bassit 1,3 , A Grakoui 2 , and RF Schinazi 1,2,3 

1 Emory University/VA Medical Center, Department of 

Pediatrics, Decatur, GA, USA; 2 Emory University/Yerkes, 

Microbiology and Immunology, Atlanta, GA, USA; 

3 Veterans Affairs Medical Center, Decatur, GA, USA 

BACKGROUND: A chimeric HCV genotype 2a 

infectious clone (C-p7) that infects hepatoma cells 

and produces a high level of infectious particles in 

Huh7.5 cell line was recently developed (Mateu et 

al., HCV 2007, Glasgow). In this in vitro C-p7 acute 

infectious system, HCV replication was inhibited by 

several 2’-Me nucleosides, and interferon alpha-2b 

(IFN) in a dose-response manner (Schinazi et al., HCV 

2007, Glasgow). 

METHODS: To assess resistance to antiviral agents, 

cloned virus containing the S282T mutation 

associated with 2’-Me nucleoside analog was created. 

Activity and toxicity of seven anti-HCV nucleoside 

analogs, IFN, and ribavirin (RBV) were evaluated 

with this new infectious cloned virus and compared 

to wild type HCV. All compounds except ribavirin 

are selective anti-HCV agents in the HCV (clone B) 

replicon. Following five days incubation, total cellular 

RNA was extracted and C-p7 viruses (wild type and 

S282T mutant), and ribosomal RNA were amplified. 

RESULTS: The C-p7 S282T point mutation in the 

coding sequence of NS5B conferred marked resistance 

to six of the seven 2’-C-methyl nucleosides evaluated. 

This mutation conferred a high level of resistance 

(10-25-fold increase in EC 90 

) to four compounds 

including 2’-C-MeC, NM107 and other 2’-methylated 

nucleosides. In contrast, 2’-F-C-MeC, PSI-6130 


showed only a 1.4-fold increase at the EC 90 

level. All 

compounds, except 2’-C-MeA (CC 50 

= 10 µM), showed 

no cytotoxicity against the C-p7 S282T mutant. As 

expected, IFN showed activity against the C-p7 S282T 

mutant, and RBV at a non-toxic concentration (< 30 

µM) showed minimal activity (EC 90 

= 23 µM). 

CONCLUSIONS: Cell-based assays with the full-length 

infectious C-p7 viruses have provided powerful and 

robust tools for developing new anti-HCV agents that 

are not cross-resistant. These results suggest that 

the genotype 2a S282T variant of full-length NS5B 

represents effective model for resistance profiling of 

NS5B polymerase inhibitors. 

Abstract 82 

Understanding the Molecular 

Basis of HBV Drug Resistance by 

Molecular Modeling 

A Sharon and CK Chu 

The University of Georgia College of Pharmacy, Athens, 

GA 30602, USA 

BACKGROUND: Despite the significant successes in 

the treatment of chronic hepatitis B, infection, the 

development of resistance against available therapy 

is a critical issue. A recent studies show the clinical 

frequency of 3TC-resistant mutants is 7.9% for 

L180M, 28.6% for M204I, 22.2% for L180M/M204I 

and 41.3% for L180M/M204V. The aim of present 

investigation is to understand the molecular basis 

of drug resistance conferred by the B and C domain 

mutations on the binding mode of five clinically 

effective anti-HBV agents [lamivudine (3TC), 

adefovir (ADV), entecavir (ETV), telbivudine (LdT), 

and clevudine (L-FMAU)]. 

METHODS: Absence of the crystal structure of 

HBV RT prompted us to use the homology modeled 

structure of HBV RT to gain insight of the drug 

resistance mechanism. Minimization, conformational 

search and induced fit docking followed by binding 

energy calculation on wild type as well as on 

mutant-HBV polymerase (L180M, M204V, M204I, 

L180M+M204V, L180M+M204I) were performed. 

RESULTS: A significant correlation has been observed 

between the fold resistance (FR: based on IC 50 

values) 

and the binding affinity of the nucleoside analogs. 

Binding mode studies reveals that the domain C 

residue M204 is closely associated with sugar/ 

pseudosugar ring positioning in the active site. The 

positioning of oxathiolane ring of 3TC is plausible 

due the induced fit orientation of the M204 residue 

in wild type, and further mutation of M204 to V204 

or I204 reduces the final binding affinity, which leads 

to the drug resistance. The domain B residue L180 is 

not directly interact with the nucleoside analogs, but 

indirectly associated with other hydrophobic residues 

such as Ala87, Phe88, P177 and M204. These five 

hydrophobic residues can directly affect the incoming 

nucleoside analog in terms of its association and 

interaction that can alter the final binding affinity 

of the molecule. There was no sugar ring shifting 

observed in the case of adefovir and entecavir, while 

exocyclic double bond of entecavir oriented to the 

backside small hydrophobic pocket (made by residues 

A87, F88, P177, L180 and M204) and enhances the 

binding affinity. LdT and L-FMAU showed backward 

shifting along with upward movement of sugar ring 

without enforcing M204 residue for induced fit 

movement which makes these molecules competitive 

inhibitor of HBV-RT, without being incorporated into 

the growing HBV-DNA chain. 

CONCLUSION: Modeling results reveal the additive 

nature of the dual mutation (L180M + M204V/I) 

causing L-nucleosides reduced binding in comparison 

to the single mutation. Structural information 

obtained from our molecular modeling studies of 

nucleosides aids to gain insight of the molecular basis 

of drug resistance of anti-HBV agents, which may 

be utilized for the future discovery of new anti-HBV 

agents (Supported by NIH AI25899). 


Abstract 83 

High Genetic Barrier to HCV 

Resistance Presented by PSI-6130 

A Uzgiris 1 , H Micolochick Steuer 2 , E Murakami 2 , H Bao 2 , 

S Cruz 1 , AG Shields 1 , S Ali 3 , WR Jiang 3 , J Symons 3 , 

PA Furman 2 , and A De La Rosa 2 

1 Siemens Medical Solutions Diagnostics, Berkeley, CA, 

USA; 2 Pharmasset Inc, Princeton, NJ, USA; 3Roche Palo 

Alto LLC, Palo Alto, CA, USA 

BACKGROUND: PSI-6130 (β-D-2′-deoxy-2′-fluoro- 

2′-C-methylcytidine) is the parent molecule of the 

prodrug R7128, an orally active inhibitor of the 

hepatitis C virus NS5B polymerase currently in clinical 

development. To date, there has been no report of 

the emergence of viral resistance to R7128 in clinical 

studies. However, reduced activity was seen with 

PSI-6130 when tested against a replicon containing 

the S282T mutation in NS5B. We have analyzed a 

proprietary database of more than 2,800 HCV NS5B 

sequences representing six HCV genotypes for the 

presence of the S282T substitution. 

METHODS: More than 2,800 HCV NS5b sequences 

from public databases and from databases at Siemens 

were combined for analysis. HCV-positive clinical 

sample data were generated with the Siemens 

TRUGENE® HCV 5′NC Genotyping Kit* and the HCV 

NS5b Assay offered through the Siemens Medical 

Solutions Diagnostics Clinical Laboratory. Sequences 

from the Los Alamos HCV database were analyzed for 

redundancy and degenerate bases. Unique sequences 

without ambiguities were selected and subtypes 

were confirmed by phylogenetic analysis. Sequential 

passaging experiments were performed with Huh7 

cells harboring the GT-1b wild-type replicon. Enzyme 

inhibition studies were performed with wild-type and 

mutant HCV RdRp and the two active triphosphate 

metabolites of PSI-6130: PSI-6130-TP and the 

triphosphate of the uridine metabolite RO2433. 

substitution at position 282 was observed in less than 

1% of the sequences analyzed. The two substitutions 

observed were the S282T (ACC and ACT) and S282R 

(AGG and CGA). The S282T substitution results 

in only a 3.8-fold reduced sensitivity to PSI-6130. 

In vitro studies showed that HCV replicons containing 

the S282T mutation were selected by long-term 

passaging (6–8 months) in the presence of PSI-6130. 

The S282T substitution reduced the replication 

capacity to 15-20% of the wild-type replicon. PSI- 

6130-TP was a more potent inhibitor of the wild-type 

and S282T mutant polymerases than was RO2433- 

TP. 

CONCLUSIONS: The S282T mutation that confers 

reduced sensitivity to PSI-6130 results in a small 

loss in antiviral activity and the substitution is not 

highly polymorphic in the wild-type HCV population. 

These in vitro data indicate that there is a high genetic 

barrier to resistance to PSI-6130. Since PSI-6130 is 

metabolized to two active triphosphate forms, PSI- 

6130-TP and RO2433-TP, it is enticing to speculate 

that the two structurally different active metabolites 

would further increase the genetic barrier of PSI- 

6130. 

*For research use only. Not for use in diagnostic 

procedures. 

RESULTS: More than 99% of the sequences analyzed 

were wild type at position 282 (AGC and AGT). A 


Abstract 84 

Determination of Lamivudine, 

Adefovir and Entecavir Resistance 

in Acute and Chronic Hepatitis B 

Virus Infections 

C Eroglu 1 , H Leblebicioglu 1 , and Members of Hepatitis 

Study Group 2 

1 Department of Infectious Diseases and Clinical 

Microbiology, Ondokuz Mayis University Medical School, 

Samsun, Turkey; 2 Hepatitis Study Group: Turkyilmaz 

R, Tutuncu E (Ankara), Sirmatel F (Gaziantep), Koksal 

I, Caylan R (Trabzon), Ozgenc O, Inan N (Izmir), Ayaz C 

(Diyarbakir), Aribas E (Konya), Sunbul M, Esen S, Bayirli 

D (Samsun), Aygen B, Yildiz O (Kayseri), Usluer G, Kartal 

ED (Eskisehir), Parlak M, Parlak E (Erzurum), Irmak H, 

Evirgen O (Van), Tulek N, Yetkin MA (Ankara), Ozsoy MF, 

Oncul O (Istanbul), Akbulut A, Cihangiroglu M (Elazig), 

Dokmetas I, Bakir M (Sivas), Ersoz G, Kaya A (Mersin), 

Bektas A (Samsun), Sencan I (Duzce), Akcam Z, Yayli G 

(Isparta), Saltoglu N, Tasova Y (Adana), Yamazhan T, 

Ulusoy S (Izmir), Ersoy Y (Malatya), Kilic D, Kaygusuz S 

(Kirikkale) Turkey 

The most widely used antivirals for treatment of 

hepatitis B virus (HBV) infection is lamivudine and 

adefovir. Reduced virologic response to therapy with 

nucleos(t)ide analogues can occur due to the emergence 

of mutation. Mutations conferring resistance to 

lamivudine, adefovir, and entecavir occur particularly 

in the polymerase gene. Importance of antiviral 

resistance to nucleos(t)ide analogues in the naïve 

HBV infected patients is not known. In this study, the 

reverse transcriptase (rt) region of polymerase gene 

has been sequenced to detect antiviral resistance. 

263 serum samples of patients with acute (n:158) and 

chronic (n:165) HBV infection have been investigated 

to detect antiviral resistance to lamivudine, adefovir, 

and entecavir. Mutations at the codons rtM204, 

rtL180, and rtV173 for lamivudine resistance, codons 

rtN236, rtI233, and rtA181 for adefovir resistance, 

and codons rtS202, rtT184, and rtI169 for entecavir 

resistance have been examined. 

Sequences have been obtained in 223 samples (118 

samples from acute and 105 samples from chronic 

HBV infection patients. Resistance for lamivudine in 

codon V173L has been revealed only in one case. No 

resistance to lamivudine at codons 180 and 204, or to 

adefovir at codons 181, 233, and 236, or to entecavir 

at codon 169, 184, and 202 have been detected. 

This study demonstrated the presence of mostly wild 

type strains in patients with acute and chronic HBV 

infection. It has been concluded that lamivudine, 

adefovir, and entecavir resistance is not an important 

problem in acute and chronic naïve HBV infected 

patients. 

Abstract 85 

Databases for Antiviral Resistance 

Monitoring in Hepatitis B 

L Yuen and S Locarnini 

VIDRL, North Melbourne and Evivar Medical Pty Ltd, 

East Melbourne, Victoria, Australia 

Mutations associated with antiviral drug resistance 

to the nucleos(t)ide analogues (NA) lamivudine 

(LMV), adefovir (ADV), telbivudine(LdT), tenofovir 

(TFV) and entecavir (ETV) have been identified for 

hepatitis B virus (HBV) using the SeqHepB system. 

SeqHepB is composed of a HBV genome sequence 

analysis program and a relational database (Yuen, 

L. et al 2007. AVR;75:64) which can then be used to 

correlate large numbers of patient clinical, routine 

pathology diagnostic data, viral mutational sequence 

information, and in vitro antiviral sensitivity and 

cross-resistance phenotypic data in an integrated and 

structured way for subsequent patient monitoring. 

The SeqHepB database currently contains routine 

pathology and specialised virology data for 2,004 

patients. Associated with these patients, there are 

340 clinical histories, 1,863 treatment histories, 

189 biopsy results, and 24,223 specimen records. In 

terms of routine pathology tests performed on the 


samples, there are 26,029 records in the database, 

and these include HBV, hepatitis C virus (HCV), and 

hepatitis D virus (HDV) related pathology test results, 

as well as routine liver function and haematology 

test results. Samples within the database are also 

associated with 4,139 HBV genomic sequence 

information corresponding to 129,405 nucleotide or 

amino acid variation data points. The mutation data 

is correlated to an extensive data set of in-house as 

well as published in vitro phenotypic data on HBV 

antiviral drug sensitivity and resistance. 

The correlation of clinical, pathological and viral 

molecular biological data using different artificial 

intelligence techniques is facilitating the analysis 

of the pathogenesis and natural history of chronic 

hepatitis B in the era of antiviral drug resistance. 

The initial correlation of these multi-disciplinary 

data has identified novel mutations associated with 

antiviral drug resistance and cross-resistance to LMV, 

ADV, LdT and ETV. A linkage that exists between a 

3-dimensional (3-D) structure viewing program and 

the database enables further analysis of potentially 

relevant mutations within the 3D model of the 

polymerase. 

