Immunomodulatory Effects of the Hepatitis C Virus (HCV) Core Protein By Rachel Elizabeth Owen A thesis submitted for the degree of Doctor of Philosophy at the University of London September 2003 The Edward Jenner Institute for Vaccine Research University College London ProQuest Number: U642330 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642330 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Hepatitis C virus establishes a persistent infection in approximately 80% of infected individuals. It is unclear why the immune system is frequently unable to mediate a response capable of controlling HCV replication. Dendritic cells (DCs) may be an extrahepatic site of viral replication and it is hypothesised that expression of the core protein within DCs may impair their functions resulting in inadequate immune responses. To investigate potential immunomodulatory effects of the core protein on DCs, replication-deficient adenoviral vectors co-expressing different versions of the core protein (full-length, truncated and a version lacking residues 125-144) with GFP were constructed. Whilst these adenoviruses were being generated, experiments were carried out with an adenoviral vector expressing all the HCV structural proteins (Ad- CE1E2). These experiments revealed no effects of the core protein on DC phenotypic or functional maturation in response to LPS, TNFa or anti-CD40 stimulation. The core protein also had no effect on the ability of DCs to take up antigen and to stimulate allogenic and antigen-specific T cell responses. The core protein has also been proposed to alter the sensitivity of cells to apoptosis; experiments performed in this thesis did not support this, as the susceptibility of DCs to apoptosis induced via the Fas-Fas ligand, TRAIL and lymphotoxin pathways was not affected by core protein expression within these cells. The susceptibility of cells to lysis by natural killer cells and CDS" cytotoxic cells was also not altered by expression of the core protein within them. These results do not support the core protein making a contribution to HCV persistence by immunomodulatory effects on DCs, although it remains possible that core protein expression at higher levels and/or at different intracellular sites within DCs could affect their functions. Acknowledgements I would like to thank my supervisor Dr Persephone Borrow for the opportunity to work in her group and for her eternal optimism, support and encouragement during my PhD. Thank you to all of the members of the viral immunology group, both past and present for their help during my PhD: Julie Lewis, Miranda Ashton, Mai Lee Wong, Valerie Walshe and Eriko Yamada for technical support and friendship. Dr Mike Riffkin and Dr Fabrizio Mattei for help with molecular biology. Dr Lite Meier and Dr Matthew Edwards for helpful discussions and advice, and to Dr Maria Montoya and Dr Simone Gloster for technical help. Thank you to Dr Steven Smith for help making adenoviruses and to Andrew Worth for teaching me confocal microscopy. Thank you to our collaborators. Dr John McLauchlan for providing me with the HCV core protein fragments and antibodies required for this project. Dr Mario Stevenson for providing Ad-GFP and Dr Berwyn Clarke for providing Ad-CE1E2. I am grateful to GlaxoSmithkine for providing GM-CSF and IL-2 and to Dr He and Dr Vogelstein for providing me with the Ad-Easy system for generating recombinant adenoviruses. Thank you to all the volunteers within the Edward Jenner Institute who donated blood for my studies. I would also like to thank my family and friends for supporting me throughout the good and not so good times of my PhD! Table of Contents ABSTRACT 2 ACKNOWLEDGEMENTS 3 TABLE OF CONTENTS 4 LIST OF TABLES & FIGURES 8 ABBREVIATIONS 12 CHAPTER 1 - INTRODUCTION 16 1.1 Introduction 16 1.2 Hepatitis C Virus 17 1.2.1 HCV Classification 17 1.2.2 HCV Genotypes 18 1.2.3 HCV Quasispecies 20 1.3. Virology 21 1.3.1 HCV lifecycle 23 1.3.2 The Structural Proteins 25 1.3.2.1 The core protein 25 1.3.2.2 The Envelope Glycoproteins 29 1.3.3 HCV Cellular Receptors 31 1.3.4 The Non-Structural Proteins 33 1.3.4.1 F Protein 33 1.3.4.2 NS2&P7 33 1.3.4.3 NS3 35 1.3.4.4 NS4 35 1.3.4.5 NS5 36 1.3.5 Models of HCV Replication 37 1.3.5.1 Molecular Clones 38 1.3.5.2 Replicons 39 1.3.6 HCV Tropism 41 1.4 Human Infection & Disease Pathogenesis 42 1.4.1 HCV Transmission 42 1.4.2 Human HCV Infection & Pathogenesis 43 1.4.2.1 Acute HCV . 