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. 138
3.3 Microbix Method for Construction of Adenoviral Vectors 141 3.3.1 Sub-cloning core protein fragments into the El region within the transfer plasmid pCA13 143 3.3.2 Subcloning GFP into the E3 region of the Adenoviral Plasmid pBHGIO 147 3.3.3 Generation of Recombinant Adenoviruses by Co-Transfection of 293 cells 152
3.4 AdEasy Method for Constructing Adenoviral Vectors 158 3.4.1 Subcloning the HCV Core Fragments into pAdTrack-CMV 161 3.4.2 Co-transformation of E.coli BJ5183 Cells with pAdTrack-CMV-Core & pAdEasy-1 165 3.4.3 Expansion of Ad-Core-195, Ad-Core-169 & Ad-Core-124 167
3.5 Ad-GFP & Ad-CEI E2 170
3.6 Analysis of GFP & Core Protein Expression in Cells Infected with Different Adenoviral Recombinants 171
3.6 Discussion 183 CHAPTER 4 - EFFECTS OF THE HEPATITIS C VIRUS CORE PROTEIN ON DENDRITIC CELL ACTIVATION AND FUNCTIONS 190
4.1. Introduction 190
4.2 Maturation of Dendritic Ceils 192
4.3. Infection of Dendritic Cells with Adenovirus 198
4.4 Effects of the HCV core protein on the ability of DCs to uptake antigen 204
4.5 Effects of the HCV core protein on DC maturation in response to different stimuli 205
4.6 Effects of the core protein on IL-12 production by Macrophages 213
4.7 Effects of the core protein on the ability of DCs to stimulate allogenic & antigen- specific T cell responses 216
4.8. Discussion 227
CHAPTER 5 - EFFECTS OF THE HEPATITIS C VIRUS CORE PROTEIN ON THE SUSCEPTIBILITY OF DCS TO INDUCTION OF APOPTOSIS VIA DIFFERENT PATHWAYS 236
5.1 Introduction 236
5.2 Effects of the HCV core protein on apoptosis induced by the TNFR family molecules 241
5.3 Effect of the HCV core protein on apoptosis induced via the perforin & granzyme pathway 256
5.4 Discussion 266
CHAPTER 6 - GENERAL DISCUSSION 274
REFERENCES 283 List of Tables & Figures
Chapter 1 Page
Figure 1.1 The HCV genome encoded polyprotein. 22 Figure 1.2 A schematic model of type 1 interferon gene induction in virally infected cells. 63 Figure 1.3 The biological properties of Interferons. 64 Figure 1.4 A schematic outline of apoptosis pathways. 75
Table 1.1 The geographical distribution of HCV genotypes & subtypes 19 Table 1.2 HCV Vaccine Strategies under consideration. 51
Chapter 2
Table 2.1.1 Biochemical Reagents 97 Table 2.1.2 Plasticware 100 Table 2.1.3 Restriction enzymes 101 Table 2.1.4 Plasmids 102 Table 2.1.5 Tissue culture reagents 102 Table 2.1.6 Cytokines 103 Table 2.1.7 Antigens & Peptides 104 Table 2.1.8 Cell lines 104 Table 2.1.9 Kits 104 Table 2.1.10 Antibodies 104
Chapter 3
Figure 3.1 HCV core fragments derived from the pGEM plasmids provided by Dr J. McLauchlan. 140 Figure 3.2 Outline of the Microbix method for generating recombinant adenoviruses 142 Figure 3.3 Plasmids used for generation of recombinant adenoviruses by the Microbix method. 144 Figure 3.4 Strategy for cloning the HCV core fragments into the transfer plasmid pCAl 3. 145 Figure 3.5 Isolation of HCV core protein fragments by restriction enzyme digestion & agarose gel electrophoresis. 146 Figure 3.6 Screening for pCAl 3-HCV-core recombinants by C/al restriction enzyme digestion. 148 Figure 3.7 Strategy for cloning GFP into the Adenoviral genome plasmid pBHGIO. 149 Figure 3.8 Isolation of the GFP-containing fragment from pABS.4 recombinants by Pad digestion. 153 Figure 3.9 Confirmation of the presence of GFP in pBHGI 0-GFP. 154 Figure 3.10 Adenoviral plaques produced from transfection of 293 cells with the plasmid pFG140 (positive control plasmid containing a circular form of the Ad5 genome) revealed by neutral red staining. 157 Page Figure 3.11 The AdEasy method for generation of recombinant adenoviruses. 159 Figure 3.12 isolation of the HCV core protein fragments from plasmids pGEM/1-169, pGEM/1-195 and pGEM/1-124,145-195 by restriction enzyme digestion & agarose gel electrophoresis. 162 Figure 3.13 Strategy for cloning HCV core fragments into the shuttle plasmid pAdTrack-CMV. 163 Figure 3.14 Screening pAdlrack-CMV-HCV-core plasmids for the presence of HCV core protein fragments by Smal digestion. 164 Figure 3.15 Screening Ad-Easy-HCV-core recombinants with Pad digestion. 166 Figure 3.16 Checking for the presence of the HCV core sequences in recombinant pAdEasy-HCV-core genomes by PCR. 168 Figure 3.17 Determination of GFP expression in 293 cells infected with Ad- GFP & Ad-CEI E2. 172 Figure 3.18 Determination of GFP expression in 293 cells infected with Ad- Core-195, Ad-Core-169, Ad-Core-124 & Ad-Empty. 173 Figure 3.19 Expression of GFP in Ad-GFP infected DCs. 175 Figure 3.20 Uninfected DCs stained for HCV core protein. 176 Figure 3.21 Staining Ad-CEI E2 infected DCs with an isotype control antibody. 177 Figure 3.22 Expression of HCV core protein in Ad-CEI E2 infected DCs. 178 Figure 3.23 Expression of HCV core protein in Ad-Core-195 infected DCs. 179 Figure 3.24 Expression of HCV core protein in Ad-Core-124 infected DCs. 180 Figure 3.25 Expression of HCV core protein in Ad-Core-169 infected DCs. 181
Chapter 4
Figure 4.1 Maturation of DCs in response to LPS determined by phenotypic surface marker staining. 193 Figure 4.2 Maturation of DCs in response to TNFa determined by phenotypic surface marker staining. 194 Figure 4.3 Maturation of DCs in response to anti-CD40 determined by phenotypic surface marker staining. 195 Figure 4.4 Timecourse of GFP expression following Ad-GFP infection of immature DCs. 199 Figure 4.5 Infection of immature and mature DCs with Ad-GFP at a range of MOIS. 201 Figure 4.6 Effect of Adenovirus on DC maturation in response to LPS, TNFa and anti-CD40 stimulation. 203 Figure 4.7 Antigen uptake capacity of immature DCs infected with Ad-GFP & Ad-CEI E2. 206 Figure 4.8 Effect of HCV core protein expression in DCs on their phenotypic maturation in response to LPS stimulation. 207 Figure 4.9 Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad- CEI E2 infected DCs in response to LPS stimulation. 209 Figure 4.10 Effect of HCV core protein expression in DCs on their phenotypic maturation in response to TNFa stimulation. 211 Page Figure 4.11 Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad- CEI E2 infected DCs in response to TNFa stimulation. 212 Figure 4.12 Effect of HCV core protein expression in DCs on their phenotypic maturation in response to anti-CD40 stimulation. 214 Figure 4.13 Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad- CEI E2 infected DCs in response to anti-CD40 stimulation. 215 Figure 4.14 IL-12 p40 production by uninfected, Ad-GFP and Ad-CEI E2 infected macrophages in response to LPS stimulation. 217 Figure 4.15 Determination of the allostimulatory capacity of uninfected, Ad- GFP & Ad-CEI E2 infected DCs by measurement of CD3^ T cell 219 proliferation. Figure 4.16 Determination of the allostimulatory capacity of uninfected, Ad- GFP & Ad-CEI E2 infected DCs by measurement of IL-2 production. 220 Figure 4.17 Determination of the allostimulatory capacity of uninfected, Ad- GFP & Ad-CEI E2 infected DCs by measurement of T cell activation marker expression. 222 Figure 4.18 Determination of the allostimulatory capacity of uninfected, Ad- GFP & Ad-CEI E2 infected DCs by measurement of T cell activation marker expression. 223 Figure 4.19 Comparison of the ability of uninfected, Ad-GFP & Ad-CEI E2 infected DCs to stimulate CD4* T cell responses to PPD, as measured by an IFNy ELISPOT assay. 225 Figure 4.20 Comparison of the ability of uninfected, Ad-GFP & Ad-CEI E2 infected DCs to stimulate CD4"^ T cell responses to PPD, as measured by a proliferation assay. 226 Figure 4.21 Comparison of the ability of uninfected, Ad-GFP & Ad-CEI E2 infected DCs to stimulate CDB^ T cell responses to a CMV epitope, as measured by an IFNy ELISPOT assay. 228
Chapter 5
Figure 5.1 A schematic outline of apoptosis pathways. 237 Figure 5.2 Induction of apoptosis in Jurkat cells by an agonistic anti-Fas antibody, measured by annexin V staining. 243 Figure 5.3 Induction of apoptosis in Jurkat cells by TRAIL, measured by annexin V staining. 244 Figure 5.4 Induction of apoptosis in Jurkat cells by LTa2p1 & LTal (32 measured by annexin V staining. 245 Figure 5.5 Induction of apoptosis in uninfected, Ad-GFP & Ad-CEI E2 infected Hep16 cells measured by annexin V staining. 247 Figure 5.6 Induction of apoptosis in uninfected, Ad-GFP & Ad-CEI E2 infected Hep3B cells measured by annexin V staining. 248 Figure 5.7 Induction of apoptosis in uninfected DCs by an agonistic anti- Fas antibody measured by annexin V staining. 249 Figure 5.8 Induction of apoptosis in uninfected DCs via the TRAIL pathway, measured by annexin V staining. 250
10 Page Figure 5.9 Induction of apoptosis in uninfected DCs by TNFa, LTaip2 & LTa2p1, measured by annexin V staining. 252 Figure 5.10 Effect of the HCV core protein on induction of apoptosis in immature & mature DCs by an agonistic anti-Fas antibody, measured by annexin V staining. 253 Figure 5.11 Effect of the HCV core protein on induction of apoptosis of immature DCs by TNFa, TRAIL, LTa2p1 & LTa1p2, measured by annexin V staining. 254 Figure 5.12 Effect of the HCV core protein on induction of apoptosis in uninfected, Ad-GFP & Ad-CEI E2 infected monocyte-derived macrophages, measured by annexin V staining. 255 Figure 5.13 NK cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected K562 target cells. 257 Figure 5.14 NK cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected hepatoma cell lines. 259 Figure 5.15 Expression of MHC class I by uninfected, Ad-GFP & Ad-CEI E2 infected Hepatoma cell lines. 260 Figure 5.16 NK cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected DCs & K562 target cells. 261 Figure 5.17 NK cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected monocyte-derived macrophages. 262 Figure 5.18 CDS'" cytotoxic T cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected B cell targets. 264 Figure 5.19 CDB"^ cytotoxic T cell lysis of uninfected, Ad-GFP & Ad-CEI E2 infected DC targets. 265
11 Abbreviations
yô gamma delta “C degrees Celsius Mg micrograms Ml micro litre 2’5’AS 2’5’ adenylate synthetase aa amino acids Ad Adenovirus Ad5 Adenovirus type 5 ADCC antibody dependant cell cytotoxicity Ad-CE1E2 Adenovirus-core-envelope-1-envelope-2 Ad-GFP Adenovirus-green fluorescent protein ALP alkaline phosphatase ALT alanine aminotransferase AP-1 activator protein -1 APC allophycocyanin ATP adenosine triphosphate BCG M. bovis Bacille Calmette-Guerin BDV Border disease virus BDVD bovine viral diahorrea disease BHK baby hamster kidney cell line BLCL B lymphoblastoid cell line bp base pair BSA bovine serum albumin 0 core CD cluster differentiation CDC Centre for disease control CIDE-B cell death inducing DFF45-like effector-B CIP calf intestinal alkaline phosphatase CMV Cytomegalovirus CO2 carbon dioxide COOH carboxylic CPE cytopathic effect CSFV classical swine fever virus CTL cytotoxic T lymphocyte DC dendritic cell DC-SIGN DC-specific, ICAM-3 grabbing, non-integrin DD death domain DISC death-inducing signalling complex DMEM Dulbecco's miminal essential medium DMSO dimethylsulphoixide DNA deoxyribonucleic acid DNTPs deoxynucleotide triphosphates dsRNA double stranded ribonucleic acid E:T effectortarget E1 envelope glycoprotein 1 E1 early region 1
12 E2 envelope glycoprotein 2 E3 early region 3 EBV Epstein Barr Virus EBNA Epstein Barr virus nuclear antigen EDTA ethylenediaminetetraacetic acid elF-2 protein initiation factor -2 EJIVR Edward Jenner Institute for Vaccine Research ELISA enzyme linked immunosorbent assay ELISPOT enzyme linked immunospot assay ER endoplasmic reticulum FACS fluorescent activated cell sorting FADD Fas-associated death domain protein FcR fragment crystallisable receptor FCS foetal calf serum FL-1 fluorescence -1 FLICE Fas-associating protein with death domain-like interleukin-1 converting enzyme FURR FLICE inhibitory proteins GAGs glycosyaminoglycans GBV-B GB-virus B GFCS good foetal calf serum GFR green fluorescent protein GM-CSF granulocyte-macrophage-colony stimulating factor Grb2 growth factor receptor bound protein 2 H2SO4 sulphuric acid HBV Hepatitis B virus HCMV Human cytomegalovirus HCV Hepatitis C Virus HIV Human immunodeficiency virus HLA human leukocyte antigen hnRNRK heterologous nuclear ribonucleoprotein K HRSF high purity salt free HRR horseradish peroxidase HSR90 heat shock protein 90 HSV Herpes simplex virus HTLV-1 Human leukaemia virus HVR1 hypervariable region 1 ICAM-1 intracellular adhesion molecule -1 IFN interferon ig immunoglobulin IL interleukin IMRDH inosine monophosphatase dehydrogenase 1RES internal ribosome entry site IRF interferon regulatory factor ISDR interferon sensitivity determining region IV intravenous JAK Janus kinase kb kilo base kDa kilodaltons LB Luria broth LCMV Lymphocytic choriomeningitis virus