The overlap of the envelope gene with the HBV 

polymerase means that antiviral drug resistant 

HBV may have an altered envelope and that this 

may have public health implications. Studies 

have shown that lamivudine resistant HBV 

(rtV173L+rtL180M+rtM204V) has a significantly 

reduced anti-HBs binding due to important changes 

in the overlapping hepatitis B surface antigen 

(sE164D+sI195M). 

Chronic hepatitis B is a disease with a complex natural 

history, and this complexity increases with the use of 

antiviral agents. The SeqHepB system is an important 

tool that will enable the physician to individualize 

patient management, to cope with the explosion 

of antiviral associated HBV mutations, and should 

prove to be a useful therapeutic guide in the clinical 

setting as new antiviral agents and combination 

thereof are trialled and implemented (Shaw, T. et al 

2006. J.Hep;44:593). 

Abstract 86 

Combination Chemotherapy 

with Nucleos(t)ide Analogue 

Reverse Transcriptase Inhibitors 

(NRTI) May Suppress Multidrugresistant 

HBV: Using In Vitro 

Assays to Identify Optimal NRTI 

Combinations and Doses 

T Shaw, T Sozzi, and S Locarnini 

VIDRL, North Melbourne, Victoria, Australia 

BACKGROUND: Prolonged treatment of chronic 

hepatitis B (CHB) nucleos(t)ide analogue reverse 

transcriptase inhibitors (NRTI) almost invariably 

engenders viral resistance and sequential NRTI 

monotherapy can promote multi-drug resistance. 

Despite the increasing incidence of drug-resistant 

HBV (which can cause life-threatening complications) 

and previous success with combination therapy 

for other chronic viral infections such as HIV 

infection, sequential monotherapy remains the 

favored treatment for CHB. NRTI that have different 

structures and which select for different spectra of 

mutations are now available, suggesting that decreases 

in the incidence of resistance is possible by design of 

combination therapy optimised to individual patients 

and HBV phenotypes. 

We decided to test these postulates using a wild-type 

HBV and a clone generated to mimic a drug resistant 

clinical isolate from a patient with CHB who responded 

poorly to sequential treatments with adefovir (ADV), 

lamivudine (LMV) and entecavir (ETV). The drug 

resistant isolate encoded multiple substitutions in 

the polymerase region including rtA181T, rtI233V, 

rtN236T and rtM250L. (See accompanying abstract 

by Ayres et al.: Selection of mutlidrug resistant 

hepatitis B virus following sequential monotherapy 

and combination therapy.) As well as exhibiting 

a severe replication deficiency relative to WT, it 

showed high level resistance to L-nucleosides and 


deoxyguanosine analogues and lower level resistance 

to adefovir and tenofovir. (See accompanying abstract 

by Sozzi et al.: Relative replication fitness and antiviral 

cross-resistance phenotypes of multidrug-resistant 

hepatitis b virus mutants implicated in non-response 

to sequential treatment with adefovir, lamivudine 

and entecavir.) 

AIMS: To compare the antiviral effects of ETV, 

ADV and tenofovir TFV alone and in combination 

replication of WT HBV and the multi-drug resistant 

rtA181T/I233V/N236T/ M250L mutant, as a basis for 

developing optimal antiviral combination therapies. 

METHODS: The inhibitory effects of ETV, ADV and 

TFV alone and in combination on in vitro replication 

of WT HBV and its multidrug resistant mutant 

derivative were compared after transfection of Huh-7 

cells. 

RESULTS: Based on EC 50 

ratios, the multi-drug 

resistant mutant displayed approximately 5, 10 and 

20-fold increases in resistance to ADV, TFV and ETV 

respectively. In general, paired drug combinations 

were additive or synergistic at low concentrations 

but antagonistic at high concentrations, presumably 

reflecting competition between ADV and TFV 

monophosphates and ETV-diphosphate for the same 

anabolic pathways. 

CONCLUSIONS: An increasing and consistent body 

of data to support the notion that combination 

therapy can decrease antiviral resistance, the 

endpoint likely to be of greatest long-term importance 

in assessing the success of treatment. In vitro 

phenotyping and antiviral assays should be useful 

for development of optimal, individually tailored 

combination therapy. It is likely to be more effective 

using combination therapy for treatment-naïve 

patients, rather than adding or replacing an antiviral 

agent after resistance develops. 

Abstract 87 

Relative Replication Capacity 

and Antiviral Cross-Resistance 

Phenotypes of Multidrugresistant 

Hepatitis B Virus 

Mutants Implicated in Nonresponse 

to Sequential Treatment 

with Adefovir, Lamivudine and 

Entecavir 

T Sozzi 1 , T Shaw 1 , FY Wong 1 , and S Locarnini 1,2 

1 VIDRL, North Melbourne, Victoria, Australia; 2 Evivar 

Medical, East Melbourne, Australia 

BACKGROUND: Prolonged treatment of chronic 

hepatitis B (CHB) nucleos(t)ide analogue reverse 

transcriptase inhibitors (NRTI) almost invariably 

engenders viral resistance. Sequencing of isolates 

from a patient with CHB who responded poorly to 

sequential treatment with adefovir, lamivudine, 

and entecavir revealed multiple substitutions in 

the polymerase region including rtA181T, rtI233V, 

rtN236T and rtM250L. Isolates were genotype D and 

HBeAg negative due to the presence of the precore [pc] 

G1896A mutation. All mutations were present on the 

same genome. (See accompanying abstract by Ayres et 

al.: Selection of mutlidrug resistant hepatitis B virus 

following sequential monotherapy and combination 

therapy.) 

AIMS: To determine antiviral drug resistance 

phenotype of the putative multi-drug resistant 

rtA181T/I233V/N236T/M250L mutant, and to 

determine how individual amino acid substations 

influence HBV replication capacity and antiviral drug 

resistance phenotypes. 

METHODS: Mutant HBV clones encoding rtA181T/ 

N236T/M250L and rtA181T/I233V/N236T/M250L, 

as well as clones encoding the individual amino 

acid substitutions in isolation were generated by 

site-directed mutagenesis from a wild type (WT)

genotype D HBV isolate. WT and derived clones 

harboured the pc mutation, which may increase 

replication efficiency but does not significantly affect 

drug resistance. The phenotypes were determined by 

comparing viral replication in transfected Huh-7 cells. 

Virus replication and its inhibition were monitored 

by measuring changes in the amounts of intracellular 

core-associated viral DNA in cell lysates using a 

commercial assay kit (Seimans Versant HBV 3.0). 

RESULTS: The putative multi-drug resistant mutant 

rtA181T/I233V/N236T/M250L showed high level 

resistance to all four L-nucleosides tested (lamivudine, 

telbivudine, clevudine and emtricitabine), as well as 

high level resistance to the deoxyguanosine analogues 

entecavir, diaminopurine dioxolane, 2’,3’-dideoxy- 

3’-fluoroguanosine and abacavir. It also displayed 

lower level, but still significant, resistance to adefovir 

and tenofovir and a severe replication deficiency 

relative to WT. Except for rtA181T and rtN236T, 

none of the individual amino acid substitutions in 

isolation conferred significant drug resistance and 

all significantly reduced replication capacity in the 

absence of antiviral selection pressure. Their role(s) 

in antiviral resistance deserves further investigation: 

under antiviral selection pressure, they presumably 

compensate for the replication defects associated 

with acquisition of drug resistance and/or confer 

some other advantage for survival. 

CONCLUSIONS: Sequential treatment with NRTI 

promotes multi-drug resistance. Development of more 

rational and efficacious anti-HBV therapy will require 

routine genotyping and phenotypes of individual 

clinical isolates. It is likely that development of multidrug 

resistance could be reduced by combination 

antiviral therapy tailored to suit individual cases. 


Co-infection with HIV 

and Other Viruses 


Abstract 88 

Treatment of HBV/HIV 

Co-infections 

MG Peters 

University of California, San Francisco, CA, USA 

Hepatitis B virus (HBV) infects an estimated 400 

million people worldwide, with 1 million deaths 

occurring annually. Transmission occurs perinatally 

and in childhood in countries of high endemicity 

but predominantly via injection drug use and sexual 

activity in the United States and other countries 

where HBV endemicity is relatively low. Given the 

shared modes of transmission, co-infection with HIV 

is common: 5%-10% of HIV-infected patients in the 

United States are co-infected with HBV. Infection with 

HDV in the US occurs in IDU. Goals of HBV therapy 

include sustained suppression of HBV replication; 

improvement in clinical liver disease; prevention of 

cirrhosis and subsequent liver failure; and prevention 

of hepatocellular carcinoma. Clearance of virus with 

eradication of cccDNA is not yet achievable. Clinical 

endpoints associated with successful therapy include 

loss of HBV DNA, loss of HBeAg and HBsAg, HBeAb 

and HBsAb seroconversion, with improvement in 

serum aminotransferases and liver histology. 

The optimal timing of HBV therapy initiation 

has been changing in patients with HBV/HIV coinfection 

due to the limited choices of HBV agents 

that are active against YMDD mutant virus and lack 

HIV activity. Treatment is indicated in those with 

elevated HBV viral load, ALT and the presence of 

fibrosis on liver biopsy. In HIV co-infected subjects, 

treatment depends on the need for HIV therapy. In 

those requiring HIV treatment, dual agents against 

HBV should be also included in the drug regimen. 

If HBV requires treatment, then consideration of 

early initiation of HIV should be entertained. If 

HIV therapy is not warranted, then options are very 

limited unless the patient is a candidate for interferon 

therapy. Adefovir may be considered, but presents 

a theoretical risk of development of HIV resistance 

to tenofovir. Telbivudine has not been studied in 

HBV HIV co-infected patients not receiving HAART. 

The following agents should be avoided without 

HAART: FTC, 3TC, tenofovir and entecavir as HIV 

resistance may emerge. Discontinuation of HIV/HBV 

therapy may lead to a flare in HBV with resultant 

liver damage. Initiation of HAART may be associated 

with flares due to immune reconstitution. Therefore 

when changing antiretroviral therapy or stopping 

HBV-active antiretrovirals liver function should be 

carefully monitored. 

Abstract 89 

Treatment of HCV in HIV-infected 

Individuals 

Z Temesgen 

Mayo Clinic, USA 

Worldwide, an estimated 40-45 million people are 

living with HIV/AIDS and approximately 170 million 

people are living with hepatitis C virus (HCV) infection. 

It is estimated that 25% to 30% of all HIV-infected 

patients also have HCV infection. Subsequent to the 

significant decrease in HIV-related morbidity and 

mortality due to highly active antiretroviral therapy 

(HAART), HCV-related liver disease has become a 

leading cause of illness and death in HIV-infected 

patients. While the impact of HCV on HIV is not well 

defined, HIV affects the natural history of HCV in a 

number of ways. HIV-HCV co-infected patients have 

higher HCV RNA levels; Progression to cirrhosis is 

faster in patients co-infected with HCV and HIV than 

those with HCV alone; Chronic HCV is a risk factor 

for drug-induced liver injury. Thus the treatment of 

HCV in patients co-infected with HIV has become 

important. 

Pegylated interferon plus ribavirin have been shown 

to be significantly more effective than standard 

interferon plus ribavirin. Sustained virologic 

response (SVR), defined as an undetectable HCV 

RNA level 6 months after the end of treatment, is 


the primary goal of therapy. The secondary goals of 

therapy are reducing fibrosis and necroinflammation. 

Virologic response rates are in general lower in coinfected 

patients compares to responses observed 

in patients infected with HCV alone; only 27-40% 

achieving an SVR compared to more than 50% SVR 

in monoinfected patients. These response rates could 

be improved by employing weight-based ribavirin 

dosing, using growth factors to avoid dose reductions 

of ribavirin or peginterferon, and prolonging the 

duration of therapy. Additionally, early virologic 

response (EVR) has utility in identifying those with 

a reasonable chance of sustained response and thus 

avoiding unnecessary toxicity in those that do not 

have such chances. The absence of a 2-log decline in the 

level of HCV RNA after 12 weeks of therapy (no EVR) 

has been shown to be a potent negative predictor of 

treatment response. Current guidelines incorporate 

these data and recommend treatment with pegylated 

interferon plus weight-based RBV for 48 weeks 

provided there has been an early virologic response 

at week 12. The optimal duration of treatment in 

general as well as the issue of different durations of 

treatment for different subgroups of HIV and HCV 

co-infected patients remain to be fully defined. 

Abstract 90 

HCV/HIV Co-infections: 

Challenges from the Perspective of 

TAG 

T Swan 

Treatment Action Group (TAG), USA 

In the late 1980s, people with HIV/AIDS and 

treatment activists demanded the opportunity to 

participate as full partners in designing public and 

privately funded clinical trials of treatments for 

HIV and its complications. After years of struggle, 

they became vital participants in all stages of HIV 

drug development, serving on scientific peer-review 

committees, protocol teams, regulatory review 

bodies, domestic and international treatment 

guidelines panels; meeting regularly with sponsors of 

public and private research efforts; and participating 

in clinical trials review and data monitoring 

committees. 

By 1996, scientific innovation, diligent research, 

and increased research funding - due in part to 

activist involvement - yielded the therapeutic breakthrough 

known as highly-active antiretroviral therapy 

(HAART). HAART has dramatically improved the 

prognosis for HIV, saving at least three million years 

of life in the US alone. 

Now, hepatitis C co-infection has emerged as a 

significant threat to health, quality of life, and survival 

of HIV positive people, among whom it is prevalent. 

Globally, four to five million people are HIV/HCV 

co-infected. In the US, approximately 30% of HIVpositive 

people are co-infected, and more than 45% 

in Southern and Eastern Europe. 

End-stage liver disease from hepatitis C is now a 

leading cause of death among HIV-positive people 

in the US and Europe. HIV accelerates hepatitis 

C progression, doubles the risk for cirrhosis, and 

shortens survival after hepatic decompensation. 

The current standard of care for HCV fails to eradicate 

the virus in the majority of co-infected people. Side 

effects are debilitating, sometimes life-threatening. 

Many are reluctant to undergo treatment, or cannot 

complete it. 