43 1.4.2.2 Chronic Hepatitis 44 1.4.2.3 Cirrhosis 45 1.4.2.4 Hepatocellular Carcinoma 46 1.4.2.5 Extrahepatic Manifestations 46 1.5 Treatment of HCV Infection & Vaccine Prospects 47 1.5.1 Current Treatment of HCV Infection 47 1.5.2 Prospects for a HCV Vaccine 49 1.6 Models of HCV Disease 55 1.6.1 The Chimpanzee Model 55 1.6.2 Alternative Non-human Primate Approaches 56 1.6.3 Mouse Models 57 1.7 The Immune Response during HCV Infection 59 1.7.1 Overview of the antiviral immune response 59 1.7.2 Innate Immune Responses, and their role in HCV infection 62 1.7.2.1 Type 1 Interferon Response 62 1.7.2.2 Other Innate Cytokines 65 1.7.2.3 Natural Killer & Natural Killer T Cells 66 1.7.2.4 Gamma Delta T Cells 68 1.7.2.5 Complement 68 1.7.3 Adaptive Immune Responses and their role in HCV infection 69 1.7.3.1 Humoral Responses 69 1.7.3.2 CD4* T Cell Responses 72 1.7.3.3 CD8"’ T Cell Responses 74 1.7.4 Immunopathological Role of the Immune Response in HCV-associated Liver Damage 80 1.7.5 Implications for Vaccine Development 82 1.8 Viral Immune Evasion Strategies 83 1.8.1 General Immune Evasion Strategies used by Viruses 83 1.8.1.1 Interference with the induction ormaintenance of host immune responses 84 1.8.1.2 Avoidance of recognition by the host immune response 85 1.8.1.3 Resistance to host effector mechanisms 87 1.8.2 HCV Immune Evasion Strategies 88 1.8.2.1 Type 1 Interferons 89 1.8.2.2 NK Cells 90 1.8.2.3 Antibody Response 91 1.8.2.4 T Cell Responses 92 1.8.3 Potential Involvement of the HCV Core Protein in HCV Evasion Strategies 94 CHAPTER 2 - MATERIALS & METHODS 97 2.1 Materials 97 2.1.1 Biochemical Reagents 97 2.1.2 Plastic ware 100 2.1.3 Restriction Enzymes 101 2.1.4 Plasmids 102 2.1.5 Tissue Culture Reagents 102 2.1.6 Cytokines 103 2.1.7 Antigens & Peptides 104 2.1.8 Cell Lines 104 2.1.9 Kits 104 2.1.10 Antibodies 104 2.2 Methods 107 2.2.1 Preparation & Quantification of Plasmid DNA 107 2.2.2 Preparation & Gel Purification of Plasmid Fragments 108 2.2.3 Subcloning Techniques 109 2.2.4 Plasmid DNA Maxipreps 109 2.2.5 Plasmid Recombination in E.coli BJ5183 Competent Cells 111 2.2.6 Polymerase Chain Reaction (PGR) 111 2.2.7 293 & 293T Cells 112 2.2.8 Generation of Recombinant Adenoviruses 113 2.2.9 CPE Assay 115 2.2.10 Generation of Ad-GFP & Ad-CEI E2 Stocks 116 2.2.11 Adenovirus Purification 117 2.2.12 Peripheral Blood Mononuclear Cell (PBMC) Isolation 118 2.2.13 Human Monocyte-Derived Dendritic Cell (DC) Production 118 2.2.14 Infection of Monocyte-derived Dendritic Cells with Adenoviruses 119 2.2.15 Assessment of GFP Expression by Flow Cytometry 120 2.2.16 Investigation of HCV Core Protein Expression by Confocal Microscopy 120 2.2.17 Stimulation of Monocyte-derived Dendritic Cell Maturation 121 2.2.18 Production of Monocle-derived Macrophages 122 2.2.19 Infection of Monocyte-derived Macrophages with Adenoviruses 122 2.2.20 Analysis of Cell Surface Marker Expression by Flow Cytometry 123 2.2.21 Dextran Uptake by Dendritic Cells 124 2.2.22 Cytokine ELISAs 125 2.2.23 Mixed Lymphocyte Reactions (MLR) 126 2.2.23.1 Proliferation 126 2.2.23.2 IL-2 Production & T Cell Surface Marker expression 127 2.2.24 Stimulation of antigen-specific T cell responses 128 2.2.24.1 IFNy ELISPOT Assay 128 2.2.24.2 Antigen-Specific Proliferation Assays 130 2.2.25 Induction of Apoptosis 131 2.2.26 Natural Killer (NK) Cell Assay 132 2.2.27 Generation of antigen-specific CD8* cytotoxic T cells 134 2.2.28 Culture of Epstein-Barr virus transformed B cell lines 135 2.2.29 CD8^ CTL Cytotoxicity Assays 135 CHAPTER 3 - GENERATION OF RECOMBINANT ADENOVIRUSES EXPRESSING THE HCV CORE PROTEIN 137 3.1 Introduction 137 3.2 Choice of HCV Core Protein and Fragments for Expression in Adenoviral Vectors.