13 LDL low density lipoprotein LE FCS low endotoxin foetal calf serum LFA-3 leukocyte function antigen-3 LPS lipopolysaccharide L-SIGN liver/lymph node-specific intracellular adhesion molecule- 3-grabbing integrin LT lymphotoxin LTPR lymphotoxin p receptor LTR long terminal repeat M Molar mAB monoclonal antibody MACS magnetic activated cell sorting MAP mitogen activated protein kinase MCP-1 monocyte-chemoattractant protein -1 M-CSF macrophage-colony stimulating factor MEM Minimum essential medium MFI mean fluorescent intensity MHC major histocompatibility complex MIP-1P macrophage inflammatory protein- 1-beta ml millilitre mm milimeter MO! multiplicity of infection mRNA messenger RNA NANBH Non A, non B hepatitis NFkB nuclear factor kappa B ng nanogram NHz amino NK natural killer NKT natural killer T cell nm nanometer NOB neutralisation of binding NS Non-structural PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PCR polymerase chain reaction PE phycoerythrin Peg Polyethylene glycol PePHD PKR-elF2a phosphorylation site homology domain PerCP peridinin chlorophyll protein PERK PKR-like ER resident kinase PKA cyclic AMP-dependant protein kinase PKR dsRNA-dependant protein kinase PPD purified protein derivative RANTES regulated on activation, normal T cell expressed and secreted RNA ribonucleic acid RNAi RNA interference rpm revolutions per minute RPMI Rose Park Memorial Institute RSV Rous sarcoma virus siRNA synthetic small interfering RNA
14 SIV Simian immunodeficiency Virus STAT signal transducers and activators of transcription SV40 Simian virus 40 TAP transporter associated with antigen processing IE tris-EDTA Th T helper TLR toll like receptors TMB tetramethylbenzidine INF tumor necrosis factor TNFa tumour necrosis factor alpha TNFR tumour necrosis factor receptor TRADD tumour necrosis factor receptor-associated death domain TRAF tumour necrosis factor receptor-associated factor TRAIL tumour necrosis factor related apoptosis inducing ligand UTR untranslated region UV ultraviolet VLP virus-like particles VZV Varicella zoster virus WHO World Health Organisation
15 Chapter 1 - Introduction
1.1 Introduction
Hepatitis C virus (HCV) was found to be the predominant cause of transfusion-
associated non-A, non-B hepatitis (NANBH) in the 1980’s. Recent estimates by the
World Health Organisation (WHO) suggest that 200 million people are now infected with this virus world-wide, with 3-4 million new infections occurring every year (Crabb,
2001). Particularly high infection rates are present in Egypt, where 19.2% of the
population is HCV seropositive (Van der Poel, 1994); Europe and the Americas are
estimated to have a HCV prevalence of 1-2.4%.
Over 80% of HCV-infected individuals fail to eliminate the infection, with many
developing complications such as cirrhosis and hepatocellular carcinoma. Currently,
treatment options are inadequate; only 20% of chronically infected individuals respond
to interferon alpha therapy (Cohen, 1999). There is no vaccine available to prevent or
help to eliminate HCV infection.
The development of prophylactic and therapeutic strategies to combat HCV infection is
hampered by problems associated with studying this virus. HCV cannot be grown in
vitro in laboratory cultures and there is still a lack of a good small animal model.
Currently chimpanzees are the only animals which can become infected by HCV and
undergo a similar disease course to that seen in humans (Walker, 1997). Therefore
many questions remain unanswered about HCV and how it causes disease.
16 1.2 Hepatitis C Virus
1.2.1 HCV Classification
HCV was found to be the cause of transfusion associated non-A, non-B hepatitis in the
1980’s. The virus was not identified until 1988 and its genome was cloned in 1989
(Choo et al., 1989). Since then, HCV has been classified as a member of the
Flaviviridae family. This family includes three genera; the Fiaviviruses, the Pestiviruses and the Hepaciviruses (hepatitis C viruses). These genera have diverse biological properties and have no serological cross reactivity. However, they do have similar virus morphology and genome organisation and they are presumed to have the same replication strategy (Rice, 1996), although HCV’s replication strategy is still unknown.
There are more than 68 members of the Flavivirus genus (Rice, 1996). Most of these are arthropod-borne and are transmitted by mosquito or tick vectors. Diseases caused by members of the Flavivirus genus of global concern include dengue fever, Japanese encephalitis. Western Nile encephalitis and yellow fever.
The Pestiviruses include three serologically related animal pathogens (Moennig and
Plagemann, 1992), bovine viral diarrhoea virus (BDVD), classical swine fever virus
(CSFV) and border disease virus (BDV).
The Hepacivirus genus contains the hepatitis C virus. Humans are the only known natural host for HCV and there is no evidence of vector-mediated transmission.
17 1.2.2 HCV Genotypes
HCV isolates that have been collected throughout the world have been found to have significant genetic diversity (Bukh et al., 1995; Okamoto and Mishiro, 1994; Simmonds,
1995). The genomic sequences of the most distantly related hepatitis C virus isolates have as much as 35% variation (Farci and Purcell, 2000). This variability is comparable to that seen in other virus families such as the Flaviviridae and the
Picornaviridae.
The HCV isolates have been grouped into six major genotypes (denoted 1-6) and further subtypes (denoted a, b, c etc) (Simmonds et al., 1993). The distribution of the major genotypes varies throughout the world; however type 1 is the most common, causing 60% of infections world-wide (Bukh et al., 1993; McOmish et al., 1994). The geographical distribution of the genotypes and subtypes are detailed in table 1.1. The most common genotypes in Europe, America and Japan are types 1, 2 and 3 (Farci and Purcell, 2000). Genotypes 2 and 3 also have a wide distribution, whilst genotype 4 is a Pan-African type and the main genotype in South Africa is type 5 and it has rarely been detected outside of Africa (Bukh et al., 1993; Farci and Purcell, 2000; McOmish et al., 1994; Simmonds et al., 1993). Similarly genotype 6 has only been found in Asia
(Bukh et al., 1993; Farci and Purcell, 2000; McOmish et al., 1994; Simmonds et al.,
1993).
Studies of the different genotypes have not indicated any major biological differences: all genotypes have been found to be pathogenic, hepatotropic and all induce chronic hepatitis C. However, there is agreement that the genotype can influence the outcome of antiviral therapy, as patients infected with genotype 1 (particularly 1b) and genotype
4 respond poorly to interferon alpha therapy compared to patients infected with
18 Genotype Subtype Geographical Reference Distribution 1 Europe America Japan (Farci and Purcell, 2000) a W. Europe N. America b 8 & E. Europe Japan 0 Indonesia 2 a Western countries (Farci and Purcell, 2000) 0 N. Italy 3 b Japan Thailand Nepal (Farci and Purcell, Indonesia 2000) c-f Nepal 4 Africa (Farci and Purcell, 2000) a Egypt 5 S. Africa (Bukh et al., 1993; Farci and Purcell, 2000; McOmish et al., 1994; Simmonds et al., 1993) 6 Asia (Bukh et al., 1993; Farci and Purcell, 2000; McOmish et al., 1994; Simmonds et al., 1993)
Table 1.1. The geographical distribution of HCV genotypes & subtypes.
19 genotypes 2 and 3 (Farci and Purcell, 2000). As yet there is no agreement on whether the genotype affects transmission, infectivity, pathogenesis and the natural history of the disease (Farci and Purcell, 2000).
1.2.3 HCV Quasispecies
Hepatitis 0 virus was first described as a quasispecies by Martell et al (Martell et al.,
1992) and further studies in chimpanzees and humans have also confirmed that HCV exists as a quasispecies (Higashi et al., 1993; Kato et al., 1992; Murakawa et al., 1992;
Tanaka et al., 1992; Yanagi et al., 1997a; Yanagi et al., 1998).
Quasispecies is a term used to describe a diverse and rapidly evolving population, the members of which are related but have distinct genomes. Viruses with RNA genomes generally exist as a quasispecies. RNA viruses encode an error-prone RNA replicase which lacks a proof reading function, thus every round of replication can results in the generation of multiple mutants with slightly different genomes. The viral quasispecies usually consist of a “master” genome that is predominant and many minor genomes that represent the variant genomes within the whole population. The average rate of mutation within the genome of RNA viruses is estimated to be 10'^ to lO"® mutations per genome and replication cycle (Domingo and Holland, 1994), therefore after a few replication cycles there is a very heterogeneous population of viruses. The average rate of mutation within the HCV genome has been estimated at 1.44 to 1.92 x 10"^ substitutions per genomic site per year (Gomez et al., 1999). This was estimated by a comparison of consensus sequences of isolates obtained from humans and chimpanzees at intervals of 8 to 13 years (Ogata et al., 1991; Okamoto et al., 1992).
20 The rapid evolution of RNA viruses is a survival strategy that enables the virus to adapt to pressure from the host environment. The existence of viral quasispecies within HCV infections could be an important mechanism used by the virus as an evasion strategy to allow the establishment of persistent infections in the face of the host immune response. Other biological implications of viral quasispecies include generation of mutants resistant to antiviral therapies and the potential to change the cellular tropism of the virus. There have been studies carried out to determine whether the diversity of the HCV quasispecies has an effect upon the outcome of infection, the progression of liver disease and the responsiveness to antiviral treatment. However, many conflicting results are reported and this area still requires further clarification.
1.3. Virology
Hepatitis C virus has a linear, positive-sense, single stranded 9.4kb RNA genome which contains a single open reading frame extending most of its length. This encodes a large polyprotein precursor that is flanked by highly conserved untranslated regions
(UTRs) at the 3’ and 5’ termini. This precursor is cleaved into individual structural and non-structural (NS) proteins at the sites indicated in figure 1.1, by host signal peptidases or virally encoded metalloproteases. Cleavage of the polyprotein takes place post-translationally. The structural proteins consist of a RNA binding nucleocapsid protein (core) and envelope glycoproteins (El and E2). The non- structural proteins consist of virally encoded proteases, a helicase, and the RNA- dependent RNA polymerase, which are required for viral replication.
21 Cleavage by NS2 Cleavage by NS3 Cleavage by host signal (+NS3) protease (+NS4) peptidase metal loproteinase
r Poly A tail 5- C l c E l E2 P7 NS2 NS3 14S4 NS5A NS5B 341nt A B 27-66nt
9030-91 )99nt. (3010-31 )33aa)
Vine n Stru )tural t lonsti uctural (NS) I Yoteins Prot 3 iins
Component of RNA binding Zn^* métallo- Ser Protease/ viral replication RNA-dependant nucleocapsid Envelope proteinase Helicase complex RNA polymerase glycoproteins
Required by IFN resistance NS3 protease & unknown role & interacts in viral with NS5A replication
Figure 1.1. The HCV genome encoded poly protein. The HCV polyprotein precursor is shown schematically. The viral structural proteins are cleaved by host cell signal peptidases into individual proteins (core and the envelope glycoproteins, E1 and E2). Cleavage of the non-structural proteins is mediated by virally encoded proteases, as indicated, in an autocatalytic manner. The functions of the individual proteins are also indicated.
22 1.3.1 HCV lifecycle
Relatively little is known about the lifecycle of HCV as the virus cannot be grown in vitro and there is a lack of small animal model systems. The first steps of a viral lifecycle involve binding to the host cell, entry and viral uncoating. Several putative receptors or co-receptors have been proposed for HCV including CD81, low density lipoprotein
(LDL), the human scavenger receptor class B type 1 and glycosyaminoglycans (these are discussed in more detail later on) which may bind to the viral envelope proteins.
However, viral entry followed by viral replication has not been demonstrated yet. It is presumed that viral entry involves host endosomes and this may be the site of viral uncoating to release the HCV genomes into the cell cytoplasm for replication and translation of viral proteins.