AIDS treatment activists have sharpened their focus 

on the urgent need for efficient hepatitis C research 

and drug development. The spectacular successes, 

and challenges in HIV drug development and clinical 

care offer important lessons for HCV. 

Effective combination therapy, and adherence 

support programs will prevent emergence of drug 

resistance. Sponsors should collaborate to support 

multi-experimental agent trials, as has been done 

recently in HIV research. 

New HCV therapies need to be studied in people 

with urgent unmet medical need: liver transplant 


candidates and recipients, HIV/HCV co-infected 

people, and nonresponders. Drug-drug interaction 

studies should be carried out early, to facilitate these 

trials, and to guide real-world use. 

Sponsors should provide pre-approval access to 

experimental agents in people who need them most. 

Expanded access is the right thing to do; it gives 

people without options access to potentially lifesaving 

treatment; broadens the pool of physicians 

with experience using new treatments; and gathers 

safety data. 

Registration trials for novel HCV therapies should help 

to define optimal treatment strategies, side effects 

management, and best practices for delivering care 

and treatment in addition to shortening treatment 

duration, and increasing efficacy to increase HCV 

treatment access, uptake, completion, and success 

rates. 

Abstract 91 

Tolerability of Darunavir/ 

Ritonavir Versus Lopinavir/ 

Ritonavir in Lopinavir/Ritonavirnaïve, 

Treatment-experienced, 

Hepatitis B or C Co-infected 

Patients in TITAN 

D Jayaweera 1 , E De Paepe 2 , JM Mrus 3 , YK Dayaram 3 , 

and F Tomaka 4 

1 University of Miami, Miami, FL, USA; 2 Tibotec BVBA, 

Mechelen, Belgium; 3 Tibotec Therapeutics, Bridgewater, 

NJ, USA; 4 Tibotec Inc., Yardley, PA, USA 

BACKGROUND: The randomized, controlled, phase 

III TITAN trial in HIV-1-infected lopinavir/ritonavir 

(LPV/r)-naïve, treatment-experienced patients 

showed that darunavir (DRV) with low-dose ritonavir 

(DRV/r) was generally well tolerated. This analysis 

compared the safety and tolerability of DRV/r versus 

LPV/r in patients with hepatitis B or C (HBV/HCV) 

co-infection. 

METHODS: Patients with HIV-1 RNA >1,000 copies/ 

mL were on stable HAART for ≥12 weeks or offtreatment 

for ≥4 weeks. Patients received DRV/r 

600/100mg bid or LPV/r 400/100mg bid, plus 2–3 

optimized NRTIs/NNRTIs. Patients with chronic 

HBV/HCV co-infection were eligible if their condition 

was stable and they would not require treatment for 

their hepatitis; those with significantly decreased 

hepatic function or hepatic decompensation were 

excluded. 

RESULTS: Of the 298 and 297 patients randomized 

to DRV/r and LPV/r (overall baseline median CD4 

count = 232 cells/mm 3 ), 52 (18%) and 37 (13%), 

respectively, had HBV/HCV co-infection at baseline. 

The most common Grade 3-4 liver-related laboratory 

abnormalities in co-infected patients were increased 

alanine aminotransferase (ALT; 12% DRV/r versus 

25% LPV/r) and increased aspartate aminotransferase 

(AST; 10% DRV/r versus 22% LPV/r). The overall 

incidence of liver-related adverse events was higher in 

co-infected patients (25% DRV/r versus 24% LPV/r) 

than in non-co-infected patients (4% DRV/r versus 

4% LPV/r). The most commonly observed (>5% in 

co-infected patients) liver-related adverse events 

were increased gamma glutamyltransferase, ALT, 

and AST. Although clinical jaundice was reported in 

6% DRV/r versus 0% LPV/r, the overall incidence of 

hyperbilirubinemia in co-infected patients was similar 

in both arms. All of these liver-related adverse events 

were reported in

Abstract 92 

The Effect of Lopinavir/Ritonavir 

on HIV/HCV Co-Infected 

Individuals 

CA Smith 1 , P Greiger-Zanlungo 2 , K Crain 3 , B Morgan 4 , 

R Stubbs 5 , and R Rode 5 

1 Mount Morris Medical, New York, NY, USA; 2 Mount 

Vernon Hospital, New York, NY, USA; 3 Penn State, PA, 

USA; 4 Morgan State, Baltimore, MD, USA; 5 Abbott 

Laboratories, Chicago, IL, USA 

Background: Over the past several years the 

Black and Hispanic communities have seen an 

increase in the prevalence and incidence of Human 

Immunodeficiency Virus (HIV) and Hepatitis C Virus 

(HCV) and the common side effect of liver toxicity of 

antiretroviral therapy. Overall, liver toxicity leads to 

treatment discontinuation in an estimated 10-15% 

of patients who initiate HAART in the United States. 

Our study reviewed use of antiretroviral therapy in 

HIV/HCV co-infected individuals and the incidence 

of severe liver toxicity after initiation of LPV/RIT 

and other highly active antiretroviral medications. 

Methods: A Retrospective chart review of HIV/ 

HCV co-infected patients on Lopinavir/Ritonavir was 

conducted over a 12 - 18 month period of time in 2 

inner city hospitals. All patients were 18 years and 

older and tested positive for HIV-1 and Hepatitis C 

viruses. The following demographic information was 

obtained: age, ethnicity, gender, and risk factor. The 

following was also obtained: LFT’s, CD4, HIV & HCV 

viral load, lipids, and antiretroviral usage. 

Results: Over 120 charts were reviewed but 76 

charts were analyzed for this study. The mean age 

was 44.1 with a range from 40 to 48. 12% (9) of had 

history of DM; 93% (70) had no history of Alcohol 

use; 48% (36) were smokers. 67% (50) were Black; 

16% (12) were Hispanic; 13% (10) were Caucasian 

and 4% (3) were other. 23% were HAV positive; 

31% positive for HBV (20% antigen positive). The 

following HCV genotypes were represented: 34% 1A; 

24% 1B; 28% of charts did not have HCV genotype 

documented. Only 6% of patients had ever been 

treated for HCV. 45% of patients were ARV naïve and 

24% were ARV experienced. 44% of patients were 

on taking Lopinavir/Ritonavir with the predominant 

ART backbone being Zidovudine +Lamivudine (29%), 

tenofovir + Emtricitabine (12%), and Abacavir + 

Zidovudine + Lamivudine. 84% of patients had a 

undetectable HIV viral load 6 months after initiation 

and less than 10% of patients had ALT and AST 

elevations. No patients discontinued LPV/RIT due to 

liver toxicity. 

Conclusion: Lopinavir/Ritonavir was the most 

frequently used protease inhibitor in this inner city 

setting of HIV/HCV co-infected individuals. Lopinavir/ 

Ritonavir is an effective option for the treatment of 

co-infected HIV/HCV infected individuals when used 

in combination with other antiretroviral agents. 

Abstract 93 

Pharmacokinetics of TMC125 

in HIV-negative Volunteers 

with Mild or Moderate Hepatic 

Impairment 

TN Kakuda 1 , M Schöller-Gyüre 2 , F Tomaka 1 , 

G De Smedt 2 , B Woodfall 2 , C Berckmans 2 , M Peeters 2 , 

and RMW Hoetelmans 2 

1 Tibotec Inc, Yardley, PA, USA; 2 Tibotec BVBA, 

Mechelen, Belgium 

BACKGROUND: TMC125 (etravirine; ETR) is a next 

generation NNRTI with potent activity against both 

wild-type HIV and viruses resistant to currently 

approved NNRTIs. TMC125 is mainly eliminated via 

the hepatobiliary route. This study aimed to assess 

the pharmacokinetics of TMC125 in HIV-negative 

volunteers with hepatic impairment compared to 

healthy controls. 

METHODS: An open-label trial was conducted. 

Volunteers with mild or moderate hepatic impairment 


(Child-Pugh A or B, respectively) and healthy 

controls matched for age, gender, race, and BMI were 

included. All volunteers received TMC125 200 mg bid 

for 7 days with a morning dose on Day 8. TMC125 

pharmacokinetics over 12 hours on Days 1 and 8 were 

determined using non-compartmental methods and 

analyzed by a linear mixed effects model. Safety and 

tolerability were assessed. 

RESULTS: TMC125 pharmacokinetics in volunteers 

with mild (n=8, median age 57 years, 5 males) and 

moderate (n=8, median age 54 years, 6 males) hepatic 

impairment were comparable to matched healthy 

controls (n=16). In volunteers with mild hepatic 

impairment, relative to controls, the least squares 

means (LSM) ratios (90% confidence intervals [CI]) 

for TMC125 exposure (AUC 12h 

) and maximum (C max 

) 

plasma concentration on Day 1 were 0.99 (0.75–1.29) 

and 0.92 (0.69–1.21), respectively. On Day 8, AUC 12h 

, 

C max 

, and minimum plasma concentration (C min 

) were 

0.87 (0.69–1.09), 0.79 (0.63–1.00), and 0.87 (0.65– 

1.17), respectively, relative to controls. For volunteers 

with moderate hepatic impairment, relative to 

controls, LSM ratios and 90% CI for AUC 12h 

and C max 

on Day 1 were 0.77 (0.55–1.08) and 0.63 (0.47–0.85), 

respectively. On Day 8, AUC 12h 

, C max 

, and C min 

were 

0.82 (0.60–1.11), 0.72 (0.54–0.96), and 0.98 (0.68– 

1.42), respectively, relative to controls. All treatment 

emergent adverse events were mild or moderate (Grade 

1 and 2) in severity. The most common adverse events 

for volunteers with mild hepatic impairment were 

fatigue and nausea (each occurring in 2 volunteers 

with hepatic impairment and 1 healthy volunteer). 

Dizziness and muscle spasms were the most common 

adverse events for volunteers with moderate liver 

impairment (each occurring in 2 volunteers with 

hepatic impairment and none in healthy volunteers). 

Headache was reported in 4 healthy volunteers, but 

not in volunteers with liver impairment. No rash was 

reported. There were no clinically significant changes 

in laboratory (including hepatic) or cardiovascular 

safety parameters. 

CONCLUSIONS: The systemic exposure to TMC125 in 

volunteers with hepatic impairment was comparable 

to that in healthy subjects. TMC125 was generally safe 

and well tolerated. No dose adjustments of TMC125 

are necessary in patients with mild or moderate 

hepatic impairment. 

Abstract 94 

Tolerability of Once-daily 

Darunavir/r versus Lopinavir/r 

in Treatment-naïve, Patients 

Co-infected with Hepatitis B 

and/or C in the ARTEMIS Trial 

R Ortiz 1 , J Fourie 2 , J Andrade-Villanueva 3 , S Dincq 4 , 

C Vanden Abeele 4 , F Tomaka 5 , and L Lavreys 4 

1 Orlando Immunology Center, Orlando, USA; 2 Chronic 

Diseases of Lifestyle Unit, Tygerberg, South Africa; 

3 Hospital Civil de Guadalajara, Guadalajara, Mexico; 

4 Tibotec BVBA, Mechelen, Belgium; 5 Tibotec Inc, 

Yardley, PA, USA 

BACKGROUND: In the randomized, controlled, Phase 

III ARTEMIS trial in HIV-1-infected treatment-naïve 

patients, once-daily darunavir/r (DRV/r 800/100mg) 

was generally well tolerated and had a lower incidence 

of treatment-related, moderate-to-severe diarrhea 

and triglyceride elevations versus lopinavir/r (LPV/r). 

For treatment-experienced patients, including those 

co-infected with hepatitis B and/or C virus (HBV/ 

HCV), DRV/r 600/100 mg bid has been shown to 

be a well-tolerated treatment option. This analysis 

examined the safety and tolerability of once-daily 

DRV/r (800/100mg) and LPV/r (800/200 mg total 

daily dose) in treatment-naïve HBV/HCV co-infected 

ARTEMIS patients. 

METHODS: Patients with HIV-1 RNA ≥5,000 

copies/mL received DRV/r 800/100mg qd or LPV/r 

800/200mg total daily dose, plus a fixed-dose 

combination tablet of tenofovir (300mg qd) and 

emtricitabine (200mg qd). Patients with chronic 

HBV/HCV co-infection were permitted to enter the 

trial if their condition was clinically stable and they 

would not require treatment for their hepatitis. HBV 

was determined by HB surface antigen testing and 

HCV by HCV antibody and PCR testing. 


RESULTS: 343 and 346 patients were randomized to 

DRV/r and LPV/r, respectively; 43 (13%) of DRV/r 

and 48 (14%) of LPV/r patients had HBV/HCV coinfection. 

The overall incidence of liver-related adverse 

events (AEs) was higher in co-infected patients 

(7/43, 16% DRV/r; 18/48, 38% LPV/r) than in nonco-infected 

patients (9/300, 3% DRV/r; 13/298, 4% 

LPV/r). In co-infected patients, the most common 

events (>5%) were liver-function abnormalities 

reported as AEs: increased ALT, increased AST and 

increased transaminases. These AEs were reported 

in ≤1% of non-co-infected patients. There were few 

clinical AEs, with the exception of HCV, which was 

reported in three (6%) LPV/r co-infected patients; all 

other AEs were reported in at most one co-infected 

patient. 

Regarding laboratory abnormalities, the observed 

incidences of increases in ALT and AST were higher 

in patients with HBV/HCV co-infection compared 

with those without co-infection in both treatment 

groups. The incidence of Grade 3 increases in ALT 

or AST was comparable in both co-infected groups. 

Grade 4 increases in ALT or AST were not observed 

in DRV/r patients with co-infection but were seen in 

6/48 (13%) and 4/48 (8%) co-infected LPV/r patients, 

respectively. In the LPV/r group, the overall incidence 

of hyperbilirubinemia was higher in co-infected 

patients (6/48, 13%) versus non-co-infected patients 

(13/298, 4%), while this was low and comparable for 

co-infected and non-co-infected DRV/r patients (1/43 

and 5/300, 2%). 

CONCLUSIONS: Although HBV/HCV co-infected 

patients had a higher incidence of liver-related AEs 

compared with non-co-infected patients, the most 

commonly observed were liver-function abnormalities 

reported as AEs. There were no Grade 4 ALT or AST 

elevations in DRV/r co-infected patients, while these 

occurred in LPV/r patients. These results support 

previous findings and suggest that safety monitoring 

of HIV-1-infected patients with HBV/HCV coinfection 

treated with once-daily DRV/r 800/100mg 

is appropriate. 

Abstract 95 

Pharmacokinetics of Multipledose 

Darunavir in Combination 

with Low-dose Ritonavir in 

Individuals with Impaired Hepatic 

Function 

F Tomaka 1 , RM Hoetelmans 2 , S Spinosa-Guzman 2 , 

E De Paepe 2 , T Stevens 1 , M De Pauw 2 , JM Mrus 3, and 

VJ Sekar 1 

1 Tibotec Inc., Yardley, PA, USA; 2 Tibotec BVBA, 

Mechelen, Belgium; 3 Tibotec Therapeutics, Bridgewater, 

NJ, USA 

BACKGROUND: Darunavir (DRV), an HIV protease 

inhibitor potent against wild-type and protease 

inhibitor-resistant HIV, is co-administered with lowdose 

ritonavir (RTV; DRV/r). This study assessed the 

pharmacokinetics and safety of DRV/r 600/100mg 

bid in HIV-negative volunteers with mild or moderate 

liver impairment compared with matched HIVnegative, 

healthy controls. 

METHODS: Liver impairment was defined according 

to Child-Pugh classification A (mild) and B (moderate). 

Volunteers received DRV/r 600/100mg bid for 6 

days with a morning dose on Day 7. Volunteers also 

received RTV 100mg on the evening of Day 7 and 

in the morning and evening of Days 8 and 9. Full 

pharmacokinetic profiles were obtained up to 72 

hours post-dose for DRV and 12 hours post-dose 

for RTV on Day 7. Safety and tolerability were also 

assessed. 

RESULTS: DRV pharmacokinetics in volunteers with 

mild (n=8) or moderate (n=8) liver impairment were 

comparable to healthy controls (n=8 in each group). 

In those with mild liver impairment, relative to 

controls, the least squares means (LSM) ratios (90% 

confidence intervals [CI]) for DRV exposure (AUC 12h 

), 

maximum (C max 

), and minimum (C min 

) plasma 

concentrations at Day 7 were 0.94 (0.75–1.17), 0.88 

(0.73–1.07), and 0.83 (0.63–1.10), respectively. 


For those with moderate liver impairment, LSM 

ratios and 90% CI for AUC 12h 

, C max 

, and C min 

at Day 

7 were 1.20 (0.90–1.60), 1.22 (0.95–1.56), and 1.27 

(0.87–1.85), respectively, relative to controls. DRV/r 

treatment was generally well tolerated, regardless of 

degree of liver impairment level. The most commonly 

reported adverse events during DRV/r treatment 

in volunteers with mild liver impairment were 

increased alanine aminotransferase, increased 

aspartate aminotransferase, and headache, each 

reported in two volunteers; these were also each 

reported in two healthy volunteers. The most 

commonly reported adverse event during DRV/r 

treatment in volunteers with moderate liver 

impairment was fatigue, reported in 2 volunteers; and 

in none of the healthy volunteers. All adverse events 

were Grade 1–2 in severity except for one Grade 3 

increase in alanine aminotransferase in a subject with 

mild liver impairment. No consistent or clinically 

relevant changes in laboratory parameters were 

observed. No adverse events led to discontinuation 

and no serious adverse events were reported. 

CONCLUSIONS: DRV pharmacokinetics in subjects 

with mild or moderate liver impairment were 

comparable to healthy controls. No serious adverse 

events, or adverse events leading to discontinuation, 

were reported. For patients with mild or moderate liver 

impairment, no dose adjustments are necessary and 

routine clinical monitoring is considered adequate. 

Abstract 96 

TMC125 Safety and Tolerability in 

Treatment-Experienced Hepatitis 

B or C Co-infected Patients in 

DUET-1 and DUET-2 

T Campbell 1 , A Mills 2 , P Morlat 3 , M Schechter 4 , 

G De Smedt 5 , M Peeters 5 , F Tomaka 6 , and B Woodfall 5 

1 University of Colorado Health Sciences Center, Denver, 

USA; 2 Private Practice, Los Angeles, USA; 3 Saint Andre 

Hospital, University Victor Segalen, Bordeaux, France; 

4 Universidade Federal do Rio de Janeiro, Brazil; 5 Tibotec 

BVBA, Mechelen, Belgium; 6 Tibotec Inc, Yardley, PA, USA 

Background: DUET-1 and DUET-2 are ongoing, 

randomized, double-blind, placebo-controlled 

trials of identical design which investigate TMC125 

(etravirine; ETR) in treatment-experienced, HIV-1- 

infected patients. We report safety results by baseline 

hepatitis co-infection status from a planned 24-week 

pooled analysis. 

Methods: Patients with documented (historical 

data and/or viral genotype) NNRTI resistance and 

with ≥ 3 primary PI mutations on stable virologically 

failing treatment were randomized to TMC125 200mg 

or placebo bid, each administered with darunavir/ 

ritonavir, optimized NRTIs +/– enfuvirtide. To be 

eligible, patients co-infected with chronic hepatitis 

B or C (HBV/HCV) had to be clinically stable with 

AST/ALT

serious hepatic AEs and hepatic AEs leading to 

discontinuation among co-infected subjects was 

comparable between the treatment groups. In both 

treatment groups, grade 3/4 AST/ALT elevations 

were more frequent in those who were co-infected; 

differences between TMC125 and placebo were 

small. 

Conclusions: Consistent with the underlying 

chronic hepatitis co-infection, hepatic AEs and 

elevated hepatic parameters were more frequent in 

co-infected patients compared with non-co-infected 

patients. The incidence and severity of hepatic AEs 

with TMC125 were generally similar to placebo, 

irrespective of hepatitis co-infection status. TMC125 

does not appear to increase hepatic toxicity in patients 

with hepatitis co-infection. 


Pharmacology and 

Drug Interactions of HBV 

and HCV Therapeutics 


ABSTRACT 97 

Preclinical Drug Metabolism in 

Viral Hepatitis Drug Discovery 

AS Ray 

Department of Drug Metabolism, Gilead Sciences, Inc., 

California, USA 

Approximately 350 and 170 million people worldwide 

are chronically infected by the hepatitis B and C 

viruses (HBV and HCV), respectively. Identification of 

more effective therapies for these viruses will greatly 

reduce morbidity and mortality due to liver disease. 

Potent inhibitors of the HCV NS3/4A protease and 

nucleoside and non-nucleoside inhibitors of the 

NS5B RNA dependent RNA polymerase (RdRp) have 

been identified in vitro. Use of metabolism assays 

and pharmacokinetic studies in animals will allow for 

some of these compounds to bridge the gap between 

the laboratory and the clinic, eventually redefining 

HCV therapy. Targeting viral replication in the liver 

presents many unique opportunities and challenges. 

The direct flow of portal blood to the liver and the 

expression of multiple influx transporters results in 

the potential for concentration of antiviral agents 

in the liver. However, the liver has a high capacity to 

convert xenobiotics into inactive metabolites through 

oxidation and conjugation and to reduce intrahepatic 

levels through efflux transport. The unique 

requirement for phosphorylation of nucleoside and 

nucleotide inhibitors makes the assessment of liver 

levels of the active nucleoside triphosphate analog 

of paramount importance in predicting efficacy. 

Nucleosides and nucleotides are effective therapies 

for HBV and a number of potent agents have either 

recently been approved by regulatory agencies or 

are in late stages of clinical development. Similarly, 

nucleoside inhibitors of the HCV RdRp promise 

to be an important part of future virally targeted 

combination therapy for HCV. Non-nucleoside and 

protease inhibitor levels in the liver can be more closely 

estimated based on their non-protein bound plasma 

concentrations by assessment of individual rates of 

liver accumulation, catabolism and excretion. Various 

strategies for predicting human pharmacokinetics 

and, ultimately, clinical efficacy will be discussed, 

highlighting examples from data generated with HBV 

and HCV inhibitors. 

Abstract 98 

Frontiers of Clinical Pharmacology 

in Hepatitis Drug Development 

CW Flexner 

Pharmacology and Molecular Sciences, and International 

Health, Johns Hopkins University, Baltimore, MD, USA 

As the number of drugs approved for the treatment 

of HBV and HCV infection continues to grow, 

the pharmacologic consequences of combination 

chemotherapy, including drug interactions, will become 

more complex. Principles of clinical pharmacology 

have informed the rational development of treatment 

regimens for HIV, and many of these principles can be 

applied to hepatitis virus infections. 

There are currently 24 antiretroviral drugs approved 

for use in the United States; three of these – 

lamivudine, emtricitabine, and tenofovir – also have 

activity against HBV. In addition, three drugs approved 

for the treatment of HBV or HCV – interferon-α, 

adefovir, and entecavir – have anti-HIV activity. 

Clearly our lives — as care providers, as patients, 

as citizens concerned about these epidemics — are 

getting more complicated. The relationship between 

drug concentrations, antiviral activity, toxicity, and 

resistance is a complex function within any individual 

patient. The makeup of a multidrug regimen, drug 

tolerance, adherence patterns, and sensitivity of the 

virus to individual drugs in the regimen all contribute 

to treatment success or failure. The availability of 

an increased number of agents makes possible the 

design of multi-drug regimens with activity against 

multiple viruses, and these regimens may be used in 

the clinic before they can be adequately evaluated in 

clinical trials. As a consequence, surprises can -- and 

will -- occur. 


Although there is an increasing interest in the 

influence of individual genetic make-up on 

drug concentrations and drug response (i.e., 

pharmacogenetics), environmental factors such as 

food effects, patient adherence behavior, interactions 

with concomitant prescription drugs, concomitant 

herbal medicines, and concomitant disease can also 

have a profound impact on treatment outcome. 

Pharmacokinetic drug interactions are becoming 

more numerous and more complex. The number of 

potential 3- and 4-drug combinations for HIV treatment 

alone now number in the thousands. It is therefore 

not possible to prospectively study all potential 

drug combinations before they might be used in the 

clinic. This is especially problematic for treatmentexperienced 

patients, whose regimens might be based 

on resistance patterns that necessitate the use of an 

obscure regimen. Several recent examples highlight 

the hazards of using untested regimens. Combination 

studies with standard doses of tenofovir (TDF) and 

didanosine (ddI) resulted in unexpectedly high 

didanosine concentrations, and may have produced 

a greater than expected frequency of didanosine 

toxicities (Robbins et al., 2003). Three studies in 

treatment naïve patients found an unacceptably high 

early failure rate with combinations of tenofovir, 

didanosine, and lamivudine, or tenofovir, abacavir, 

and lamivudine (Jemsek et al., 2004; Landman et 

al., 2004). In addition, a recent report highlights an 

unexpected association between early non-response 

to HCV treatment and simultaneous use of abacavir 

(Bani-Sadr et al., 2007). Ribavirin is an inhibitor of 

inosine 5′-monophosphate dehydrogenase, and is 

antagonistic in vitro with the antiretroviral nucleosides 

didanosine, zidovudine, and didanosine. A clinical 

pharmacological interaction between abacavir and 

ribavirin has been postulated but not proven. 

The mechanisms for these unexpected outcomes are 

not yet completely understood. In the case of the TDF 

and ddI interaction, there appears to be a previously 

unexpected pharmacokinetic interaction between 

these drugs, perhaps involving an inhibition by TDF 

of ddI degradation by the enzyme purine nucleoside 

phosphorylase (Ray et al., 2003). TDF and ddI do not 

interfere with intracellular phosphorylation of the 

other agent when both are applied to human cells 

(Robbins et al., 2003). In the case of the TDF-ddI 

and TDF-abacavir failures, the final answer is not yet 

known. However, these regimens may be associated 

with early emergence of the nucleoside resistance 

mutation K65R (Landman et al., 2004), resulting in 

an unexpectedly high rate of early genetic resistance. 

This may represent a pharmacodynamic, rather than 

a pharmacokinetic interaction. 

Polypharmacy is a growing complication of HIV, HCV, 

and HBV infections. As the number of agents effective 

in the management of these infections continues 

to grow, patients and physicians are expected to 

deal with the increasingly complex potential for 

pharmacokinetic and pharmacodynamic drug 

interactions (Piscitelli and Gallicano, 2001). The HIV 

protease inhibitors and the nonnucleoside reverse 

transcriptase inhibitors are especially susceptible 

to pharmacokinetic drug-drug interactions. The 

potent inhibition of CYP 3A4 by ritonavir produced 

the unexpected finding that ritonavir increased the 

AUC and Cmax of fluticasone, when administered 

as a nasal spray, by approximately 350-fold and 25- 

fold respectively. This significant increase in plasma 

fluticasone resulted in a significant decrease (86%) 

in plasma cortisol. This has produced features of 

Cushing’s syndrome in some patients, and the 

possibility of secondary adrenal insufficiency when 

fluticasone is stopped (Andrade and Flexner, 2000). 

A better understanding of mechanisms of drug 

interaction should lead us in the future to improved 

strategies for managing complex regimens in our 

patients. 

Suggested Reading: 

Andrade A, Flexner C. HIV-related drug metabolism 

and cytochrome P450 enzymes. AIDS Clin Care. 

2000;12:91-95. 

Bani-Sadr F, Denoeud L, Morand P, Lunel-Fabiani F, 

Pol S, Cacoub P, Perronne C, Carrat F; Agence Nationale 

pour la Recherche contre le SIDA et les Hepatites 

Virales HC02-Ribavic Study Team. Early virologic 

failure in HIV-co-infected hepatitis C patients treated 


with the peginterferon-ribavirin combination: does 

abacavir play a role? J Acquir Immune Defic Syndr. 

2007;45:123-5. 

Flexner C. HIV drug development: the next 25 years. 

Nature Reviews Drug Discovery 2007; in press. Online 

version published October 12, 2007. 

Jemsek J, Hutcherson P, Harper E. Poor virologic 

responses and early emergence of resistance in 

treatment naïve, HIV-infected patients receiving a 

once daily regimen of didanosine, lamivudine, and 

tenofovir DF. In Program and Abstracts of the Eleventh 

Conference on Retroviruses and Opportunistic 

Infections. San Francisco, CA, February, 2004. 