The exact process of HCV replication remains poorly characterised due to the lack of model systems. However, similarities to the replication strategy of related Fiaviviruses are expected for HCV. HCV replication is thought to take place in the cytoplasm of infected cells. The HCV proteins and genome template along with host factors are thought to form a replication complex in association with perinuclear membranes
(Egger et al., 2002; Hijikata et al., 1993; Ishido et al., 1998; Koch and Bartenschlager,
1999; Lin et al., 1997; Neddermann et al., 1999). Recently it has been suggested that
HCV replication occurs on lipid rafts that have been recruited from the intracellular membranes separate from the endoplasmic reticulum (ER) (Shi et al., 2003). Viral proteins have also been detected in the Golgi apparatus and ER, so these are also likely sites involved in HCV replication (Serafino et al., 2003). It is likely that HCV replication occurs by the synthesis of a full-length negative-strand RNA initially, which acts as a template for positive-strand RNA synthesis, resulting in the production of
23 progeny genomes. These new genomes are translated and viral proteins and genomes packaged into new virions.
The 3’-most 150 nucleotides of the HCV genome (part of the 3’ UTR) have been found to contain RNA signals which are essential for replication (Yi and Lemmon, 2003). This region contains a poly U/UC tract, which forms part of a stem loop structure. Mutations within this region stopped replication, proving it is essential for replication (Friebe and
Bartenschlager, 2002). The stem loop I is thought to facilitate recognition of the 3’ end of the viral RNA by the RNA replicase (Yi and Lemon, 2003). The 5’ UTR also contains structures essential for HCV replication. It contains a 5’ proximal stem loop RNA element and an 1RES (internal ribosome entry site) element which modulate replication and translation (Luo et al., 2003). It is thought that domains I and II of the 1RES are necessary for replication (Kim et al., 2002b). The 5’ UTR of HCV is also thought to have strong promoter activity (Dumas et al., 2003). Translation of the polyprotein is mediated by the 5’ 1RES in a cap-independent manner and is stimulated by the non structural proteins (He et al., 2003; Li et al., 2003a), however the regulatory processes involved remain poorly understood.
HCV polyprotein processing is mediated by three proteases, two virally encoded and one host cell protease that is thought to be a signal peptidase present in the lumen of the endoplasmic reticulum. It is thought that this signal peptidase is responsible for the cleavage of the viral glycoproteins. Complete cleavage between E1 and E2 occurs rapidly during or straight after translation, however cleavage between E2 and p7/NS2 can be incomplete. This results in two forms of E2 (fully processed E2 and uncleaved
E2-p7), however the significance of these two forms in virion assembly are unknown.
Production of the mature capsid protein (core) results from cleavage of the COOH- terminus but it is not known what catalyses this cleavage (Reed and Rice, 2000). The cleavage of the non-structural proteins involves two viral proteases: the zinc stimulated
24 NS2-3 protease and the NS3 serine protease. The NS2-3 protease only seems to cleave at the NS2/3 site, by an autocatalytic mechanism (Grakoui et al., 1993a; Hijikata et al., 1993). Cleavage at the NS3/4 site is also an autocatalytic event, whilst processing at the NS4A/4B, NS4B/5A and NS5A/5B sites is not thought to be autocatalytic but still occurs rapidly.
As with viral RNA replication, little is known about the assembly and release of virions.
The packaging signals of the HCV genome have not yet been characterised, although the core protein is thought to be required for the formation of viral nucleocapsids containing the genomic RNA. The envelope proteins are inserted into the membrane of the ER and become glycosylated in the lumen of the ER. During virion assembly, the core protein which forms the viral capsid is thought to associate with the cytoplasmic side of the ER membrane. It is likely that viral nucleocapsids acquire the envelope glycoproteins by budding into the endoplasmic reticulum, as El and E2 have ER retention signals (Reed and Rice, 2000). Virions then pass through the host secretory pathways to be released at the cell surface from the infected cell. HCV viral particles are thought to be about 50nm in diameter (He et al., 1987; Shimizu et al., 1996).
1.3.2 The Structural Proteins
1.3.2.1 The core protein
The core protein forms the viral nucleocapsid, which is enveloped during virion morphogenesis. The mature core protein is 191 amino acids long, with the last 20 amino acids forming a signal peptide for El. The core protein has a molecular weight of 21 to 23kDa (Hijikata et al., 1991b) and can be seen as two forms called p21 and p23. These have been found in all HCV strains examined. Initial polyprotein cleavage
25 generates p23, which is thought to be an immature version of the core protein. This undergoes additional processing to yield p21 (Grakoui et al., 1993c; Harada et al.,
1991; Hijikata et al., 1991b). The core protein that has been isolated from infectious sera corresponds to p21 so this is likely to be the mature core present in viral particles
(Yasui et al., 1998). The production of the p21 form of the core protein by C-terminal cleavage of p23 is considered to be crucial for viral assembly and replication and it is the C-terminal sequence which regulates processing of the core protein (Kato et al.,
2003). The core protein is cleaved from El by cellular signal peptidases in the ER.
Further proteolytic processing can result in the generation of truncated forms of the core protein. These can be truncated at residues 182, 178 (Hussy et al., 1996), 171
(Liu et al., 1997) or 153 (Lo et al., 1994). The regulation and functional significance of core protein cleavage is not clear, however unlike the full-length core protein, truncated proteins shorter than 179 amino acids are translocated into the nucleus where they may have different functions.
The full-length core protein has two distinct domains, a NH 2 domain that is highly charged and allows binding to ribosomes and cellular DMA and RNA (Santolini et al.,
1994), and a COOH domain that is hydrophobic and responsible for binding the core protein to the ER membrane within cells. The NH 2-terminal domain contains several nuclear localisation signals, thus when the COOH-terminal domain is removed, the truncated proteins are translocated into the nucleus. In natural infections of humans and chimpanzees core protein expression has only been found in the cytoplasm of infected cells. It is rarely found in the nucleus of infected cells, although Kawamura et al (Kawamura et al., 1997) did detect nuclear core expression in a core transgenic mouse line. However, nuclear expression of the core protein may have a role in regulating cellular gene functions.
26 The core protein is thought to have several cytoplasmic functions, including binding viral RNA (Hwang et al., 1995; Santolini et al., 1994), multimerisation with other capsid proteins (Matsumoto et al., 1996; Nolandt et al., 1997) and interactions with El (Lo et al., 1996), all of which have implications in virion assembly. The core protein has been shown to associate with ribosomal sub-units (Santolini et al., 1994) which may be important in the uncoating process. In addition the core protein associates with lipid droplets, co-localising with apolipoprotein A2 (Barba et al., 1997). This may account for HCV-induced steatosis due to changes in lipid metabolism. Steatosis results when there is an accumulation of fat droplets in the cytoplasm of cells when there is interference with normal fat metabolism and mainly occurs in the liver. Steatosis has also been observed in a line of core transgenic mice (Moriya and Yotsuganagi, 1997).
The role of core expression in the nucleus of cells is controversial and several different functions have been proposed. The core protein has been reported to modulate the activity of promoters of both cellular and viral genes; some promoters are activated by core whilst others are suppressed (Alisi et al., 2003; Chang et al., 1998; Kwun and
Jang, 2003; Ohkawa et al., 2003; Oka et al., 2003; Ray et al., 1997; Ray et al., 1998b;
Shih et al., 1993). Cellular promoters that may be modulated by the HCV core protein include c-myc, c-fos, p53 and p21 and viral promoters include the Rous sarcoma virus
(RVS) LTR, HIV LTR, SV40 and HBV promoters. Modulation of promoter activity has the ability to disrupt the normal function of the host cells and may be related to the development of hepatocellular carcinoma in some hepatitis C infected patients. HCV core protein has also been described to have transforming properties under certain conditions (Chang et al., 1998; Li et al., 2003b; Ray et al., 1996; Tsuchihara et al.,
1999). This may be related to the ability of the core protein to suppress the promoter activity of the p53 gene (Ray et al., 1997) and one of the target genes p21 (Ray et al.,
1998b). A study by Tsutsumi et al (Tsutsumi et al., 2002) found that components of some signalling pathways (c-jun, N-terminal kinase & AP-1) were enhanced in core
27 transgenic mice which, they propose may have a role in hepatocellular carcinoma.
Moriya et al (Moriya et al., 1998) have found the core protein to induce hepatocellular carcinoma in a core protein transgenic mouse line, again suggesting that the core protein may have a role in the development of hepatocellular carcinoma in humans, although this remains to be confirmed. The core protein has also been found to modulate hepatocyte cell cycle, which may also be involved in the development of hepatocellular carcinoma (Nguyen et al., 2003).
It is not clear whether the core protein binds directly to promoter elements or has an indirect effect by binding transcription factors. The latter may be the case as for example, HCV core protein has been shown to bind heterologous nuclear ribonucleoprotein K (hnRNPK) (Hsieh et al., 1998) which is involved in cellular pre- mRNA splicing and nuclear RNA transport. The core protein has also been shown to bind to RNA helicase (You et al., 1999).
The core protein has also been shown to interact with members of various signalling pathways. For example, core can activate or suppress the transcription factor NFkB in different cell lines (Marusawa et al., 1999; Shrivastava et al., 1998; Tsutsumi et al.,
2002). It can also activate AP-1 and Elk-1 which are part of the MAP kinase signalling pathway (Fukuda et al., 2001; Shrivastava et al., 1998). The core protein has also been shown to induce tyrosine phosphorylation and STAT-1 activation in hepatic cells, which may impair cytokine signalling pathways (Bode et al., 2003). These interactions may have an important role in the disruption of cell regulation and the transformation of cells.
It has also been shown that the core protein can interact with the cytoplasmic domains of several members of the tumour necrosis factor receptor (TNFR) family. These
28 include the lymphotoxin p receptor (LTpR) (Chen et al., 1997; Matsumoto et al., 1997) and TNFR-1 (Zhu et al., 1998) which are important molecules in cytokine signalling pathways. If the interaction of core with these receptors results in altered signal transduction or sensitivity to cytokines, this could have important implications with regard to disruption or modulation of the host immune response, which is discussed in more detail later. The core protein may also bind other members of the TNFR family including the Fas antigen which may sensitise cells to Fas-mediated apoptosis
(Ruggieri et al., 1997). There is also a report of the core protein inducing CC chemokine expression (MCP-1 and RANTES) which could affect the recruitment of cells of the immune system to virally infected cells (Meng Soo et al., 2002).
There are many conflicting reports of pathogenic roles of the HCV core protein. This may be due to the different cell culture systems or mouse strains used to study the core protein. The role of the core protein during viral replication and its role in liver pathology in a natural infection both remain incompletely understood.
1.3.2.2 The Envelope Glycoproteins
The envelope glycoproteins El and E2 have been shown to be N-terminally glycosylated at multiple sites (Grakoui et al., 1993c; Hijikata et al., 1991a; Hsu et al.,
1993). Both proteins have a COOH-terminal domain that is thought to be inserted into the membrane of the ER whilst the bulk of the protein is translocated into the ER lumen
(Flint and McKeating, 2000). El and E2 are thought to form heterodimers
(Deleersnyder et al., 1997; Dubuisson et al., 1994; Dubuisson and Rice, 1996) which are held together by non-covalent interactions, although heterogeneous disulphide linked aggregates have also been observed (Dubuisson et al., 1994; Grakoui et al.,
1993c). The retention of non-covalently linked heterodimers of El and E2 in the ER has been shown by Kien et al (Kien et al., 2003). The formation of E1-E2 complexes
29 can be monitored by conformation dependant monoclonal antibodies and these studies suggest that the COOH-terminal domain of E2 is necessary for correct folding of E1
(Cocquerel et al., 1998; Cocquerel et al., 2002; Cocquerel et al., 2003; Michalak et al.,
1997; Patel et al., 2001). The transmembrane region of E2 appears to be multifunctional and has a role as an anchor. It also contains signalling sequences, so is likely to have a role in localisation and assembly of virions (Cocquerel et al., 2001).
E2 has been found to bind and inhibit PKR-elF2a phosphorylation site homology domain (PePHD) and the PKR-like ER resident kinase (PERK), which could interfere with signalling within host cells and induce interferon (IFN) resistance (Pavio and Lai,
2003; Pavio et al., 2002).
The envelope glycoproteins are proposed to have a role in the binding and uptake of
HCV into host cells. The E2 glycoprotein is thought to act as the virion attachment protein. It has been shown to bind to several putative host cell receptors (see below).
It is a major target for antibodies in the infected host. The E2 protein contains a hypervariable region (HVR1) (Kurosaki et al., 1993; Ogata et al., 1991) within the NH 2- terminal region between amino acids 1 to 27 of the E2 protein (384-410 of the polyprotein). This variability appears to be driven by antibody selection of immune escape variants (Farci et al., 1994; Farci et al., 1996; Kato et al., 1993; Sekiya et al.,
1994; Shimizu et al., 1996; Weiner et al., 1992). The HVR1 varies within isolates and between genotypes, although no association between HVR1 sequence and particular genotypes have been made. This variability may be a mechanism that HCV uses to evade host immune responses and is discussed in detail later.