Landman R, Peytavin G, Descamps D, et al. Low 

genetic barrier to resistance is a possible cause of 

early virologic failures in once-daily regimen of 

abacavir, lamivudine, and tenofovir: the Tonus Study 

. In Program and Abstracts of the Eleventh Conference 

on Retroviruses and Opportunistic Infections. San 

Francisco, CA, February, 2004. 

Nettles RE, Kieffer TL, Parsons T, Johnson J, 

Cofrancesco J, Gallant JE, , Carson K, Siliciano RF, 

Flexner C. Marked intraindividual variability in 

antiretroviral concentrations may limit the utility of 

therapeutic drug monitoring. Clin Infect Dis 2006; 42: 

1189-1196. 

Piscitelli SC, Gallicano KD. Interactions among drugs 

for HIV and opportunistic infections (review). N Engl 

J Med. 2001;34:984-96. 

Ray AS, Mahmoudi A, Fridland A. Mechanism of 2’, 

3’-dideoxyinosine’s drug interaction with allopurinol, 

ganciclovir, and tenofovir disoproxil fumarate 

(abstract). Antiviral Res. 2003;57:A50. 

Robbins BL, Wilcox CK, Fridland A, Rodman JH. 

Metabolism of tenofovir and didanosine in quiescent 

or stimulated human peripheral blood mononuclear 

cells. Pharmacotherapy 2003 Jun;23(6):695-701. 

Washington CB, Flexner C, Sheiner LB, Rosenkranz 

SL, Segal Y, Aberg JA, Blaschke TF, for the ACTG 378 

Study Team. Effect of simultaneous versus staggered 

dosing on pharmacokinetic interactions of protease 

inhibitors. Clin Pharmacol Ther 2003; 73: 406-416. 

Abstract 99 

Current and Future: HCV 

Maintenance Therapy 

KL Lindsay 

University of Southern California, 1640 Marengo Street, 

Suite 103, Los Angeles, CA 90033 USA 

Background: Three large maintenance peginterferon 

therapy trials were initiated in the late 

1990’s to evaluate the effectiveness of long-term 

low-dose peginterferon alfa treatment of patients 

who had failed to develop a virological response 

to peginterferon + ribavirin combination therapy. 

Patients with clinically compensated liver disease 

and advanced hepatic fibrosis were included, 

and randomized to receive either peginterferon 

monotherapy or no treatment in two studies or 

colchicine in the third study. 

Status of peginterferon maintenance 

trials: Primary study endpoint data from the EPIC-3 

study are anticipated in 2009. A planned interim 

analysis of the COPILOT study in 2005 demonstrated 

a reduction in portal hypertension outcomes in 

patients treated with peginterferon compared to 

those in the colchicine group. Primary outcome data 

is now available from the HALT-C trial, in which 1050 

patients who had not responded to PEG IFN + RBV 

therapy, with liver biopsy Ishak fibrosis scores ≥ 3 

were randomized to receive either peginterferon alfa- 

2a 90 mcg weekly (n=517) or no treatment (n=533) 

for 3.5 years. Clinical outcomes were monitored, and 

liver biopsies were repeated 1.5 and 3.5 years after 

randomization. For patients without cirrhosis at 

baseline, a ≥ 2 point increase in the fibrosis score on 

follow-up liver biopsies defined a histologic outcome. 


At the end of the study the frequency of outcomes 

in the treated group and the control group was not 

different (34.1% vs. 33.8%, hazard ratio = 1.01; 95% 

CI = 0.84-1.26; p=0.91). However, mean serum ALT 

and HCV RNA levels decreased significantly with 

treatment (both p

CONCLUSIONS: An HCV replicon over-expressing 

the MDR1 protein, Pgp, was established as a screen 

for monitoring whether HCV inhibitors are Pgp 

substrates. A positive correlation was demonstrated 

between the more classical caco bidirectional assay 

and the HCV replicon Pgp system. Advantages of 

employing the robust HCV replicon Pgp assay include 

the requirement of lower compound concentrations 

as well as providing a direct measure of the effect 

of Pgp efflux on the potency of any HCV replication 

inhibitor. 

Abstract 101 

Pharmacologic Evaluation of 

Novel Small Molecule HCV 

Inhibitors Affecting NS5Adependent 

Functions 

CR Wobbe, NJ Cao, H-J Cho, R Fathi, A Lam, G Li, 

Y Liao, K Nawoschik, A Sandrasagra, G Westby, R Wu, 

Z Yang, and Q Zhu 

XTL Biopharmaceuticals, Valley Cottage, NY, USA 

Background: HCV NS5A is considered to be a 

promising target for antiviral intervention, but lack 

of a functional biochemical assay has limited efforts 

to discover compounds that target this essential viral 

protein. To date, only one compound targeting NS5A 

has been advanced to clinical trials. By applying a 

chemistry technology known as Diversity Oriented 

Synthesis and phenotypic screening against the 

cell-based HCV replicon, we have identified and 

optimized novel, selective and highly potent small 

molecule inhibitors of HCV replication that appear to 

act on NS5A and have favorable pharmacokinetic and 

pharmacodynamic properties. 

do not affect the growth of a range of other RNA 

and DNA viruses in culture. Transient treatment 

of replicon cells results in a rapid, > 6-log reduction 

in HCV RNA with no rebound following compound 

removal. Biochemical and genetic studies suggest 

that these molecules interact with domain I of NS5A. 

Compounds administered orally to rats at 25 mg/kg 

preferentially accumulate in liver, achieving C max 

of 

1-10 µg/g and C 8hr 

(a surrogate for C min 

) of 0.25 – 5 µg/g, 

hundreds of times above the replicon EC 90 

, and have 

elimination half-lives of 4-11 hr, suggesting that they 

may be suitable for b.i.d. dosing. No adverse clinical, 

hematologic, clinical chemistry or histopathologic 

changes were observed in rats receiving 250 mg/kg/d 

of leads in multiple-dose toxicology studies. Leads are 

not active in the Ames test or in assays for inhibition 

of major CYP-450 enzymes or hERG. Analysis of 

the metabolism of these compounds in microsomes 

and animals indicates that they are very stable, with 

metabolic half-lives in the range of hours, less than 

10% conversion to oxidative metabolites and less 

than 1% conversion to glucouronides. 

Conclusion: These data support the hypothesis 

that NS5A is a pharmacologically tractable target 

for blocking HCV replication and identify a family 

of novel small molecule compounds that target 

NS5A and, as such, could complement protease and 

polymerase inhibitors currently in development for 

treatment of HCV infection. 

Methods and Results: Lead molecules have 

nanomolar EC 50 

s against genotype 1a and 1b 

replicons, are active against replicons that are 

resistant to protease and polymerase inhibitors, are 

not toxic to a wide range of cultured cell lines and 


Optimizing the Outcome of 

Therapeutics for HBV and HCV 


Abstract 102 

Measuring and Mapping 

Partnership Integration for 

Optimizing Assessment and 

Treatment of Hepatitis C 

G Butt 1, 2 , W Hill 1 , L McGuinness 1 , and M Krajden 1, 2 

1 British Columbia Centre for Disease Control, Vancouver, 

British Columbia, Canada; 2 University of British 

Columbia, Vancouver, British Columbia, Canada 

Background: In response to the hepatitis C (HCV) 

epidemic in British Columbia, collaborative projects 

were established in four geographic areas with high 

prevalence rates: Campbell River, Kamloops, Prince 

George and Surrey. Service delivery began with 

initial partnerships between public health nurses 

and local physicians. Nurse leadership broadened 

the partnerships to include other disciplines. The 

resulting model of service delivery provided integrated 

prevention and care services linked through informal 

interprofessional partnerships. A recent evaluation 

of HCV treatment outcomes from the project sites 

demonstrated viral clearance rates comparable to 

published clinical trials. The aim of this study is to 

describe and measure the breadth, depth and extent 

of interprofessional partnerships for each project 

site. 

Methods: Leaders from each project site developed 

a list of their inter- and intra-agency partners with 

whom they had contact within six months. Partners 

were mailed a self-adminstered survey package that 

included the Integration of Human Services Measure © 

which asked them to rate their present and desired 

level of service integration with each member of the 

partner list using a 5-point Likert scale. Descriptive 

statistics were calculated for each partner. The 

geographic location and mean scores for present and 

desired level of integration were displayed on a map 

of BC using GIS mapping techniques. Ethics approvals 

were obtained from revelant ethics review boards. 

Results: Survey response rates ranged from 34% 

to 53%. The number of partners ranged from 29 to 

68. Public health, provincial government agencies 

and specialist physicians had higher mean scores for 

present and future integration. Three sites operating 

for >5 years had the greatest number and diversity 

of partners and the least difference between current 

and desired integration mean ratings (Kamloops 

[difference=0.22], Prince George [difference=0.12] 

Campbell River [difference=0.37] versus Surrey 

[difference=0.45]). The majority of partners were 

located near the local project sites, however, all sites 

had linkages with other project sites, province-wide 

partnerships, and some national partnerships. 

Conclusion: This study demonstrates the diverse 

number and type of resources that are required to 

provide appropriate assessment and treatment for 

HCV. GIS mapping provided an effective mechanism 

to show the broad range of informally-linked 

partnerships. Therapeutic outcomes similar to 

research trials were achieved in partnerships with 

low current mean integration scores that were close 

to their desired state of integration. 

Abstract 103 

Development of a Novel Mouse 

Model to Evaluate Single and 

Combination Therapy Against 

Hepatitis B Virus 

MA Feitelson 1 , MM Clayton 1 , B Sun 1 , and RF Schinazi 2 

1 Department of Biology, College of Science and 

Technology, Temple University, Philadelphia, PA, USA; 

2 Emory University/VA Medical Center, Decatur, GA, USA 

BACKGROUND: Woodchuck hepatitis virus infected 

woodchucks have been used for preclinical 

development of drugs against hepatitis B virus 

(HBV). However, there is no simple in vivo model to 


evaluate small amounts of compounds against wild 

type HBV. In addition, drug resistance is a problem 

in the development and application of new drugs 

against HBV infection. 

METHODS: To develop a model that addresses 

these limitations, HepAD38 cells, in which HBV 

replication is regulated by tetracycline, were grown as 

subcutaneous tumors in nude mice. Mice developing 

viremia were then left untreated, given tetracycline 

in the drinking water, and in some mice, tetracycline 

was replaced with PBS, lamivudine (3TC), clevudine 

(CLV), tenofovir dipivoxil fumarate (TDF), or CLV 

plus TDF combination therapy. Virus DNA titers 

were measured by real-time PCR during and after 

drug treatment. Toxicity was monitored by alanine 

amino transferase (ALT) measurements and by tumor 

weight in treated and control animals. 

RESULTS: In water fed and PBS injected mice, virus 

titers reached to ~10 9 copies/ml serum within 35 days 

of HepAD38 injection, while in tetracycline treated 

mice, virus titers remained at 10 4 -10 5 . HBV DNA levels 

were suppressed by 3TC, TDF and CLV, as expected, 

with the latter two drugs showing more sustained 

virus suppression compared to 3TC. Combination 

therapy with up to 27 fold lower concentrations of 

CLV plus TDF was much more effective than either 

drug alone in suppressing virus titer, which remained 

depressed for at least 3 weeks after the end of 

treatment. There was no demonstrable toxicity to 

HepAD38 cells in drug treated mice. 

CONCLUSIONS: Hence, a robust tetracycline controlled 

system for HBV replication in vivo has been 

demonstrated, validated with single drugs against 

HBV, and shown to be useful in assessing combination 

therapy. This system will be useful for preclinical 

assessment of small amounts of other single or 

multiple compounds against HBV in vivo before 

expensive human trials are initiated. 

Abstract 104 

Association of Pretreatment 

Serum Interferon-γ-inducible 

Protein 10 (Ip-10) Levels with 

Sustained Virological Response 

to Peginterferon Plus Ribavirin 

Therapy for Chronic Hepatitis 

C (CHC) with Virus Genotype 1 

Infection 

M Fukuda 1 , H Yatsuhashi 1 , R Nakao 1 , S Hashimoto 1 , 

A Nishikawa 1 , N Hai 1 , M Tateyama 1 , Y Motoyoshi 1 , 

S Nagaoka 1 , N Taura 1 , S Abiru 1 , K Yano 1 , A Komori 1 , 

K Migita 1 , H Fujioka 1 , H Sakai 2 , E Takezaki 3 , 

H Morimoto 4 , T Muro 5 , Y Hijioka 6 , M Yagura 7 , 

N Masaki 8 , and H Ishibashi 1 

1 National NHO Nagasaki Medical Center, Clinical 

Research Center, Nagasaki, Japan; 2 National NHO 

Beppu Medical Center, Oita, Japan; 3 National NHO 

Higashihiroshima Medical Center, Hiroshima, Japan; 

4 National NHO Kanazawa Medical Center, Ishikawa, 

Japan; 5 National NHO Oita Medical Center, Oita, Japan; 

6 National NHO Osaka-Minami Medical Center, Osaka, 

Japan; 7 National NHO Tokyo Hospital, Tokyo, Japan; 

8 International Medical Center of Japan, Tokyo, Japan 

Background and aim: Interferon-γ inducible 

protein 10 kDa (IP-10 or CXCL10) is a CXC chemokine 

that, unlike other CXC chemokines, lacks chemotactic 

activity for neutrophils, but rather targets 

T lymphocytes, NK cells, and monocytes. Recently, 

baseline levels of IP-10 before the initiation of 

therapeutic intervention for hepatitis C virus (HCV) 

infection were reported to have close relationship to 

a sustained virological response (SVR) in Caucasian 

patients with HCV genotype 1 infection (Lagging M, et 

al, Hepatology 2006). Such molecules which associate 

with IFN response may have a different behavior 

among ethnic groups. We aimed this study to examine 

a possible association between the serum IP-10 level 

and virological response to the peg-interferon plus 

ribavirin (PegIFN/RBV) therapy in Japanese patients 

with CHC infected with genotype 1 HCV. 


Methods and patients: Serum IP-10 levels were 

determined by enzyme linked immunosorbent assay. 