The El protein has been proposed to act as the virion fusion protein as its C-terminal hydrophobic region (amino acids 331-383) has been shown to be responsible for membrane association and inducing changes in membrane permeability (Ciccaglione et al., 2001; Ciccaglione et al., 2000) However another study by Takikawa et al
30 (Takikawa et al., 2000) suggests that induction of cell fusion requires E1 and E2 as well as appropriate host cell components (see below).
1.3.3 HCV Cellular Receptors
Many research groups are trying to identify the receptor(s) HCV uses to infect host
cells, and there is a great deal of controversy over this issue. A number of different host
molecules have been shown to bind recombinant HCV glycoproteins, virus-like
particles and pseudoparticles and have thus been proposed to have a role in the HCV
attachment and entry process. These include; CD81, (Bartosch et al., 2003a; Levy et al., 1998; Petracca et al., 2000; Pileri et al., 1998; Roccasecca et al., 2003; Triyatni et al., 2002), low density lipoprotein (LDL) (Bartosch et al., 2003a; Hijikata et al., 1991a;
Manzini et al., 1998; Miyamoto et al., 1992; Monazahian et al., 1999; Prince et al.,
1996), glycosyaminoglycans (GAGs) (Barth et al., 2003; Germi et al., 2002; Penin et al., 2001), the human scavenger receptor class B type 1 (Bartosch et al., 2003a;
Scarselli et al., 2002), lactoferrin (Nozaki et al., 2003; Yi et al., 1997) and more recently
DC-SIGN (DC-specific, I CAM-3 grabbing, non-integrin) and L-SIGN (Liver/lymph node specific intracellular adhesion) (Lozach et al., 2003; Pohlmann et al., 2003).
All of these receptors have been found to bind HCV envelope proteins with different
affinities, for example, the binding of GAGs and CD81 is moderate compared to LDL
(Germi et al., 2002). Attempts have been made to correlate expression patterns with
the cell types infected with HCV (Bartocsh et al., 2003b; Meola et al., 2000). However
this cannot yield definitive conclusions about the roles played by these putative
receptors. There may be multiple receptors or additional factors such as co-receptors
which have not been identified as yet involved in the binding and entry of HCV into
31 susceptible host cells. It is also conceivable that HCV may use different receptors to infect different cell types.
In the absence of in vitro HCV infection systems, it has been difficult to explore the role(s) played by these multiple putative receptor components in HCV attachment and entry. However, the recent development of HCV infectious pseudoparticles by Bartosh et al, (Bartosch et al., 2003b) has allowed such interactions to be examined. The pseudoparticles were produced by putting unmodified and functional HCV glycoproteins on retroviral and lentiviral core particles with green fluorescent protein, which allowed infectivity to be determined. Primary hepatocytes and hepatocellular carcinoma cells were the major targets of infection in vitro and high infectivity required the E1 and E2 glycoproteins. However, CD81 nor LDL were sufficient to mediate HCV cell entry, again indicating that other unknown host cell factors are needed for infection.
These pseudo-particles may mimic early steps of HCV infection, but the various steps involved in HCV entry (fusion, internalisation and cell penetration) are likely to be mediated by different molecules which remain to be identified.
It is important to identify the receptors HCV uses for entry into host cells to allow inhibition of infection. Some of the interactions of HCV proteins with host cell receptors that have been identified so far have roles other to/or in addition to roles in the HCV attachment and entry process. For example, CD81 interactions have implications in modulating the host immune response (discussed in more detail later). The interaction with DC-SIGN may be involved with transmission of HCV, as has been observed in HIV infection (Geijtenbeek and van Kooyk, 2003; Nobile et al., 2003; Soilleux, 2003) where
HIV-1 uses binding to DC-SIGN to enable its transport to other lymphoid cells and tissues.
32 1.3.4 The Non-Structural Proteins
The non-structural proteins are mainly proteins with enzymatic activity which play an important role in viral replication. However, the precise roles of many of the non- structural proteins remain to be identified.
1.3.4.1 F Protein
A frameshift signal during translation of the core region of the HCV polyprotein has been identified which results in production of the core protein and a small 1.5kDa protein called the F protein (Xu et al., 2001). This frameshift signal has been found to be a triple frameshift (Choi et al., 2003) which occurs between codons 8 and 11 (Xu et al., 2001). The F protein has been found to have more diversity than the type 1b core protein, but there are no differences between HCV isolates or between isolates from hepatocellular carcinoma patients and non-hepatocarcinoma patients (Ogata et al.,
2002). Little is known about the biological function of the F protein, although it has been shown to be a labile protein that is associated with the endoplasmic reticulum (Xu et al., 2003) and has a cytoplasmic location (Roussel et al., 2003).
1.3.4.2 NS2&P7
NS2 is a hydrophobic protein with a molecular mass of 23kDa. Its major function is the cleavage of its own COOH terminus with NS3. This cleavage is dependant on the presence of zinc (Hijikata et al., 1993; Thibeault et al., 2001) and is autocatalytic, resulting in the formation of an active NS2-3 protease. A cellular co-factor may be involved in this event, as it has been shown that the efficiency of processing depends on the presence of microsomal membranes (Grakoui et al., 1993b; Santolini et al..
33 1995). A study by Waxman et af (Waxman et al., 2001) has suggested that the cellular chaperone protein HSP90 is essential for NS2/3 activity, as the HSP90-containing complex is needed for maturation of the HCV polyprotein that encodes the enzymes required for replication. The essential requirement for NS2 as a protease for HCV replication (Whitney et al., 2002), makes it a target for the development of a drug which can inhibit viral replication.
NS2 has been found to have multiple transmembrane domains and glycosylation studies have shown that the NH 2 and COOH termini of NS2 are located in the lumen of the ER (Yamaga and Ou, 2002). It remains to be determined whether NS2 has a separate role in HCV replication. It may play a role in virion assembly, as it has been co-precipitated with the envelope glycoproteins (Dubuisson et al., 1994). NS2 has also been found to interact with liver-specific pro-apoptotic CIDE-B protein (a novel family of apoptotic inducing factors) and inhibit the induction of apoptosis (Erdtmann et al.,
2003). This could be a potential evasion strategy used by HCV, which are discussed in more detail later.
The small hydrophobic protein p7 is located between E2 and NS2 and may be generated when a host signal peptidase insufficiently cleaves E2/NS2, resulting in E2 and p7 being produced (Lin et al., 1994). The function of this protein is not fully understood but it appears to be necessary for particle infectivity in related viruses
(Harada et al., 2000). Recent studies have found this protein can be inhibited by amantidine, suggesting that it may is function as an ion channel (Griffin et al., 2003;
Pavlovic et al., 2003). As it is a membrane protein it has also been proposed that it could potentially have a functional role in interacting with different components of the secretory pathway (Carrere-Kremer et al., 2002). The sequence of p7 has been found to be quite variable; this could thus be another region apart from the HVR1 that may
34 contribute to viral persistence by mutating to achieve immune evasion (Rispeter et al.,
2000).
1.3.4.3 NS3
NS3 is a hydrophilic protein approximately 70kDa in size. It has two functional domains, a serine protease domain and an NTPase/helicase domain. The serine protease activates the cleavage of other non-structural proteins (NS4A/B and NS5A/B), whilst the NTPase/helicase is involved in viral replication, acting by unwinding regions of secondary structure in the template dsRNAs and RNA-DNA heteroduplexes. NS3 has been shown to work as a dimer and can exist in three states with differing affinities for nucleic acid and ATP binding (Locatelli et al., 2002). NS3 alone has been shown to be a poor helicase on RNA, but its activity is greatly promoted by the presence of the co-factor NS4 (Pang et al., 2002). In contrast, NS3 has been shown to be a highly processive helicase on DNA, suggesting that it may have an ability to affect host DNA, as HCV replication does not involve a DNA intermediate (Pang et al., 2002). NS3 is critical for HCV replication; this has been shown by the introduction of mutations which produced cell culture adapted replicons capable of in vitro replication (Bukh et al.,
2002; Grobler et al., 2003). NS3 is also proposed to regulate signal transduction by cyclic AMP-dependant protein kinase (PKA) and influence the survival and proliferation of the host cell (reviewed by Reed and Rice, 2000).
1.3.4.4 NS4
NS4A is a hydrophobic protein of 54 amino acids, for which several functions have been identified, it is involved in activation and stabilisation of the serine protease NS3 and anchorage of NS3 and possibly other members of the replication complex to perinuclear membranes (Hijikata et al., 1993; Kim et al., 1996). NS4A has been shown
35 to augment the proteolytic activity of NS3 by a protein-protein interaction (Back et al.,
2000). NS4A has also been shown to form a complex with NS4B/NS5A (Lin et al.,
1997) and it also interacts with NS5A, regulating its phosphorylation. It has been observed that NS4A preferentially localises in the cisternae of the ER surrounding mitochondria and it is suggested that this localisation of NS4A may be related to an unknown subcellular-compartment related function (Mottola et al., 2002).
NS4B is poorly characterised and is approximately 27kDa in size, it is relatively hydrophobic and is found to be co-localised in the ER with other HCV non-structural proteins (Hugle et al., 2001). It is thought to have a role in the viral replication complex as it can interact with NS5B (the RNA dependant RNA polymerase) (Piccininni et al.,
2002) and it may be required for NS5A phosphorylation (Neddermann et al., 1999;
Tanji et al., 1995).
NS4A and NS4B may also have effects upon infected host cells. It was observed that
NS4A and NS4B inhibited cellular protein synthesis by targeting HCV 1RES initiated translation, which may be a novel function for aiding survival of the virus (Kato et al.,
2002).
1.3.4.5 NS5
NS5A is hydrophilic and present in two forms (56 & 58kDa) which result from differential phosphorylation (Kaneko et al., 1994; Tanji et al., 1995). It is associated with other virally encoded proteins as part of the replication complex, although its role in replication has not been defined. NS5A has also been shown to interact with a wide range of molecules within different signalling pathways, for example, Grb2 (He et al.,
2002) and the interferon-stimulated dsRNA-dependant kinase (PKR) (Noguchi et al.,
2001), and disrupts their signalling, which may contribute to viral persistence and
36 pathogenesis. NS5A is proposed to function as an inhibitor of PKR (Gale et al., 1997), which is a major part of the host antiviral immune response; hence NS5A is thought to contribute to HCV resistance to interferons. Enomoto et al (Enomoto et al., 1995;
Enomoto et al., 1996) found a region of NS5A whose sequence correlated with the effectiveness of interferon treatment in HCV infected individuals. It has been called the interferon-sensitivity determining region (ISDR) and mutations in this region determine the responsiveness to interferon (Hung et al., 2003). This is discussed in more detail later. NS5A has also been found to bind to TRADD (Tumour necrosis factor receptor- associated death domain) and prevent TNF-mediated apoptosis (Majumder et al.,
2002). In contrast it can also bind and interact with the core protein which may have a role in inducing apoptosis (Goh et al., 2001).
NS5B is a 68kDa protein, which has been shown to act as an RNA dependant RNA polymerase that acts as a functional oligomer for RNA synthesis (Wang et al., 2002). It is essential for replication and has been shown to interact with NS5A, NS3 and NS4A to form the viral replication complex (Ishido et al., 1998; Shirota et al., 2002). NS5B has been shown to produce initiation dinucleotide products from the 3' terminal sequence of plus and minus strands of the HCV genome (Reigadas et al., 2001; Shim et al., 2002) for viral replication. Structures within NS5B have been identified as being essential for its ability to act as a polymerase and these are being targeted for new antiviral strategies (Carroll et al., 2003; Gu et al., 2003; Stuyver et al., 2003).
1.3.5 Models of HCV Replication
HCV remains a difficult virus to study because of the lack of an in vitro tissue culture system to propagate the virus. There is also no small animal model of HCV infection;
37 currently chimpanzees remain the only animal susceptible to HCV infection (discussed
in more detail later).
Several cell lines and primary cells from humans and chimpanzees have been reported to support HCV replication. These include continuous hepatoma cell lines, B and I cell
lines and primary hepatocytes (Bartenschlager and Lehmann, 2000). However, none of these have been found robust enough to allow the classical virological, biochemical
or genetic analysis of the HCV replication system. Attempts were made to generate
and propagate HCV infection in susceptible cell lines by introducing synthetic full-length genomic RNA derived from an infectious clone, but these attempts failed, possibly due to the lack of appropriate host cell factors. However, in vivo infection has been achieved with molecular clones.
1.3.5.1 Molecular Clones
Molecular clones of HCV were generated from HCV genomic RNA isolated from
infectious serum. Initial attempts to infect chimpanzees with molecular clones derived from strain H77 HCV RNA failed. This was due to mutations introduced into the genome during cloning which affected the viability of each clone in vivo. Success was achieved with the identification of a consensus sequence for different genotypes. Now, genotype specific molecular clones exist for genotype 1b (Beard et al., 1999; Yanagi et al., 1998) and genotype la (Yanagi et al., 1997b). Chimeric genomic clones also exist for typelb-la and type1a-2a (Yanagi et al., 1999; Yanagi et al., 1997b; Yanagi et al.,
1998) Molecular clones can transmit infection in chimpanzees and are capable of
propagating virions (Hong et al., 1999; Kolykhalov et al., 1997).