Serum IP-10 levels were measured in 40 healthy 

persons and 402 patients (202 men (50.2%) and 200 

women (49.8%); mean age 57.3 years old (SD; 10.2, 

range 17-79)) who initiated PegIFN/RBV therapy. 

The fibrosis stage and the grade of inflammation were 

determined in liver biopsy specimens taken before 

therapy. 

Results: IP-10 levels in 402 CHC patients were 

significantly higher than those in 40 healthy persons 

(522.8 (373.7) vs. 97.9 (24.9) pg/ml, respectively; 

P

mean attendance being 15 subjects per week (range 

3-32). Overall, 10 (8%) did not medically qualify for 

treatment, 39 (30%) were lost to follow-up and 8 

(6%) had completed or initiated treatment for HCV 

infection prior to attending the group. We observed a 

high uptake of HCV treatment among attendees, with 

30% of subjects (39/129) currently under evaluation 

and 26% (33/129) having initiated or completed 

treatment for HCV infection. In a comparison of 

subjects that had initiated or completed treatment 

for HCV infection (n=33) and those lost to follow 

up (n=39), those having received treatment for 

HCV infection had a higher median attendance [34 

meetings (Interquartile range, IQR = 11-33) vs. 2 

meetings (IQR=1-3, P3 clinic visits (97% vs. 23%, P

higher (P

Abstract 108 

Changes of IFN-inducible Gene 

(IP-10, PKR, MxA) Expressions 

During Pegylated Interferon Alfa- 

2b Plus Ribavirin Therapy in HCV 

Genotype 1 Infected Japanese 

Patients 

R Nakao, H Yatsuhashi, M Fukuda, A Nishikawa, 

S Hashimoto, N Hai, M Tateyama, Y Motoyoshi, 

S Nagaoka, N Taura, K Yanagi, K Yano, S Abiru, 

A Komori, H Fujioka, K Migita, M Nakamura, 

and H Ishibashi 

Clinical Research Center, National Hospital Organization 

Nagasaki Medical Center, Omura, Nagasaki, Japan 

BACKGROUND: Recently, it has been reported that 

pretreatment interferon(IFN)-gamma-inducible protein 

10 kDa (IP-10 or CXCL10) might be a predictive 

factor for sustained viral response to PEG-IFN plus 

RBV therapy in patients infected with hepatitis C virus 

(HCV) genotype 1. In order to clarify the role of IP-10 

in the treatment of IFN, we evaluated the changes of 

IFN-related gene expression during treatment and 

examined its relationship with the viral response to 

IFN. 

METHODS: A total of 81 HCV genotype 1 patients 

treated with PEG-IFN alfa-2b plus RBV. Serum IP- 

10 levels were measured by ELISA before therapy, 

during treatment (week 1, 2, 4, 12, and 24), end of 

treatment and 24 weeks after cessation of therapy. In 

addition, we examined IP-10, IFN receptor (IFNAR2), 

myxovirus resistance-A (MxA), RNA-dependent 

protein kinase (PKR), IFN regulatory factor-1 (IRF1), 

IRF2 (IRF2) and signal transducers and activators 

of transcription factor (STAT3) gene expression in 

peripheral blood mononuclear cells in 58 patients. 

The primary study end point was the early virologic 

response (EVR) – an undetectable serum HCV-RNA 

level at treatment week 12. 

patients with non-EVR were significantly higher than 

those with EVR (pretreatment: 486 v 384 pg/ml, P < 

0.05, at week 1: 645 v 428 pg/ml, P < 0.01, at weeks 2: 

444 v 318 pg/ml, P < 0.05, post-treatment: 418 v 186 

pg/ml, P < 0.001, respectively). The expression levels 

of IFNAR2, IRF1, IRF2 and STAT3 were significantly 

down-regulated by PEG-IFN plus RBV therapy 

while the expression levels of MxA and PKR were 

significantly up-regulated. At pretreatment, there 

was no difference in IFN-related gene expression 

between the EVR group and the non-EVR group. At 

week 2 compared with baseline, the change of PKR 

and IP-10 expression elevated significantly higher in 

non-EVR than in EVR (PKR: 2.5 v 1.7 fold, P < 0.05, 

IP-10: 1.0 v 0.7 fold, P < 0.05). 

CONCLUSIONS: Unexpectedly, the non-EVR group 

had a stronger response of IFN-related molecules 

upon PEG-IFN plus RBV administration than EVR 

group. It has been reported that elevated serum IP- 

10 levels were significantly associated with a poor 

response to antiviral therapy (J Infect Dis 2006, 

Hepatology 2006, Gut 2006). On the other hand, in 

long-term IFN-gamma treatment, suppression of IFNalfa 

signaling is mediated partly through elevation in 

STAT1 protein expression. These high levels of IFNgamma 

and STAT1 protein may suppress the effect 

of IFN-alfa therapy and, consequently contribute to 

IFN-alfa treatment failure in CHC patients (Biochem. 

J. 2004). We suggest that resistance to PEG-IFN plus 

RBV therapy may not be due to a disturbance in IFN 

cell signaling, but high levels of IP-10 and IFN-gamma 

negatively regulate IFN-alfa-activated signals. 

RESULTS: At pretreatment, week 1, 2 and 24 weeks 

after cessation of therapy, serum IP-10 levels in 


Abstract 109 

Baseline Characteristics and Early 

Virologic Response to Telbivudine 

Predict 2‐Year Outcomes for 

Patients with HBeAg-negative 

Chronic Hepatitis B 

T Poynard 1 , GV Papatheodoridis 2 , M Tong 3 , N Tsai 4 , 

and M Buti 5 

1 Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 

2 Athens University Medical School, Athens, Greece; 

3 Huntington Medical Research Institutes, Pasadena, CA, 

USA; 4 John A Burns School of Medicine, Honolulu, HI, 

USA; 5 Hospital Valle de Hebrón, Barcelona, Spain 

BACKGROUND: The ability to predict long-term 

treatment response based on baseline demographics 

and early responses to therapy may help physicians 

individualize patient management and optimize 

outcomes in chronic hepatitis B. For HBeAg-negative 

patients, trends suggest better outcomes for patients 

with low baseline HBV DNA levels (

Abstract 110 

Predictors of Sustained Viral 

Response in the Retreatment of 

Previous Interferon/Ribavirin 

Nonresponders: Results from the 

EPIC 3 Program 

T Poynard 1 , E Schiff2 , R Terg 3 , R Moreno Otero 4 , 

S Flamm 5 , W Schmidt 6 , T Berg 7 , F Goncales Jr 8 , 

J Heathcote 9 , M Diago 10 , T McGarrity 11 , P Bedossa 12 , 

W Deng 12 , P Mukhopadhyay 12 , L Griffel 12 , 

M Burroughs 12 , C Brass 12 , and J K Albrecht 12 

1 Service d’hepatologie, Hôpital La Pitié Salpêtrière, Paris, 

France; 2 University of Miami School of Medicine, Miami, 

Florida, USA; 3 Hospital Municipal de Gastroenterologie 

Dr. Bonorino Udaondo, Capital Federal, Argentina; 

4 Hospital Universitario de la Princesa, Madrid, Spain; 

5 Northwestern University, Chicago, Illinois, USA; 

6 University of Iowa Hospitals and Clinics, Iowa City, 

Iowa, USA; 7 Virchow Klinikum der Charite, Berlin, 

Germany; 8 Hospital das Clinicas da Unicamp Cidade 

Universitaria, Campinas, Brazil; 9 University Health 

Network, Toronto, Ontario, Canada; 10 Hospital General 

Universitario de Valencia, Valencia, Spain; 11 Milton S. 

Hershey Medical Center, Hershey, Pennsylvania, USA; 

12 Schering-Plough Research Institute, Kenilworth, New 

Jersey, USA 

BACKGROUND: EPIC 3 includes a prospective 

trial designed to assess the efficacy and safety of 

retreatment of subjects with chronic hepatitis C and 

fibrosis (METAVIR F2-F4) who were nonresponsive 

to previous treatment with any interferon (IFN) alfa 

plus ribavirin with peginterferon (PEG-IFN) alfa-2b 

and ribavirin. The overall sustained virologic response 

(SVR) rate was 23% in these subjects and 57% among 

those with undetectable hepatitis C virus (HCV) RNA 

at treatment week (TW) 12. The aim of this analysis 

was to define predictors of SVR in these subjects. 

TW12, TW24, and TW48 and at follow-up weeks 12 

and 24 using a quantitative TaqMan assay (Schering- 

Plough Research Institute, Kenilworth, New Jersey, 

USA; lower limit of detection 125 IU/mL). 

RESULTS: Undetectable HCV RNA at TW12 was the 

most important predictor of SVR; 57% of subjects 

with undetectable HCV RNA at TW12 attained SVR. 

In contrast, only 6% of those with a 2 log 10 

or more 

drop in HCV RNA and with detectable virus at TW12 

attained SVR; no subjects with less than a 2 log 10 

drop at TW12 attained SVR. Results of a multivariate 

analysis indicate that other important predictors of 

SVR were fibrosis score (F2, 30% SVR; F3, 25% SVR; 

F4 17% SVR; P

Abstract 111 

Addition of Lamivudine to 

Patients with Chronic Hepatitis 

B who are Viremic on Adefovir 

Dipivoxil Lowers Plasma HBV 

DNA Levels 

M Tong 1 and J Ryan 2 

1 Huntington Medical Research Institute, Pasadena, CA, 

USA; 2 Gilead Sciences, Foster City, CA, USA 

Background: There is limited information on 

whether it is beneficial to add lamivudine (LAM) 

to patients with chronic hepatitis B (CHB) who are 

viremic while receiving adefovir dipivoxil (ADV) 

monotherapy. 

Methods: We performed a retrospective search of 

the HMRI (Huntington Medical Research Institute) 

database to identify viremic patients (HBV DNA ≥ 

3 log 10 

copies/mL by PCR-based assay) who received ≥ 

6 months of treatment with ADV, and who had LAM 

added to their treatment. Patients with or without 

LAM-resistance were included. The primary analysis 

was change in HBV DNA at 6 months after adding 

LAM; we also determined the proportion of patients 

with undetectable HBV DNA, and e-antigen loss or 

seroconversion at any time during the follow-up 

period. 

Results: 15 patients were identified who met 

inclusion criteria; 12 patients (80%) had a prior history 

of LAM use and 9 patients (60%) were LAM-resistant 

at the time of LAM addition (baseline). Median age 

was 50 yrs, 80% were male, 87% were Asian; 40% were 

HBeAg-positive. Median HBV DNA prior to starting 

ADV monotherapy was 6.4 log 10 

copies/mL. At the 

time LAM was added to ADV, median HBV DNA was 

4.7 log 10 

copies/mL, and patients had received ADV 

monotherapy for a median duration of 16 months. 

At 6 months following the addition of LAM, median 

HBV DNA declined by 2.1 log 10 

copies/mL; all 

patients became undetectable (LLD < 160 copies/mL) 

after a median of 6 months follow-up. HBeAg loss/ 

seroconversion was seen in 40% of HBeAg-positive 

patients. Responses in patients with LAM-resistance 

were similar to those in patients without evidence of 

resistance. 

Conclusions: In this small cohort, adding LAM 

to ADV in patients with persistent viremia on ADV 

monotherapy appeared to be effective in controlling 

HBV replication, regardless of the presence of LAMresistance. 

Abstract 112 

A Systematic Review of the 

Effectiveness of Pegylated 

Interferon, Lamivudine, Adefovir 

and Entecavir for the Treatment 

of Hepatitis B 

G Woo 1 , Y Nishikawa, J Heathcote 1,2 , M Sherman 2 , 

T Einarson 1 , W Ungar 1,3 , M Krahn 1,2,4 

1 University of Toronto, Ontario, Canada; 2 University 

Health Network, Ontario, Canada; 3 Hospital for Sick 

Children Research Institute, Ontario, Canada; 

4 Toronto Health Economics and Technology Assessment 

Collaborative, Ontario, Canada 

Background: Treatment options for chronic 

hepatitis B (CHB) are constantly evolving. An upto-date 

and comprehensive analysis comparing 

the effectiveness of all available treatments is not 

currently available. 

Aims: To systematically review the effectiveness 

of pegylated interferon (PEG), lamivudine (LAM), 

adefovir (ADF) and entecavir (ENT) in treating CHB. 

Methods: Pubmed, Embase, Cochrane, and Econlit 

were searched for randomized controlled trials 

assessing the efficacy of the selected drugs for treating 

CHB published in the English language from the dates 

of inception to the end of December 2006. Patients 


were considered to have CHB if they had elevated 

ALT levels and active viral replication. Monotherapy, 

combination and sequential therapies were included. 

The treatment duration was one year. The search 

was performed with the aid of a librarian. Two 

independent reviewers independently assessed the 

relevance of the studies; discrepancies were solved 

through debate and assessment by a third reviewer. 

We included studies that documented: patient 

randomization, patient baseline characteristics, 

defined eligibility criteria, patient blinding, adequate 

information to assess one of our targeted clinical 

endpoints and reporting of drop-outs within the 

one year treatment period. Among trials that met 

our inclusion criteria, we abstracted data describing 

normalization of ALT, HBV DNA, sustained 

biochemical response, HBeAg seroconversion, 

histological improvement, drop-outs and adverse 

events. Data was collected and checked by two 

independent reviewers. Intention-to-treat data 

were combined using a random-effects metaanalysis, 

with missing data considered as treatment 

failures. Outcomes were expressed as relative risks 

versus placebo or active drug with 95% confidence 

intervals. 

Results: The initial search yielded 2064 references, 

127 were excluded due to inadequate blinding, 

allocation concealment, randomization and reporting 

of outcomes; 20 studies were included. Trials involved 

5573 patients (4121 males, 1309 females), ranging 

in size from 200-814 patients. Mean age was 40.7. 

Monotherapy comparisons available were LAM, 

ADF, and ENT versus placebo, and LAM versus PEG, 

ADF and ENT. Eleven trials studied HBeAg-positive 

patients, four trials studied HBeAg-negative patients, 

and four trials studied both. Due to small numbers of 

trials for comparison led to pooling of HBeAg-positive 

and HBeAg-negative studies. 