Molecular clones have also been used to map critical regions of the HCV genome
required for infection. Bukh et al (Yanagi et al., 1999) mapped critical regions of the 3’
38 UTR and found regions within the 3’ UTR that were essential for HCV viability in vivo. It is proposed that these conserved regions are required to stabilise RNA for translation or to act as replicative signals for the viral polymerase (Reed and Rice, 1998).
Molecular clones have also been useful in showing that the enzymatic activities of the non-structural proteins are essential for replication (Kolykhalov et al., 2000).
1.3.5.2 Replicons
The development of the subgenomic replicon system by Lohmann et al (Lohmann et al., 1999) represented a major breakthrough for studying HCV replication. However the major problem with this system is that virions are not assembled and released from cells. The subgenomic replicon system consists of non-structural genes under control of the T7 RNA polymerase promoter. These constructs are transfected into the human hepatoma cell line, Huh-7 and replicons are selected by antibiotic resistance. High copy numbers of the replicons (1000 molecules per cell), which were 10,000 fold higher than seen in the in vitro infection system were achieved (Bartenschlager, 2002). The replicon system initially gave a very low frequency of replication (Lohmann et al.,
1999), however mutations were found within NS5B which gave rise to a higher frequency of replication, increasing the number of resistant colonies 1000-fold over the original system (Lohmann et al., 2001).
This system can also be used to study HCV replication by reverse genetics, by introducing mutations and looking at the effects of these on RNA replication, thus allowing the function of a particular gene, protein or RNA sequence to be determined.
Recently mutations in all the non structural proteins have been found that can have an impact on replication of the replicons. Some mutations enhance replication co operatively when combined with other highly adaptive mutations, for example, mutations in NS3. Other mutations are not compatible with each other, for example,
39 mutations in NS4B-5A and NS5B (Lohmann et al., 2003). It has become more apparent through the use of replicons that host cell factors play an important role in
HCV replication (Lohmann et al., 2003; Murray et al., 2003).
The original replicons lacked the structural proteins of HCV, but recently Pietschmann et al (Pietschmann et al., 2002) have constructed a replicon with the full-length HCV genome. The structural proteins were expressed and the envelope glycoproteins folded productively. The core protein was also expressed and was found on the surface of lipid droplets in the ER and cis-Golgi compartments. However, no viral particle assembly or virion release was observed; again this is likely to be due to the lack of host cell factors. Replicons have now also been constructed for other strains of
HCV including strain H77 (type 1a) (Blight et al., 2003).
Replicons have been used to show that IFN treatment can decrease replication by dramatically decreasing levels of RNA (Bartenschlager, 2002). The replicon system also has a valuable role in assessing the ability of novel compound to inhibit HCV replication. They have been used to test synthetic small interfering RNA (siRNA) molecules, showing that they have antiviral properties (Randall et al., 2003; Wilson et al., 2003). RNA interference (RNAi) is an antiviral mechanism which induces the degradation of double stranded RNA; this has also been shown to inhibit the replication of HCV replicons (Kapadia et al., 2003).
Cell-free replicon systems have also been developed (All et al., 2002; Hardy et al.,
2003). These used lysates from Huh-7 replicon transfected cells, which were capable of making replicative intermediates from endogenous HCV RNA templates. This system could also be used to identify and test compounds capable of inhibiting RNA synthesis.
40 Another recent advance has been the production of a chimeric yellow fever virus- hepatitis C virus, where the HCV structural genes replaced the yellow fever structural genes (Molenkamp et al., 2003). This chimeric virus expressed E1 and E2 heterodimers, but again there was no assembly of viral particles or release of HCV virus like particles.
1.3.6 HCVTropism
Liver hepatocytes are the principal site of HCV replication (Chang et al., 2003; Choo et al., 1989; Kolykhalov et al., 1997; Nakamoto et al., 1994; Negro et al., 1999; Tsutsumi et al., 1994); however some studies have found that cell types in other tissues may also be infected. The cellular tropism of HCV is very controversial and research is hampered by technical problems in detecting hepatitis C virus.
HCV RNA has been found in epithelial cell of parotid glands (De Vita et al., 1995), bone marrow (Gabrielli et al., 1994; Sansonno et al., 1996), lymph nodes, pancreas (Laskus et al., 1998) and PBMCs (Gong et al., 2003; Lerat et al., 1998; Muller et al., 1993). It is not clear at which stage of infection cells other than hepatocytes become infected:
Muller et al, (Muller et al., 1993) detected HCV RNA in PBMCs only from chronically infected carriers. It is also unclear how efficiently HCV undergoes productive replication in extrahepatic sites. One hypothesis suggests that pluripotent CD34'' haematopoietic cells may act as viral reservoirs. Sansonno at si, (Sansonno et al., 1998) have demonstrated these cells can support productive HCV infection in 80% of chronically infected patients.
The tropism of HCV for cell types in the peripheral blood and lymphoid tissues is of particular interest, as it may contribute to HCV persistence and/or immunological
41 abnormalities associated with chronic HCV infection. Muller et al (Muller et al., 1993) detected HCV RNA in the liver, serum, total peripheral blood mononuclear cells
(PBMCs) and B cells in chronically infected patients but not in the T cells or natural killer (NK) cells. Subsequently, Lerat et a! (Lerat et al., 1998) detected positive and negative sense HCV RNA in PBMCs in 15-100% of samples tested. They found polymorphonuclear leukocytes, monocytes, macrophages and B cells to be infected, but not T cells. In contrast, Hu et a! (Hu et al., 2003) have suggested that HCV can replicate in T and B cells. NS5A expression has also been detected in PBMCs (Gong et al., 2003), again suggesting HCV replication can occur within these cells. Bain et a!
(Bain et al., 1999) detected HCV RNA in monocyte-derived dendritic cells in 40% of chronic HCV carriers and reported that the viral sequences were distinct from those detected in the liver or serum. Navas et a! (Navas et al., 2002) and Goutagny et a!
(Goutagny et al., 2003) have also found DCs to be susceptible to HCV infection. It has been suggested that there may be selection of specific HCV viral quasispecies during viral replication in different cell types, leading to PBMC and liver adapted isolates
(Shimizu et al., 1997).
1.4 Human Infection & Disease Pathogenesis
1.4.1 HCV Transmission
The main routes of HCV transmission are intravenous drug abuse and iatrogenic means including blood transfusion in developing countries where blood is not screened. Blood transfusion products also used to represent a major source of HCV infection in developed countries prior to 1990, when new blood screening tests were introduced. The Centre for Disease Control (CDC) estimates the number of new infections in the USA fell from 230,000 per year in the 1980’s to 36,000 in 1996
(Cohen, 1999) after the introduction of blood screening tests. Nosocomial transmission
42 has been documented, for example, during dialysis (Katsoulidou et al., 1999) or during surgery (Esteban et al., 1996; Ross et al., 2000). Neediestick injuries are a major route of transmission in healthcare workers (Thomas et al., 1993). The high prevalence of
HCV in Egypt was due to the use of parenteral anti-schistosomal therapy (Frank et al.,
2000). There has also been documentation of maternal-foetal spread, however this is
infrequent and is mainly associated with HIV co-infection (Ohoto et al., 1994; Thomas et al., 1998). Sexual transmission is reported to be inefficient (Wyld et al., 1997). HCV
has also been detected in saliva (Couzigou et al., 1993) but again this is an inefficient
route of transmission (Couzigou et al., 1993; Sagnelli et al., 1997).
1.4.2 Human HCV Infection & Pathogenesis
Following HCV exposure clinical symptoms can occur within 7-8 weeks although this
can vary greatly between patients (2-26 weeks) (Marcellin, 1999). The majority of
people do not in fact have any symptoms during acute HCV infection. Approximately
20% of infected individuals undergo an acute infection and recover from a self-limited
hepatitis. The remaining 80% of infected individuals cannot eliminate the virus and
become persistently infected. Chronic HCV infection is ultimately associated with
development of cirrhosis in 14-50% of individuals and a further 10-30% of these
develop hepatocellular carcinoma (Koshy and Inchauspe, 1996).
1.4.2.1 Acute HCV
Diagnosis of acute HCV infection is rare as many patients are asymptomatic. The first
marker of HCV infection is the presence of serum HCV RNA that can be detected by
PCR. This can be detected as early as 1 week after infection and levels can reach 10®-
10® genomes per ml (Farci et al., 1991; Hino et al., 1994; Puoti et al., 1992). Anti-HCV
43 antibodies can be detected in the acute phase of HCV, but seroconversion is delayed in many patients for several weeks (Marcellin, 1999). In patients who develop acute hepatitis, serum alanine aminotransferase (ALT) levels increase just before the onset of clinical symptoms. At their peak responses ALT levels can be increased 10 times over normal levels, however generally they are only moderately increased.
20% of acutely infected patients undergo a self-limiting hepatitis where HCV RNA is undetectable and anti-HCV antibody levels have decreased but are still detectable many years later (Marcellin, 1999). The serum ALT levels return to normal after an initial increase. It is unknown whether the virus is completely eliminated from the host or whether it remains at very low levels in hepatocytes or other cells which may have become infected. However, upon re-infection these patients have an increased chance of developing cirrhosis.
1.4.2.2 Chronic Hepatitis
Most HCV infected individuals progress to develop a chronic infection. Two patterns of chronic HCV have been observed, differing in the ALT levels. Some patients have chronic hepatitis with normal ALT levels whilst others have chronic hepatitis with raised
ALT levels.
In patients with normal ALT levels (approximately 25%, this varies depending on the study (Alberti et al., 1992; Conry-Cantelena et al., 1996; Prieto et al., 1995; Serfaty et al., 1995; Shakil et al., 1995), HCV serum RNA and antibodies against HCV are detectable (Marcellin et al., 1997). These patients are asymptomatic and have a good prognosis. They may have a balance between HCV replication and host immune control of the virus, resulting in a low cell mediated immune response against HCV infected hepatocytes and little liver damage. These patients have been found to have
44 some degree of histological abnormality when liver biopsy samples are taken; 24% are normal, 54% have mild chronic hepatitis and 21% have moderate chronic hepatitis but only 1% have cirrhosis (Marcellin, 1999). The progression to fibrosis in these patients is very slow (Mathurin et al., 1998).
The second group of patients who have raised serum ALT levels (approximately 75%) can be split into two further groups; those with mild chronic hepatitis and those with moderate/severe chronic hepatitis. Approximately 50% of patients are in each group.
Patients with mild chronic hepatitis have mild histological lesions when liver biopsy is performed (Marcellin, 1999). They generally have non-specific symptoms and a slow progression of disease, with a low long-term risk of cirrhosis (Takahashi et al., 1993).
Patients with moderate or severe hepatitis have marked necro-inflammatory lesions or extensive fibrosis when liver biopsies are taken (Marcellin, 1999). It has been observed that there is an overall correlation between the degree of increase in ALT levels and the degree of histological activity. However there are exceptions so, ALT levels are not an ideal marker for disease severity (Haber et al., 1995; Healy et al.,
1995).
1.4.2.3 Cirrhosis
Cirrhosis is the most common and most serious outcome of HCV infection and affects
15-20% of HCV infected individuals (Lauer and Walker, 2001). The progression from chronic active hepatitis to cirrhosis is slow and the average onset of cirrhosis occurs 21 years after initial infection (Kiyosawa et al., 1990). The viral load and HCV genotype have not been found to affect the rate of progression (Bevegnu et al., 1997; Zeuzem et al., 2000). However, co-factors such as a high alcohol intake (Mendenhall et al., 1991;
Pares et al., 1990) or co-infection with hepatitis B virus (Colombo et al., 1989; Fong et al., 1991) have been associated with enhanced disease progression. Cirrhosis is a
45 major cause of liver transplants (approximately 30% of transplants, (Charlton et al.,
1998). Re-infection of the liver graft with HCV normally occurs, but the patient usually has a mild chronic hepatitis and their survival is no different to that of non-HCV liver transplant patients.
1.4.2.4 Hepatocellular Carcinoma
Once cirrhosis has become established patients are at a higher risk of hepatocellular carcinoma (Colombo et al., 1991; Ikeda et al., 1993; Tsukuma et al., 1993).
Hepatocellular carcinoma without cirrhosis is rare. The incidence of hepatocellular carcinoma is between 5-10%, with Western countries having a lower incidence than
Asia (Marcellin, 1999). Treatment of hepatocellular carcinoma is complicated and insufficient, leading to liver transplantation in these patients.
1.4.2.5 Extrahepatic Manifestations
HCV infection has been associated with a range of extrahepatic manifestations. The disease most commonly associated with HCV infection is mixed cryoglobulinemia, where serum cryoglobulins consisting of immune complexes of anti-HCV antibodies
(IgG and IgM), HCV RNA, immunoglobulins, rheumatoid factor and complement are found (Sansonno et al., 2003; Trendelenburg and Schifferli, 2003). 30-50% of chronic
HCV patients are found to have cryoglobulinemia (Marcellin, 1999), however the majority are asymptomatic. These cryoglobulins can cause a variety of clinical manifestations in patients including purpura (82%), arthralgia (42%) and Raynauld’s phenomenon (22%) (Agnello, 1997). There is evidence that HCV is an important cause of cryoglobulinémie nephropathy (D'Amico and Fornasien, 1995; Roth, 1995).