No treatment was superior for all outcome measures. 

Monotherapy was found to be superior to placebo. 

Comparisons of single drugs favored treatment with 

ADF or ENT over LAM or PEG. In direct comparisons, 

LAM was superior to PEG with better clinical 

outcomes and fewer adverse events and patient 

dropouts. Combination and sequential treatments 

were not superior, but comparisons were limited by 

our one-year follow-up. 

Conclusion: Monotherapy with ADF or ENT are 

the most attractive treatment options within the first 

year of treatment. Further research on combination 

and sequential therapies may provide better options 

but presently insufficient evidence exists to support 

this approach. 

Abstract 113 

Safety Recommendations for 

Laboratory Values in Specific 

Product Characteristics (SPC) of 

Peginterferon Alfa-2a (PEG) and 

Ribavirin (RBV) - What Happens 

Under Real Life Conditions? 

E Zehnter 1 , S Mauss 2 , K Boeker 3 , T Lutz 4 , S Racky 5 , 

W Schmidt 6 , R Ullrich 7 , R Heyne 8 , A Schober 9 , C John 10 , 

KH Hey 11 , B Möller 8 , B Bokemeyer 12 , B Kallinowski 13 , 

S Pape 14 , U Alshuth 15 , and D Hüppe 16 

1 Center of Gastroenterology, Dortmund, Germany; 

2 Center of HIV and Hepatogastroententerology, 

Duesseldorf, Germany; 3 Center of Gastroenterology, 

Hannover, Germany; 4 Center of Infectiology, Frankfurt, 

Germany; 5 Center of Gastroenterology, Bad Schwalbach, 

Germany; 6 Center of Gastroenterology, Berlin, Germany; 

7 Center of Gastroenterology, Krefeld, Germany; 

8 Livercenter, Berlin, Germany; 9 Center of 

Gastroenterology, Goettingen, Germany; 10 Center of 

Gastroenterology, Berlin, Germany; 11 General Practice, 

Paderborn, Germany; 12 Center of Gastroenterology, 

Minden, Germany; 13 Center of Gastroenterology, 

Schwetzingen, Germany; 14 Center of Gastroenterology, 

Paderborn, Germany; 15 BU Hepatitis/HIV/Infectiology, 

Roche Pharma AG, Grenzach-Wyhlen, Germany; 

16 Center of Gastroenterology, Herne, Germany 

BACKGROUND: Recommendations of SPCs derived 

from forced conditions in pivotal trials. A very 

important part concerns haematological laboratory 

data at start and during therapy. Information about 

adherence to SPC recommendations are missing. 


METHODS: Until May 2006 data of 4377 patients 

in different phases of CHC treatment with PEG 

and RBV were recorded in a German observational 

trial conducted by BNG and Roche. In the following 

treatment behaviour is described, if laboratory 

data achieve recommended cut-off values of SPC. 

RESULTS: A small part of pts had baseline values 

under recommended numbers of haematological 

parameters: Neutrophils (Neu)

81/2234 (3.6%) by histology (H). In 310 patients 

(3.0%) cirrhosis was clinically diagnosed (C) and 

classified according to Child Pugh: A 86.5%, B 10.0%, 

C 3.5%. Patients diagnosed by sonography were on 

average 58.2 yrs., 60.6% male, with BMI 26.3 kg/ 

m², thrombocytes 137,989 /µl, duration of infection 

19.2 yrs., source of infection (multiple answers 

possible): blood products 31.4%, IVDU 23.7%, 

medical intervention 8.7%, unknown 35.3%. Current 

alcohol abuse was reported for 12.2% of patients. 

Distribution of genotypes: G1 75.3%, G2/3 21.1%, G4 

3.1%. Treatment-rate with Peginterferon alfa-2a and 

Ribavirin was (according to diagnosis tool): 31.4% 

(S), 75.5% (H), 42.6% (C). SVR rate of patients with 

cirrhosis (S) was 37.3% (28/75). Reasons against a 

therapy were (S): decompensated disease 33.6%, other 

therapy 10.2%, concomitant disease/continuous drug 

abuse 25.7%, patient’s request 22.2%, age 8.3%. 

CONCLUSIONS: About 3-5% of the described patient 

group already had a liver cirrhosis at screening. The 

patients are older and have been infected for longer 

than average. Most common way of infection was 

medical intervention. Although these patients are 

in urgent need of hepatitis C therapy, more than 

1/5 cannot be convinced to start. Success rate of 

therapy in this real-life setting was similar to the 

numbers observed in clinical studies. However, due 

to decompensation or other diseases, for the majority 

of patients interferon therapy is not an option any 

more. 


Late Breaker 

Session 


Abstract 115 

Long Term Follow-Up of HCV 

Patients Completing Peg-IFN Plus 

Ribavirin Treatment – Is There a 

Cure? 

MP Manns 

Medical School of Hannover, Hannover, Germany 

The goal of treatment in chronic hepatitis C is to 

achieve undetectable HCV RNA in serum 24 weeks 

after the end of therapy. It has been a debate whether 

this means cure of this chronic viral infection. 

Information on HCV RNA longterm after the end 

of therapy is limited. Therefore the information is 

valuable for long term follow up studies of patients 

that were treated as part of large pivotal trials 

with interferon based therapies. Data are available 

from several pivotal trials with pegylated and nonpegylated 

interferon alpha 2a and 2b alone or in 

combination with ribavirin. 

Non-pegylated interferon alpha 2b (McHutchison 

et al, EASL 2006) : 1071 treatment naïve patients 

and patients relapsed after the end of therapy were 

treated with interferon alpha 2b (Intron A) +/- 

ribavirin (Rebetol) and who completed 24 weeks of 

follow up were recruited. HCV RNA was measured by 

PCR (NGI PCR with sensitivity of 100 copies/ml and 

after 03/2001 with Taqman SP assay and a sensitivity 

of 29 IU/ml). Of the 1071 pts enrolled in the long 

term study 492 had achieved SVR and 579 were 

non-responders (NR). 61 % of SVR and 28 % of NR 

patients completed 5 years of follow up with a median 

duration of 283 and 131 weeks, respectively. Only 5 

of the 492 SVR patients had a definite relapse on long 

term follow up. There were an additional 7 patients 

with possible recurrent disease with inconsistent 

HCV RNA results. Thus the probability of SVR for the 

5 year posttreatment follow up period was between 

97 – 99 % for these patients. 

Pegylated Interferon alpha 2b (Schering-Plough, 

personal communication; Manns et al, submitted): 

567/1695 (33 %) of patients from two pivotal trials 

(Lindsay et al , Hepatology, 2001; Manns et al, Lancet 

2001) treated with Peg-IFN alpha 2b +/- ribavirin 

who completed 24 week follow up were assessed 

annually for up to 5 years for evidence of disease 

progression and sustained HCV RNA negativity 

(Taqman SP PCR, sensitivity 29 IU/ml ). Three 

SR subjects relapsed during the 5 year FU period, 

all within the first two years. The Kaplan-Meier 

estimate for continued SVR over 5 years is 99%. 

Pegylated Interferon alpha 2a (Swain et al , EASL 2007): 

997 patients treated with pegylated interferon alpha 

2a (Pegasys) alone or in combination with ribavirin 

(Rebetol) were followed for a mean of 4.1 years after 

the end of treatment. They were from 4 studies with 

monotherapy of PEG-IFNalpha2a with elevated ALT, 

4 studies with combination therapy and elevated 

ALT, 1 study with combination therapy and normal 

ALT, and 1 study with HCV/HIV co-infection treated 

with either mono- or combination therapy. 989/997 

patients (99%) stayed HCV RNA negative (range 0.4 – 

7 years) after treatment cessation. 8 patients became 

positive between 1.1 and 2.9 years after completing 

therapy. Six of these 8 patients had high viral load 

pre-treatment. 

Conclusions: For interferon alpha 2a and 2b, 

pegylated or non-pegylated, with or without ribavirin, 

HCV RNA negativity 24 weeks after the end of therapy 

is an excellent predictor of long term clearance 

of HCV RNA from serum. These data confirm the 

concept of cure in the treatment of chronic hepatitis 

C by interferon based therapies. 


Abstract 116 

Delta Hepatitis: Treatment 

Options for a Largely Neglected 

Disease 

MP Manns and H Wedemeyer 

Dept. of Gastroenterology, Hepatology and 

Endocrinology, Hannover Medical School, 

Carl-Neuberg-Straße 1, 30625 Hannover, Germany 

The hepatitis delta virus (HDV) is a satellite virus 

that requires co-infection with hepatitis B virus 

(HBV) for its replication. Delta hepatitis represents 

possibly the most severe form of chronic hepatitis. 

In chronically infected HBV carriers, HDV can result 

in fulminant acute hepatitis or severe chronic active 

hepatitis, progressing to cirrhosis in at least two 

thirds of patients at a younger age, and increasing the 

risk of hepatocellular cancer threefold and mortality 

twofold compared with chronic HBV infection alone. 

Worldwide, around 15 million people are infected 

with HDV and are therefore at risk of the potentially 

severe sequalae of infection. The epidemiology of 

HDV infection has changed in recent decades, with 

reports of decreased prevalence in some countries, 

linked to the introduction of vaccination against 

HBV. In contrast, the epidemiology of delta hepatitis 

had changed significantly in Germany with large 

numbers of patients migrating to Germany from 

Eastern Europe and countries of the former Soviet 

Union. Subsequently, the anti-HDV prevalence 

among HBsAg-positive individuals remained constant 

between 8% and 14% since 1997 at our center. 

Treatment options for delta hepatitis are limited. 

So far, no nucleoside or nucleotide analogue used 

for the treatment of viral hepatitis has shown any 

efficacy against HDV. Interferon alpha may cure delta 

hepatitis in single patients but requires prolonged 

administration with high doses. Three pilot studies 

with PEG-IFN alpha-2b (1.5 μg/kg/week) have 

demonstrated a sustained HDV-RNA response of 

17%-43%. We did investigate the efficacy of pegylatedinterferon 

alfa-2a and/or adefovir dipivoxil in 90 

patients with delta hepatitis recruited in Germany, 

Turkey and Greece treated for 48 weeks. An at least 

2xlog 10 

-decline of HDV-RNA was observed in 39% 

of patients treated with PEG-IFNa-2a plus adefovir, 

44% of patients treated with PEG-IFNa-2a and 

placebo and 8% treated with adefovir. A sustained 

HDV-RNA response was observerd in roughly one 

quarter of patients receiving PEG-IFN either alone 

or in combination with adefovir. Interestingly, the 

combination therapy group was superior in HBsAg 

reduction which may also have significant impacts 

for the treatment of HBV infection without delta 

hepatitis. 

In summary, delta hepatitis is a largely underestimated 

severe liver disease in Europe affecting mainly 

immigrants. PEG-IFN alpha treatment should be 

considered and the role of combination and prolonged 

therapies requires further investigation. 

Abstract 117 

Biochemical Mechanism of 

Entecavir as an Antiviral 

Polymerase Inhibitor 

RA Domaoal 1 , M McMahon 2 , CL Thio 2 , CM Bailey 1 , 

J Tirado-Rives 4 , A Obikhod 3 , M Detorio 3 , KL Rapp 3 , 

RF Siliciano 2 , RF Schinazi 3 , and KS Anderson 1 

1 The Yale University School of Medicine, Department 

of Pharmacology, New Haven, Connecticut 06520- 

8066, USA; 2 Department of Medicine, Johns Hopkins 

University School of Medicine, and Howard Hughes 

Medical Institute, Baltimore MD 21205, USA; 3 Emory 

University School of Medicine/Veterans Affairs Medical 

Center, Decatur, Georgia, 30033, USA; 4 Yale University, 

Department of Chemistry, New Haven, Connecticut 

06510, USA 

The novel 2’-deoxyguanosine analog Entecavir (ETV) 

is a potent inhibitor of hepatitis B virus (HBV) 

replication and is recommended for treatment in 

human immunodeficiency virus type 1 (HIV-1) and 

HBV co-infected patients because it had been reported 

that ETV is HBV-specific. Recent clinical observations, 


however, have suggested that ETV may indeed 

demonstrate anti-HIV-1 activity. To investigate this 

question at a molecular level, kinetic studies were used 

to examine the interaction of ETVTP with wild type 

(WT) HIV-1 reverse transcriptase (RT) and the NRTI 

resistant mutation M184V. Using single turnover 

kinetic assays, we found that HIV-1 WT RT and 

M184V RT could use the activated ETV triphosphate 

metabolite as a substrate for incorporation. The 

mutant displayed a slower incorporation rate, a lower 

binding affinity and a lower incorporation efficiency 

with ETVTP compared to WT RT, suggesting a kinetic 

basis for resistance. Our results are supported by 

cell-based assays in primary human lymphocytes 

that show inhibition of WT HIV-1 replication by ETV 

and decreased susceptibility of the HIV-1 containing 

the M184V mutation. This study has important 

therapeutic implications as it establishes ETV as an 

inhibitor for HIV-1 RT and illustrates the mechanism 

of resistance by the M184V mutant. 

Abstract 118 

Advancing RNAi-based 

Therapeutic from Discovery to 

Clinic 

C Pachuk 1 and R Gish 2 

1 Nucleonics Inc., Horsham PA, USA; 2 California Pacific 

Medical Research Institute, San Francisco, CA, USA 

The presentation will provide a brief overview of 

Nucleonics’ RNA interference technology, preclinical 

and clinical development of its eiRNA-based anti- 

HBV therapeutic, NUCB1000. Unlike current small 

molecule HBV therapies, RNAi-based drug product 

NUCB1000 has been shown to suppress viral antigen 

expression in addition to inhibiting viral replication. 

NUCB1000 has been designed to be effective against 

all HBV genotypes and has demonstrated activity 

against known drug resistant mutants. Because 

NUCB1000 targets multiple RNA sequences, it is not 

predicted to select for viable escape mutants over the 

course of therapy. The presentation will detail product 

development, including delivery, efficacy, safety 

in preclinical models and the clinical development 

program. NUCB1000 is currently undergoing safety 

evaluations in Phase-1b clinical studies in CHB 

patients. 