This is where the serum cryoglobulins precipitate in the kidneys and an immune mediated reaction causes renal damage.
46 HCV is also implicated in other diseases such as B cell non-Hodgkin’s lymphoma
(reviewed by Haufater et al., 2000) and Sjogren’s syndrome (Almasio et al., 1992;
Haddad et al., 1992) where a high HCV seroprevalence has been reported.
1.5 Treatment of HCV Infection & Vaccine Prospects
1.5.1 Current Treatment of HCV Infection
The treatments for hepatitis C infection presently consist of interferon (IFN), either alone or in combination with the antiviral drug ribavirin. However, less than half of those infected respond favourably to the current therapies (McHutchinson et al., 1998;
Poynard et al., 1998). There is therefore a great demand for new and more effective treatments.
During the mid-1980’s interferons (IFNs) were shown to decrease serum ALT and HCV
RNA levels in infected patients (Davis et al., 1989; Di Bisceglie et al., 1989; Hoofnagle et al., 1986). Interferon has direct antiviral effects produced via induction of genes including 2’5’ adenylate synthetase (2’5’AS) and dsRNA dependant protein kinase
(PKR). It may also act indirectly by modulating innate and adaptive immune responses
(Goodbourne et al., 2000). Type 1 interferon is the treatment given to HCV-infected individuals. In the 1990’s interferon treatment became widely available, yet the success of treatment varied greatly. It was found that genotype 1 infections with high
HCV RNA levels responded better to IFN therapy for a longer duration than genotype 2 or 3 infections (Davis, 2000). Recently IFN therapy has been combined with ribavirin.
Ribavirin is an inhibitor of inosine monophosphatase dehydrogenase (IMPDH) which is a key enzyme in the purine biosynthetic pathway, thus affecting viral replication. In addition to its antiviral activity ribavirin may also reduce generalised pro-inflammatory
47 responses and reduce bystander damage to hepatocytes (Davis, 2000). Ribavirin alone reduces serum ALT levels in 40% of chronic HCV patients, but it does not decrease serum HCV RNA levels (Bodenheimer et al., 1997; DiBisceglie and Shindo, 1992;
Dusheiko et al., 1996; Reichard et al., 1991).
Sustained responses in patients treated with IFN alone are 6-15% after 6 months and
13-25% after 12 months (Davis, 2000). This increases to 30-40% in patients treated with IFN and ribavirin combination therapy (Davis et al., 1998; McHutchinson et al.,
1998; Poynard et al., 1998). Augmentation of T cell responses has also been detected in successful IFN/ribavirin therapy (Sreenarashimhaiah et al., 2003). In addition to the fact that response rates are low, there are many other problems with the current therapy, the main one being the expense. In the USA, the average lifetime cost of IFN, ribavirin, lab testing and other visits is $10-12,000 and only improves the patient's life expectancy by one year (Wong, 1999). The side effects of treatment are also unpleasant, with patients suffering from flu-like symptoms and depression which can lead to problems with compliance with treatment.
Improvements to the existing IFN therapies are being made by the addition of polyethylene glycol to IFNa. This extends the half life of the IFN and the duration of therapeutic activity. Pegylated IFNs have been shown to have higher response rates than the conventional monotherapy (Heathcote et al., 2000; Zeuzem et al., 2000).
New therapies are also being developed, such as inhibitors of viral replication, anti inflammatory drugs and immune modulators. Potential inhibitors of viral replication include other IMPDH inhibitors which act in a similar way to ribavirin. Ribozymes and anti-sense oligonucleotides that are targeted at preventing viral replication are also being explored as potential HCV therapies. Macejak et al (Macejak et al., 2001) have developed a nuclease resistant ribozyme directed against the HCV 5’UTR that forms
48 the 1RES required for translation. The ribozyme catalyses RNA cleavage and splicing reaction and can reduce viral replication by 95-99% when in combination with IFN.
Other groups are also exploring the use of ribozymes as therapy for HCV (Lieber et al.,
1996; Welch et al., 1996; Welch et al., 1998).
1.5.2 Prospects for a HCV Vaccine
A prophylactic vaccine for HCV would be a great benefit to public health, even though transmission has been reduced since the introduction of blood screening tests. It would benefit health care workers, haemodialysis patients, and patients with diseases
requiring frequent blood transfusions or blood products, IV drug users and partners of
HCV positive patients. Given that current treatments are not effective in all HCV
infected patients and almost 3% of the world's population harbours HCV, there is also
an urgent need for a therapeutic HCV vaccine to be developed.
The requirements for prophylactic and therapeutic HCV vaccines may differ. Ideally,
prophylactic vaccines should induce sterilising immunity which may be difficult to
achieve. However, it may not in fact be necessary. It may be enough to prevent the
development of chronic infection, as this is the major cause of morbidity and mortality
in HCV infection. A vaccine which allowed a “transient infection” (sub-clinical or of
limited acuity), whilst preventing the development of a chronic infection could be as
beneficial. Therapeutic vaccines could likewise aim to eliminate the infection or reduce
the severity of infection-associated disease. They could potentially be used in
combination with other antiviral therapies.
Many problems are associated with HCV vaccine development. It is unclear what kind
of immune response vaccines may need to elicit to combat this infection. The type of
49 response required may differ depending on the aims of the vaccine, for example, sterilising immunity may depend heavily on neutralising antibodies, whilst studies in chimpanzees and humans have indicated that good control of viral replication may also require innate responses and CD4‘" and CDS'" T cells responses (discussed in more detail later). A further complication is that vaccine-induced immune responses should not cause immunopathological liver damage. It is thought that much of the liver damage occurring during HCV infection may be due to an inappropriate host immune response rather than direct damage caused by the virus.
The genetic variability of HCV presents a further challenge to vaccine development. A
HCV vaccine should ideally provide protection against all the major circulating genotypes, as a narrow specificity would limit the use of the vaccine geographically.
HCV vaccine development is also hampered by the lack of an in vitro replication system and a small animal model. Chimpanzees are the only animal known to date to be infected with HCV apart from humans. Altogether developing a HCV vaccine is a great challenge to the scientific community.
There are several approaches currently under consideration for HCV vaccine development. These are outlined in table 1.2 and are currently being assessed in in vitro, mouse model systems and the chimpanzee model. However, as yet no approach has been approved for human clinical trials.
Traditional vaccines rely upon the whole organism being inactivated or attenuated
(usually by heat or formalin) before inoculation. This is not possible for HCV as very little virus can be isolated and there is no in vitro culture system available to generate large enough quantities of virus for inactivation.
50 Approach HCV Protein in Vaccine Reference Recombinant Proteins E1/E2 & Adjuvants (Choo et al., 1994) (MF59/muramyldipeptide)
E1/E2 in peptide vaccine (Esumi et al., 1999) produced in insect cells
Core in ISCOMS (Polakos et al., 2001)
Core in virosomes (Hunziker et al., 2001 )
NS3 in liposomes (Jiao et al., 2003)
Virus-Like Particles (VLPs) CE1E2 (Baumert et al., 1998; Baumert et al., 1999)
CE1E2 (Lechmann et al., 2001)
CE1E2 (Qiao et al., 2003) CE1E2 (Murata et al., 2003)
Subunit Vaccine HVR-1 from E2 & cholera (Nemchinov et al., 2000) toxin produced in TMV
Peptide Vaccine C, NS4, NS5, E2 (Hiranuma et al., 1999)
DNA Vaccine CE1E2, C&IL-4/IL-2 or (Geissier et al., 1997) GM-CSF
C (Tokushige et al., 1996)
C (Major et al., 1995)
E2 (Forns et al., 1999; Forns et al., 2000)
E1/E2 (Lee et al., 1998)
C/E2 (Inchauspe et al., 1998)
NS3/NS4/NS5 & GM-CSF (Cho et al., 1999)
NS3 (Brinster et al., 2001)
51 Approach HCV Protein in Vaccine Reference Live/Killed Viral Vectors E2 in Rhabdovirus (Siler et al., 2002)
Recombinant Viral Vectors CE1E2 in Adenovirus (Lasarte et al., 1999) (Bruna-Romero et al., 1997) (Makimura et al., 1996) (Shirai et al., 1994)
CE1E2 in Vaccinia (Large et al., 1999)
C/E1/E2/NS2/NS3 & (Pancholi et al., 2000; NS3/NS4/NS5 in Pancholi et al., 2003) Canarypox
0 or E2 in Semliki Forest (Vidalin et al., 2000) Virus
NS3 in Semliki Forest Virus (Brinster et al., 2002)
DNA Prime-Boost C & recombinant C (Hu et al., 1999)
E2 & recombinant E2- (Song et al., 2000) HSVIgpD
C/E1/E2/NS2/NS3 & (Pancholi et al., 2000; NS3/NS4/NS5 in Pancholi et al., 2003) Canarypox
C or E2 in Semliki Forest (Vidalin et al., 2000) Virus
NS3 in Semliki Forest Virus (Brinster et al., 2002)
Table 1.2. HCV Vaccine Strategies under consideration. The different HCV vaccine strategies outlined in this table are being tested in in vitro, mouse model systems or in the chimpanzee model. No candidate vaccines have yet been approved for human clinical trials.
52 The development of virus-like particles (VLP) produced in in vitro cultures has provided an alternative approach to a HCV vaccine. VLPs are generated using a baculovirus expression system to express the HCV structural proteins (Baumert et al., 1998;
Baumert et al., 1999). These have been inoculated into mice and were shown to induce strong antibody and cell mediated responses. However, the immunogenicity depended upon the particle formation. Even so these are an attractive vaccine candidate requiring further study.
Another approach would be the development of a subunit vaccine as used for example, the hepatitis B vaccine. This was the initial approach taken by Chiron for the development of a HCV vaccine (Choo et al., 1994). They used purified recombinant core and envelope glycoproteins injected intramuscularly in an oil/water emulsion.
Chimpanzees were inoculated 18 times and three weeks after vaccination they were challenged with a homologous virus. Some of the animals did not become infected
(those with the highest antibody response), whilst some did become infected and later resolved the infection, undergoing an acute course of infection. Control un-vaccinated chimpanzees developed a chronic infection. Re-challenged of protected animals with a heterologous virus after a further boost with the recombinant proteins resulted in none developing chronic infections. These studies showed that sterilising immunity was not achieved but this may be a valid approach for preventing the development of chronic disease.
Peptide vaccines consist of chemically synthesised peptides which could act as immunogens. However, the major obstacle to this approach is that a single peptide without CD4* T cell help may be a poor immunogen. This may be overcome by the use of potent adjuvants. It may also be necessary to include several epitopes and a mixture of peptides. This approach is being evaluated for HCV, by using T cell
53 epitopes (both CD4* and CD8* within the core protein and two non-structural proteins
(NS4 & NS5) and the E2 glycopotein (Hiranuma et al., 1999).
Another approach being investigated is that of DNA vaccination. Purified plasmid DNA encoding antigens of interest under the control of a eukaryotic promoter is inoculated into the host where the host cells express the antigens of interest intracellularly, therefore mimicking a natural irfection. The advantage of this system is the ease at which DNA can be manipulated and that several antigens can be incorporated into the vaccine to allow priming of different arms of the host immune response. Cytokine genes can also be included to ect as adjuvants enhancing the immune response. The possibility of including other imrrune modulating molecules (for example, co-stimulatory molecules like CD80, CD86 andCD40L) is also being explored.
Recombinant viruses can also be used as vectors for vaccination. These have the advantages of giving high levels of recombinant protein expression in the host cells and of inducing local immune activation. Several vectors are being explored for use in a
HCV vaccine, including replication-deficient adenoviruses and vaccinia virus. Both of these have been used to express the HCV structural proteins (Bruna-Romero et al.,
1997; Large et al., 1999; Lasarte et al., 1999; Makimura et al., 1996; Shirai et al.,
1994). Other potential viral vectors include non-replicating canarypox virus, fowlpox virus (Paoletti et al., 1995), attenuated vaccinia strains (e.g. Modified Vaccinia Ankara) and alphavirus vectors such as Semiliki forest virus (Smerdou and Liljestrom, 1999).
The most popular approach currently is the combination of single modality vaccines into heterologous prime-boost strategies. Frequently DNA vaccination followed by a purified recombinant protein boost or boosting by a viral vector (e.g. Canarypox vectors) expressing the protein of interest.
54 1.6 Models of HCV Disease
As discussed previously, there is a lack of an in vitro model system for studying HCV
and for propagating the virus. Thî lack of a small animal model system also hampers
HCV research. Currently, chimpanzees remain the only animal susceptible to HCV
infection. However efforts to improve this model system and to develop alternative
transgenic mouse model systems are being undertaken by many research groups.
1.6.1 The Chimpanzee Model
Chimpanzees are the only animal susceptible to HCV infection and have been used for
over 20 years in attempts to ideniify and characterise HCV. For example, they were
used to characterise the NANB hepatitis agent (later identified as HCV), and have been
used to study HCV transmission, immunity and pathogenesis. Chimpanzee
experiments played an important role in determining the size of the HCV virions (Alter
et al., 1978; Hollinger et al., 1978; Tabor et al., 1978) and biochemical properties of the
virus (Bradley et al., 1983; Feinstone and Mihalik, 1983; Heinrich et al., 1982; Purcell et
al., 1985; Tabor and Gerety, 1980; Yoshizawa et al., 1982). Chimpanzees have also
provided material for the isolation and characterisation of the HCV genome (Choo et
al., 1989). They have also played an important role in testing the infectivity of
molecular clones (Kolykhalov et al., 1997; Yanagi et al., 1997a). In addition, they have
been used to study the immune response to HCV. HCV-specific CD4‘" and CD8* cells
have been identified in both chimpanzees and humans and correlates of immunity and
protection are being determined using the chimpanzee model.
Chimpanzees are 98.5% genetically similar to humans and undergo a similar clinical
course to humans infected with HCV. Both have detectable HCV RNA days after
55 exposure, with increases in ALT levels. Both develop chronic infections with evidence of long-term liver damage. However chimpanzees do not have the same degree of
liver damage as humans (Bradley et al., 1981; Seef, 1995) and they are less likely to develop hepatocellular carcinoma, although there has been a report of hepatocellular
carcinoma occurring in chimpanzees (Muchmore et al., 1988).
The advantages of using chimpanzees to study HCV include the ability to induce
infections with well characterised HCV inocula, the opportunity to obtain liver tissue
during the course of infection and the availability of samples prior to infection and in the
early stages of infection to allow the early events of infection to be analysed. However, there are also disadvantages; chimpanzees are rare animals and have been an endangered species since 1988, therefore are limited in availability. They are also expensive and require appropriate non-human primate research facilities as they are
difficult animals to handle. In spite of these disadvantages the chimpanzee model will
have a valid place in future studies of HCV as it facilitates well controlled experiments.
Chimpanzees can be used in the evaluation of new therapies or vaccines as well as in further studies of HCV transmission, clinical outcomes of infection and immune
responses evoked during infection.
1.6.2 Alternative Non-human Primate Approaches
Several other species of non-human primates have been inoculated with infectious
serum from a HCV infected chimpanzee in an attempt to identify other species
susceptible to infection. These include the cynomolgus monkey, the rhesus monkey,
the green monkey, the Japanese monkey and the doguera baboon; however none of
these were susceptible (Abe et al., 1993). Tupaias (tree shrews) (Xie et al., 1998),
marmosets (Feinstone and Alter, 1981; Watanabe et al., 1987) and tamarins
56 (Karayiannis et al., 1983) have been shown to be possibly susceptible to HCV infection, however follow up studies of these are absent, conflicting or discouraging
(Garson et al., 1997).
1.6.3 Mouse Models
Mouse models offer several advantages over non-human primate models; primarily,
mice are easy to breed, maintain and manipulate. There are genetically defined and transgenic and knockout strains widely available. However, there are disadvantages to
using mice to study HCV, the most important being that mice are not susceptible to
HCV infection. They are also not genetically similar to humans, although most mouse genes have a human homologue.
Several groups have generated transgenic mice that express HCV structural proteins in the liver. Mice expressing core (Matsuda et al., 1998), core-E2 (Pasquinelli et al., 1997)
and core-E1-E2 (Kawamura et al., 1997) have been made. However, none of these
mice have shown histological or biochemical evidence of liver disease. Other groups
have found that expression of core in transgenic mouse lines caused steatosis and
some mice developed hepatic tumours (Moriya et al., 1998; Moriya and Yotsuganagi,
1997). These conflicting reports may be due to differences in the genetic backgrounds
of the mouse strains used, or in the level of protein expression achieved. Most
transgenic mice express the HCV proteins to high levels, which does not occur in
human infection. The mice may also have different cellular locations of the HCV
proteins.
The transgenic mice discussed above are also immunotolerant to the HCV proteins as
they are exposed to them from birth, therefore they cannot be used to study immune
57 responses to the viral proteins. To overcome this problem, Wakita et al (Wakita et al.,
1998) developed a transgenic mouse in which the cre/loxP system was used to
conditionally express the HCV structural proteins, to which antibodies were generated.
These mice may be useful for studying the role of immune responses generated to
HCV in the pathological changes occurring in the liver, but their use in studying viral
replication is limited as they don't produce replicating virus.
To allow studies of HCV pathogenesis, infection and replication to be carried out,
researchers are now turning to xenograft models, creating mice with human or
chimpanzee hepatocyte growth by transplantation or repopulation. Mercer et a!
(Mercer et al., 2001) have infected transgenic mice which have been repopulated with
human liver with serum from a HCV positive human. These mice can support HCV
replication and are capable of producing infectious viral particles that can transmit HCV
infection to other mice. Mice have been infected with several HCV genotypes (types
la, 1b, 3a and 6a) and rapid increases in viral titres and long term persistence of
infection occurred. These mice may prove to be valuable in studying HCV replication
and may be useful for evaluating potential therapies and vaccine candidates.
However, at the moment there are several drawbacks to this system. There is a high
mortality rate in the mice and microsurgical skills and equipment are required. The
access to human hepatocytes may also be limiting in the generation of the mice.
In summary, the animal models currently available for studying HCV all have
drawbacks, although advances with transgenic and xenograft mouse systems are
encouraging. In vitro tissue culture systems are also developing and will have a role to
play in studying HCV. Until a good system is available researchers will rely upon the
chimpanzee model to provide insights into HCV infection and immunity and it will be
essential in the evaluation of potential vaccine candidates.
58 t. 7 The Immune Response during HCV infection
The immune response to HCV has been studied both in humans and in experimentally infected chimpanzees, which like HCV-infected humans, develop an acute infection with mild hepatitis that may either resolve or progress into a chronic infection (reviewed by Walker, 1997). Efforts have been made to characterise and compare immune responses associated with clearance of acute HCV infection, good and poor containment of chronic HCV infection and HCV-associated pathology, with the goal of addressing questions including: how do some patients spontaneously clear HCV and what is a protective immune response? In chronically infected patients, why do the induced responses fail to eliminate the virus? How does the virus evade or impair host immune responses? What role do different arms of the immune response play in immunopathological damage during a chronic HCV infection? There is a particularly great need to define protective immune responses as this will enable the design of vaccines and therapies which are capable of inducing the required responses without causing further immunopathology. Many of these questions still remain unanswered due to the difficulties associated with studying the immune responses to HCV infection.
These include the paucity of acutely presenting patients, the drawbacks of the chimpanzee model and lack of good small animal models for HCV infection (as discussed previously), the lack of neutralisation assays and the variability of the virus.
1.7.1 Overview of the antiviral immune response
The immune system recognises and responds to infection with viruses and other pathogens, becoming activated to mount an immune response, which aims to contain or eliminate the infection, and may provide protection against re-infection.
59 Following viral infection, the immune system recognises viral components or infection- associated cell stress by receptors such as Toll-like receptors (TLR) which are pattern recognition receptors that recognise conserved sequences on pathogens (pathogen associated molecular patterns) resulting in rapid activation of host responses. These early recognition events trigger host cells to release cytokines, the most important of which are type 1 interferons (IFNa and IFN(3), for example, TLR3 recognises viral dsRNA and triggers interferon alpha production (Williams and Sen, 2003). Cytokine
release results in the activation of cells within the innate immune system, especially
macrophages and dendritic cells which through further cytokine release and direct cell cell contact interactions orchestrate full activation of the innate immune response. This also leads to the activation of an appropriate adaptive immune response.
The role of the innate immune defences is to provide rapid control of viral replication in the initial stages of infection. The effector mechanisms involved in innate immunity are
not pathogen-specific. One example is the direct antiviral effects of type I interferons, which activate several target genes in host cells including 2’5’ adenylate synthetase
(2’5’AS), which activates RNAse L to degrade viral RNA and the dsRNA dependant
protein kinase (PKR) which inhibits viral replication. Another example is the activity of
natural killer (NK) cells. They recognise infected cells in a pathogen non-specific
manner, and both lyse virally infected cells in a MHC-unrestricted manner via perforin and granzyme release and also secrete cytokines such as IL-12 and IFNy which that
have direct antiviral effects and/or further activate immune responses. Macrophages
and dendritic cells also contribute to innate immune responses by the production of
cytokines that have direct antiviral effects and enhance other innate immune
mechanisms.
60 Factors released and cells activated during the innate immune response are important
in activating appropriate adaptive responses. DCs in particular act as a bridge between
innate and adaptive responses (Banchereau et al., 2000). The adaptive response is
characterised by specificity and memory and is mounted more slowly. It is mediated by
T and B lymphocytes, which have surface receptors that recognise specific pathogen
components. B cell receptors bind to their target antigens alone, enabling them to
recognise “free” pathogens, whereas T cell receptors recognise a complex composed
of a pathogen-derived peptide presented by a host cell surface protein (MHO class I or
class II), which targets them to interact with other cells. T cell activation requires the
presentation of cognate peptide and MHC to the T cell by a DC. The DC triggers the
activation, clonal expansion and differentiation of antigen-specific CD4* and CD8* T
cells. Activated CD4"^ and CD8* T cells differentiate with effector functions. CD4" T
cells provide help to CD8* T cells and B cells by cell-cell contact and the further release
of cytokines, which results in the activation of CD8* cytotoxic T cells and induction of
class switching in antigen-activated B cells for antibody production. Antibodies
generated against the virion surface provide a barrier to the spread of the virus
between cells and tissues, as they can block infectivity of viral particles. Antibodies
also mediate the destruction of virally infected cells by mechanisms including
complement-dependent lysis or antibody-dependent cell mediated cytotoxicity (ADCC).
CD8* T cells are the major effector cell during many viral infections. They focus at the
site of viral replication and destroy virally infected cells, and also release cytokines that
can “cure" infected cells by non-lytic means. Once the immune response has
established control of the viral infection, the majority of activated and expanded cells
die, whilst some remain as memory cells. The memory cells are present at a higher
frequency than prior to the infection and are more readily triggered should the virus be
encountered again, thus memory cells allow a faster response to control the infection
upon re-exposure. Pre-existing antibodies may also block or control the spread of
infection when a pathogen is encountered for the second time.
61 1.7.2 Innate Immune Responses, and their role in HCV infection
■I
1.7.2.1 Type 1 Interferon Response
Type 1 interferons are a family of cytokines induced in response to the presence of pathogens, especially viruses. They are the earliest known host induced defences, and are produced within hours of infection. Type 1 interferons include the alpha interferons (IFNa) and interferon beta (IFN(3). They are released by multiple cell types in response to viral infections but particularly high quantities are made by plasmacytoid dendritic cells (Siegal et al., 1999). Figure 1.2 shows the pathways involved in type 1 interferon production in cells.
Type 1 interferons mediate their multiple pleiotropic effects by binding to surface receptors and triggering the induction of other genes. One set of IFN-inducible genes create an antiviral effect within cells, as shown in figure 1.3. Some of the IFN-inducible genes that have antiviral effects include 2’5’ adenylate synthetase (2’5’ AS), dsRNA dependant protein kinase (PKR) and the Mx proteins. 2’5’ AS undergoes a conformational change when IFN and dsRNA bind and becomes activated. It then polymerises ATP and other nucleotides in 2’5’ linkages which create 2’5’ oligonucleotides that activate endoribonucleases (RNase L). These degrade viral RNA by cleavage. There are several isoforms of 2’5' AS which may act against different viruses and have differing abilities to induce an antiviral state (O'Connell, 1997). PKR is a cyclic AMP-independent kinase which is activated by autophosphorylation in the presence of dsRNA. The activated PKR phosphorylates cellular proteins including protein initiation factor (elF-2). This inhibits viral protein synthesis, thus inhibiting viral replication.
62 B
receptor
IRF-7^ ^lR F-3^^ / p p „ licBa ^ ^\ /iK B a \ t r 1» NF-icB NF-k B ATF2/c^l!\
isc ;f 3
IFN-B !-► IFN-B IRF-7
!-► IF N -04 (—► 1FN-€U>
Figure 1.2. A schematic model of type 1 interferon gene induction in response to virus infection (adapted from Mamane etal, 1999). (A) Following recognition of virus infection (e.g. TLR3 recognising dsRNA), activation of different transcription factors including IRF-3 occurs through distinct pathways. These activate the immediate-early production of IFN-p and IFN-a4. (B) Secreted IFN-p and IFN-a4 bind to the type I IFN receptor on the cell surface in an autocrine or paracrine fashion. This results in the activation of the JAK- STAT signalling pathways, leading to the expression of IRF-7. IRF-7 activation results in amplification of IFN gene expression, with further production of IFN-p and IFNa4 plus production of the other subtypes of IFN-a.