Abstract 119 

Ultra-deep Pyrosequencing of 

HBV Quasispecies in Nucleoside 

Analog Treated and Untreated 

Patients 

S Margeridon-Thermet 1 , N Shulman 1 , A Ahmed 1 , 

T Liu 1 , C Wang 1 , B Simen 2 , J Simons 2 , M Egholm 2 , 

B Gharizadeh 3 , and RW Shafer 1 

1 Department of Medicine, Stanford University, Stanford, 

CA, USA; 2 454 Life Sciences, Branford, Connecticut, USA; 

3 Stanford Genome Technology Center, Stanford, CA, 

USA 

BACKGROUND: HBV nucleoside analog (NA) 

resistance has become increasingly common. There 

is a high level of cross-resistance among available 

NA with mutations selected by one NA contributing 

resistance to others. Because HBV is a quasispecies, 

we sought to determine whether it is possible to 

detect minor NA-resistant variants that are not 

detected by standard direct-PCR dideoxynucleoside 

(Sanger) sequencing. 

METHODS: 15 cryopreserved plasma samples from 

HBV-infected patients with well-characterized 

treatment histories were sequenced by Sanger 

sequencing and by ultra-deep pyrosequencing (UDPS; 

454 Life Sciences) using the FLX platform. For UDPS, 

four pairs of primers tailed with 454 adaptors and 

a patient-specific bar code were used to amplify 

(Expand High Fidelity PCR System) 1,226 bp of HBV 

encompassing the known NA-resistance mutations. 

A plasmid HBV clone was sequenced as a control to 

determine the sequencing error rate. 


RESULTS: Plasma samples were obtained from 

4 treated and 11 untreated patients. The treated 

patients had received either 3TC (n=1), 3TC/adefovir 

(n=1), or 3TC/adefovir/entecavir (n=2). The median 

plasma HBV DNA was 6.7 log copies/ml (range: 5.2 to 

8.5 log copies/ml). A minimum of 400 viral templates 

was submitted for UDPS. Each of the 64 amplicons 

from the 16 HBV samples were sequenced on a single 

PicoTiter plate. A median 16,000 reads of >200 

nucleotides were obtained per HBV sample providing 

a median sequence coverage of 3,200 per bp (range: 

1,170 to 6,459). The single-read mismatch error rate 

was estimated to be 0.1% in non-homopolymeric 

regions and 0.45% in homopolymeric regions 

providing sensitivity for detecting minor variants of 

between 0.2% to 2.0% depending on the region and 

extent of sequence coverage. 

Three of four treated patients had drug-resistant 

variants detected by UDPS but not Sanger sequencing. 

In the 3TC-treated patient, L180M and M204V were 

detected by Sanger sequencing and UDPS; L173V 

was detected only by UDPS (16% of reads). In the 

3TC/adefovir-treated patients, N236T was detected 

only by UDPS (6% of reads). In one of the threedrug 

treated persons M204V/I and L180M/V were 

detected by UDPS and Sanger sequencing, but L80V 

(1.5% of reads) and A181T (0.8% of reads) were 

detected only by UDPS. Among the 11 untreated 

patients, low-levels (2% to 24%) of the accessory 

drug resistance mutations V214A and I233V were 

detected in 3 patients. G to A hypermutation, which 

has the potential to complicate sequence analysis, was 

present at low levels in several patients and validated 

by clonal sequencing. 

Conclusions: UDPS quantified known HBVresistance 

mutations at a level of sensitivity not 

previously possible. UDPS will provide new insight 

into HBV population genetics in NA-treated and 

untreated HBV-infected patients. 


Author Index 

Author Abstract Page 

Afdhal, NH. ..................40.. . . . . . . . . . . . . . . . 41 

Alter, H. ...................... 3.. . . . . . . . . . . . . . . . . . 7 

Anderson, KS................117.. . . . . . . . . . . . . . . 130 

Ayres, A .. . . . . . . . . . . . . . . . . 79, 80.. . . . . . . . . . . . . 85, 86 

Balfe, P.......................54.. . . . . . . . . . . . . . . . 59 

Bartenschlager, R.. . . . . . . . . . . . . 04.. . . . . . . . . . . . . . . . . . 8 

Bassit, L.. . . . . . . . . . . . . . . . . . . . . 81.. . . . . . . . . . . . . . . . 87 

Blatt, LM.. . . . . . . . . . . . . . . . . . . . 58.. . . . . . . . . . . . . . . . 63 

Brass, V .. . . . . . . . . . . . . . . . . . . . . 07.. . . . . . . . . . . . . . . . 10 

Butt, G.. . . . . . . . . . . . . . . . . . . . . 102.. . . . . . . . . . . . . . . 115 

Chisari, FV....................19.. . . . . . . . . . . . . . . . 21 

Chu, CK .. . . . . . . . . . . . . . . . . . . . . 82.. . . . . . . . . . . . . . . . 88 

Chung, KW .. . . . . . . . . . . . . . . . . . 29.. . . . . . . . . . . . . . . . 30 

Crabbé, R.. . . . . . . . . . . . . . . . . . . . 56.. . . . . . . . . . . . . . . . 61 

Dash, S.. . . . . . . . . . . . . . . . . . 13, 14.. . . . . . . . . . . . . 14, 15 

Dayaram, YK.. . . . . . . . . . . . . . . . . 91.. . . . . . . . . . . . . . . . 99 

De Francesco, R...............24.. . . . . . . . . . . . . . . . 25 

De La Rosa, A .. . . . . . . . . . . . . . . . 83.. . . . . . . . . . . . . . . . 89 

Dennin, RH.. . . . . . . . . . . . . . . . . . 64.. . . . . . . . . . . . . . . . 68 

Di Bisceglie, AM.. . . . . . . . . . . . . . 39.. . . . . . . . . . . . . . . . 41 

Eroglu, C .. . . . . . . . . . . . . . . . . . . . 84.. . . . . . . . . . . . . . . . 90 

Evans, MJ .. . . . . . . . . . . . . . . . . . . 53.. . . . . . . . . . . . . . . . 59 

Ewart, G......................65.. . . . . . . . . . . . . . . . 69 

Feitelson, MA .. . . . . . . . . . . 66, 103.. . . . . . . . . . . . 70, 115 

Fenton, K.....................01.. . . . . . . . . . . . . . . . . . 3 

Flexner, CW...................98.. . . . . . . . . . . . . . . 107 

Fried, MW.. . . . . . . . . . . . . . . . . . . 77.. . . . . . . . . . . . . . . . 83 

Fukuda, M.. . . . . . . . . . . . . . . . . . 104.. . . . . . . . . . . . . . . 116 

Gastaminza, P.................15.. . . . . . . . . . . . . . . . 16 

Gish, R.. . . . . . . . . . . . . . . . . . . . . 118.. . . . . . . . . . . . . . . 131 

Glebe, D.. . . . . . . . . . . . . . . . . . . . . 47.. . . . . . . . . . . . . . . . 51 

Grakoui, A.. . . . . . . . . . . . . . . . . . . 06.. . . . . . . . . . . . . . . . . . 9 

Grebely, J....................105.. . . . . . . . . . . . . . . 117 

Hausman, DF .. . . . . . . . . . . . . . . . 67.. . . . . . . . . . . . . . . . 71 

Hostetler, KY.................26.. . . . . . . . . . . . . . . . 27 

Husa, P......................106.. . . . . . . . . . . . . . . 118 

Husova, L. ...................68.. . . . . . . . . . . . . . . . 72 

Jacobson, I. ..................23.. . . . . . . . . . . . . . . . 25 

Kim, HJ.. . . . . . . . . . . . . . . . . . . . 107.. . . . . . . . . . . . . . . 119 


Kiss, A. . . . . . . . . . . . . . . . . . . . . . . 57.. . . . . . . . . . . . . . . . 62 

Klade, CS.. . . . . . . . . . . . . . . . . . . . 48.. . . . . . . . . . . . . . . . 52 

Koike, K.. . . . . . . . . . . . . . . . . . . . . 49.. . . . . . . . . . . . . . . . 53 

Korba, BE.....................21.. . . . . . . . . . . . . . . . 23 

Koziel, MJ.. . . . . . . . . . . . . . . . . . . 44.. . . . . . . . . . . . . . . . 49 

Lau, GKK.. . . . . . . . . . . . . . . . 30, 43.. . . . . . . . . . . . . 30, 45 

Law, M.. . . . . . . . . . . . . . . . . . . . . . 45.. . . . . . . . . . . . . . . . 49 

Lee, CH......................31.. . . . . . . . . . . . . . . . 31 

Lefkowitz, EJ .. . . . . . . . . . . . . . . . 59.. . . . . . . . . . . . . . . . 64 

Lemon, SM .. . . . . . . . . . . . . . . . . . 18.. . . . . . . . . . . . . . . . 21 

Liang, TJ .. . . . . . . . . . . . . . . . . . . . 05.. . . . . . . . . . . . . . . . . . 8 

Lin, K .. . . . . . . . . . . . . . . . . . . . . . . 28.. . . . . . . . . . . . . . . . 29 

Lindsay, KL .. . . . . . . . . . . . . . . . . . 99.. . . . . . . . . . . . . . . 109 

Liu-Young, G.. . . . . . . . . . . . . . . . . 10.. . . . . . . . . . . . . . . . 11 

Locarnini, S.. . . . . . . . . . . . . . 02, 85.. . . . . . . . . . . . . . 3, 90 

Love, R.. . . . . . . . . . . . . . . . . . . . . . 69.. . . . . . . . . . . . . . . . 73 

Luscombe, CA.. . . . . . . . . . . . . . . . 32.. . . . . . . . . . . . . . . . 32 

Manns, MP .. . . . . . . . . . . . 115, 116.. . . . . . . . . . . 129, 130 

Marcellin, P.. . . . . . . . . . . . . . . . . . 63.. . . . . . . . . . . . . . . . 67 

Margeridon-Thermet, S........119.. . . . . . . . . . . . . . . 131 

Martinot-Peignoux, M..........11.. . . . . . . . . . . . . . . . 12 

Matsuura, Y...................27.. . . . . . . . . . . . . . . . 28 

McHutchison, JG.. . . . . . . . . 61, 70.. . . . . . . . . . . . . 65, 73 

McPhee, F .. . . . . . . . . . . . . . . . . . 100.. . . . . . . . . . . . . . . 110 

Morrey, JD...................71.. . . . . . . . . . . . . . . . 74 

Murakami, E.. . . . . . . . . . . . . . . . . 33.. . . . . . . . . . . . . . . . 32 

Nakao, R....................108.. . . . . . . . . . . . . . . 120 

Nelson, D.....................62.. . . . . . . . . . . . . . . . 66 

Neumann, AU.. . . . . . . . . . . . . . . . 22.. . . . . . . . . . . . . . . . 24 

Oh, J-W.. . . . . . . . . . . . . . . . . 72, 73.. . . . . . . . . . . . . 75, 76 

Olsen, DB....................60.. . . . . . . . . . . . . . . . 65 

Omata, M....................42.. . . . . . . . . . . . . . . . 44 

Park, SJ .. . . . . . . . . . . . . . . . . . . . . 16.. . . . . . . . . . . . . . . . 16 

Pachuk, C....................118.. . . . . . . . . . . . . . . 131 

Pedersen, IM.. . . . . . . . . . . . . . . . . 09.. . . . . . . . . . . . . . . . 11 

Perelson, AS..................08.. . . . . . . . . . . . . . . . 10 

Peters, MG....................88.. . . . . . . . . . . . . . . . 97 

Polyak, S.....................74.. . . . . . . . . . . . . . . . 77 

Poynard, T.. . . . . . . . . . 41, 109, 110.. . . . . . . . 43, 121, 122 


Author Index 


Ray, AS.......................97.. . . . . . . . . . . . . . . 107 

Reddy, KR .. . . . . . . . . . . . . . . . . . . 12.. . . . . . . . . . . . . . . . 13 

Ryan, J.. . . . . . . . . . . . . . . . . . . . . 111.. . . . . . . . . . . . . . . 123 

Sandrasagra, A .. . . . . . . . . . . . . . . 34.. . . . . . . . . . . . . . . . 33 

Shafer, RW. ..................20.. . . . . . . . . . . . . . . . 22 

Shimotohno, K.. . . . . . . . . . . . . . . 55.. . . . . . . . . . . . . . . . 60 

Shin, JW. ....................50.. . . . . . . . . . . . . . . . 53 

Simister, PC...................78.. . . . . . . . . . . . . . . . 84 

Smith, CA. ...................92.. . . . . . . . . . . . . . . 100 

Sozzi, T. ................. 86, 87.. . . . . . . . . . . . . 91, 92 

Swan, T. .....................90.. . . . . . . . . . . . . . . . 98 

Taylor, J.. . . . . . . . . . . . . . . . . . . . . 75.. . . . . . . . . . . . . . . . 77 

Temesgen, Z .. . . . . . . . . . . . . . . . . 89.. . . . . . . . . . . . . . . . 97 


Thommes, PA.................35.. . . . . . . . . . . . . . . . 34 

Tomaka, F .. . . . . . . . . 93, 94, 95, 96.. . 100, 101, 102, 103 

Tran, CV.................. 36, 37.. . . . . . . . . . . . . 35, 36 

Ujino, S......................38.. . . . . . . . . . . . . . . . 37 

Vaillant, A .. . . . . . . . . . . . . . . . . . . 25.. . . . . . . . . . . . . . . . 26 

Visvanathan, K.. . . . . . . . . . . . . . . 46.. . . . . . . . . . . . . . . . 50 

Wen, YM .. . . . . . . . . . . . . . . . . . . . 51.. . . . . . . . . . . . . . . . 54 

Wobbe, CR...................101.. . . . . . . . . . . . . . . 111 

Woo, G.. . . . . . . . . . . . . . . . . . . . . 112.. . . . . . . . . . . . . . . 123 

Yi, M.........................17.. . . . . . . . . . . . . . . . 17 

Zehnter, E .. . . . . . . . . . . . . 113, 114.. . . . . . . . . . . 124, 125 

Zheng, B.....................52.. . . . . . . . . . . . . . . . 55 

Zimmerman, KA...............76.. . . . . . . . . . . . . . . . 78 


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