63 IFNap IFNy
R e a lto rs
Signal transduction CTL killing
Transcriptional Induction ^ Adhesion molecules ....^ ...... = t Caspases À—i- procaspases i Cell death MHC class I mRNA degradation 1 2’5’ASa 4#—h 2’5’ASi 1 Translational arrest j-PKRa PKRi proteasome / Viral dsRNA Viral proteins
Primary target cell N / Virus replication & protein synthesis
Figure 1.3. The biological properties of interferons. Interferons bind to specific surface receptors on primary target cells and induce the transcription of a variety of genes that mount an antiviral state in infected cells. These gene products often depend on viral dsRNA as a co-factor to ensure they are only active under infectious conditions. PKR & 2’5’AS are synthesised as inactive precursors (PKRi & 2’5’ASi, respectively) and are activated by dsRNA (PKRa & 2’5’ASa, respectively). Once activated they shut down translation. IFNs also induce a pro-apoptotic state by inducing the action of caspases from procaspases. IFNs also upregulate the synthesis of proteins involved in the processing and presentation of viral antigens to CD8+ CTL, enhancing recognition of infected cells by CTL .
64 Type 1 interferons also have regulatory roles as well as conferring an antiviral state on cells. They regulate the acti\/ation, proliferation and differentiation of cells involved in innate and adaptive responses (Biron, 1998; Biron, 1999; Biron, 2001), for example, enhancing MHC expression, NK activity and antibody-dependant cell mediated cytotoxicity and aiding cell migration. They also bias the adaptive response towards a type 1 response (Le Bon and Tough, 2002).
Relatively little is known about type 1 interferon responses in early HCV infection as the majority of patients do not present until a chronic infection has been established. The paucity of model systems also hampers study of responses during acute HCV infection.
Interferons probably play an important role in control of HCV replication as the virus attempts to impair IFN production and to evade interferon effector pathways (discussed in more detail later). IFN production is not completely blocked, as 2’5’ AS induction has been observed in HCV infections (Giannelli et al., 1993; Pawlotsky et al., 1995).
However type I interferon production is clearly suboptimal during chronic HCV infection, as indicated by the relative success of IFN therapy in some patients with chronic HCV.
1.7.2.2 Other Innate Cytokines
The production of other cytokines and chemokines by macrophages and dendritic cells during innate immune responses also play a role in controlling viral replication and the subsequent activation of adaptive immune responses. These include tumour necrosis factor alpha (TNFa) and interleukin -1 (IL-1) which are pro-inflammatory cytokines that will promote an inflammatory response and the recruitment of activated cells to the site of infection (McGuinness et al., 2000). Innate cytokines such as IL-12, IL-15 and IL-18 act in combination with type 1 interferons to promote the activation of innate effector cells such as natural killer cells, which play an important role in controlling viral infections. IL-12, IL-15 and IL-18 also all promote type 1 adaptive immune responses.
65 There is an increase in the numbers of innate cells in the liver during HCV infection, including infiltrating macrophages that secrete TNFa, IL-ip, IL-12, IL-18 and the chemokine MIP-ip, all of which will promote an inflammatory response. In contrast,
IL-12 has been shown to have therapeutic efficacy in HCV infection (Rossol et al.,
1997), suggesting it may play a (likely indirect) role in controlling HCV infection.
1.7.2.3 Natural Killer & Natural Killer T Cells
Natural killer (NK) cells are non-T, non-B lymphocytes that do not have antigen-specific receptors. They comprise of approximately 10% of human peripheral blood mononuclear cells, and are present at a higher frequency in the liver. They are activated early during innate immune responses by cytokines including type 1 interferons, IL-12, IL-15 and TNFa. NK cells provide a rapid response to viral infections, with the peak of their activity at 2-4 days post infection. Their activity declines as the adaptive antigen-specific CDS'" T cell response is induced (around day
7 post-infection). The major function of NK cells is the lysis of virally infected cells via a perforin-dependent mechanism. They also secrete cytokines including IFNy which controls viral replication by non-lytic mechanisms. NK cells have recently been found to interact with DCs (Ferlazzo et al., 2000; Gerosa et al., 2002; Piccioli et al., 2002), an interaction that is likely to be important in the regulation of both innate and adaptive responses.
Natural killer T cells (NKT) have a restricted T cell receptor expression and some are
CDId restricted. Like NK cells, they also have cytotoxic activity against virally infected cells and can also be triggered to release large amounts of pro-inflammatory cytokines.
The importance of NK cells in certain viral infections has been demonstrated in mice and humans. Mice depleted of NK cells have higher levels of replication of viruses
66 such as murine cytomegalovirus and vaccinia virus (Bukowski et a!., 1985). Humans who have suppressed NK function in Chediak-Hegashi syndrome also have an increased susceptibility to certain viral infections, particularly herpes virus infections
(Padgett et al., 1968; Wolff, 1972).
Relatively little is known about the early NK cell response during acute HCV infection as the majority of patients do not present until a chronic infection has been established.
However, these cells are likely to be important effectors in controlling early viral replication.
The majority of studies on NK cells in HCV infection have been carried out in chronically infected patients. A higher frequency of NK and a lower frequency of NKT and T cells have been detected in the liver in end-stage liver disease than in chronically infected and normal individuals (Bosivert et al., 2003). This is in contrast to the peripheral blood where T cells are the largest population, followed by NK, NKT and B cells. NK cells have been shown to be capable of lysing HCV infected hepatocytes
(Yonekura et al., 2000) which would prevent the spread of virus to neighbouring cells.
The cytotoxic capacity of NK cells has been studied in chronic HCV infections and several studies have found them to have a significant decrease in cytotoxicity (Bonavita et al., 1993; Castiglione et al., 2001; Corado et al., 1997; Gabrielli et al., 1995; Par et al., 1995) as the cells expressed lower levels of perforin compared to cells from normal individuals. NKT cells are also found at lower levels in HCV patients in their peripheral blood (Lucas et al., 2003) and the reduced numbers may be due to activation-induced cell death (Kakimi et al., 2000). The precise role of NKT cells in HCV infection remains unknown.
67 1.7.2.4 Gamma Delta T Cells
Gamma delta (yô) T cells are a primitive T cell with a T cell receptor consisting of gamma and delta chains (as opposed to alpha and beta chains in the T cell receptor of conventional T cells). They represent a small proportion of T cells (approximately 5%).
They are detected in the peripheral blood and also in the liver. They release cytokines to aid both innate and adaptive immune responses and may have cytolytic activity against infected cells.
Increased numbers of yô T cells have been detected in HCV infected liver (Agrati et al.,
2001a; Nuti et al., 1998) and these cells have been found to have an activated/memory phenotype (Agrati et al., 2001a) and secrete IFNy (Agrati et al., 2001a; Agrati et al.,
2001b). Tseng et al (Tseng et al., 2001) also found yô T cells to release TN Fa and IFNy when CD81 was bound to the cells. CD81 is a potential receptor for HCV; therefore
CD81 ligation by HCV on yô T cells may activate the cells. Higher numbers of yô T cells have also been found in the periphery in asymptomatic HCV carriers than in chronic patients, which suggests a compartmentalisation of the cells (Par et al., 2002).
The yô T cells have been found to have cytolytic activity against primary hepatocytes,
K562, Daudi and Huh-7 cell lines (Poccia and Agrati, 2003), therefore have the
potential to lyse hepatocytes infected with HCV. The precise role of yô T cells in HCV
infection remains unknown, but they may contribute to immunopathological damage in the liver (discussed below).
1.7.2.5 Complement
There are over twenty complement proteins in the serum. Upon activation, these
proteins react in a cascade which generates products that mediate and amplify immune
68 and inflammatory reactions. There are two complement pathways: the classical pathway which involves antibodies for activation, and the alternative pathway in which antibodies are not involved. Both pathways can result in the generation of a membrane attack complex which lyses cells. Complement mediates antiviral effects by the production of an acute inflammatory reaction leading to the recruitment of leukocytes and the lysis of infected cells. The complement membrane attack complex may also directly damage or lyse membranes of enveloped viruses, allowing the destruction of viral nucleic acids (Almeida and Waterson, 1969). Complement also enhances neutralisation of virions by antibodies which may prevent viral attachment, therefore making them more readily destroyed by phagocytosis.
The role of complement in HCV infection is unclear, however it has been observed that the HCV core protein can interact with a complement receptor and impair T cell responses (Kittlesen et al., 2000; Yao et al., 2001a; Yao et al., 2001b).
Failure to eliminate HCV may be due to a deficiency of innate immune responses early on in infection, resulting in the establishment of a persistent infection.
1.7.3 Adaptive Immune Responses and their role in HCV infection
1.7.3.1 Humoral Responses
Antibodies play an important role in controlling virus infections as they provide a major barrier against spread of infection to neighbouring cells and are particularly important in restricting the spread of pathogens in the blood stream. The first antibodies secreted are IgM, which are multivalent and of low affinity. Following class switching, high affinity
IgG classes of antibodies are produced. These IgG antibodies persist and provide
69 protection against re-infection. At mucosal surfaces, IgA plays a major role in
preventing infection. Antibodies neutralise viral activity by blocking viral binding and entry into cells, or by blocking replication at a post-entry step. They also target virions for complement lysis and opsonise viral particles for enhanced uptake by
macrophages. They combat infected cells too, targeting them for complement-
mediated lysis and destruction by ADCC, although they generally play a limited role in the elimination of established viral infections. Nonetheless, B cell activity is essential for the long term control of other chronic viral infections such as LCMV (lymphocytic choriomeningitis virus) (Planz et al., 1997). Once infection has been eliminated, pre existing antibodies have a role in preventing re-infection on later exposure. The
presence of antibodies is indicative of prior exposure to a pathogen.
In HCV infection, antibodies are normally detected within 7-8 weeks of inoculation of experimentally infected chimpanzees (Schmilovitz-Weiss et al., 1993) and are detectable between 7-31 weeks post infection in human serum (Chien et al., 1992).
This is quite late compared to other viruses (Alberti et al., 1988; Battegay et al., 1993;
Koup and Ho, 1994; Modlin, 2000). Individuals also vary greatly in the specificity and timing of their antibody responses.
Antibodies reactive to all structural and non-structural proteins have been detected in
HCV-infected individuals (Bukh et al., 1995; Lemon and Honda, 1997; Neumann et al.,
1998; Simmonds, 1995). A qualitative and quantitative analysis of the humoral
response to HCV in both acutely and chronically-infected patients (Chen et al., 1999)
revealed that antibody responses to HCV antigens were of relatively low titre, with the
exception of antibodies against the core protein, and were delayed in appearance until
the chronic phase of infection. Further, again with the exception of anti-core
antibodies, antibodies to HCV antigens were highly restricted to the IgGI isotype.
70 Most seropositive patients are still viraemic and no correlation between the clinical outcome of infection and the antibody response has been observed. No serological marker was of prognostic value in differentiating a resolved acute infection from an infection which chronically progressed. Likewise, a study in chimpanzees also reported poor antibody responses during acute HCV infection in both resolvers and non resolvers (Cooper et al., 1999). However, another study suggested that the time of appearance of E2-HVR1-specific antibodies was significantly different in patients who resolved infection compared to those who progressed to a chronic infection. A high antibody response to the E2-HVR1 correlated with clearance of acute HCV infection
(Zibert et al., 1997).
There is difficulty in measuring HCV-specific neutralising antibodies as the virus cannot be grown in vitro. An assay which is frequently used is neutralisation of the binding of
E2 to CD81 expressed by MOLT-4 cells (“NOB” assay) (Pileri et al., 1998; Rosa et al.,
1996), but it is not clear how “NOB” activity relates to the neutralisation of viral infectivity in vivo. It is reported that chronically infected patients lack or have low levels of neutralising antibodies (Rosa et al., 1996); but amongst very rare patients who naturally resolve chronic HCV, very high “NOB” titres are frequently found for an extended period of time (Ishii et al., 1998).
The antibody response wanes with time in both chimpanzees and humans in the absence of viraemia (Bassett et al., 1998; Dittmann et al., 1991; Farci et al., 1991;
Goeser et al., 1994; Takaki et al., 2000). HCV-specific antibodies were shown not to prevent re-infection of chimpanzees with homologous or heterologous strains of virus in some studies (Farci et al., 1992; Prince et al., 1992). By contrast, a protective role for
E2-specific neutralising antibodies was demonstrated by both passive protection (Farci et al., 1996) and active immunisation experiments (Choo et al., 1994) in chimpanzees.
However, neutralising antibodies are frequently directed against epitopes in the HVR1,
71 thus may offer limited cross-protection against heterologous HCV isolates (Choo et al.,
1994).
1.7.3.2 CD4^ T Cell Responses
T cell responses are critical during viral infections; defective T cell function is frequently associated with chronic virus infections in both mice and humans (Oxenius et al., 1998;
Reignat et al., 2002). T cells play a more important role in clearance of established viral infections than antibody. CD4'' T cells recognise antigenic peptides presented in the context of MHC class II. Virus-specific CD4'’ T cells have a direct effector role in controlling viral infections by the production of soluble antiviral factors such as IFNy and by cytotoxic mechanisms (Littaua et al., 1992), though these are generally less important than CD8* T cytotoxic mechanisms, probably because of their lower frequency and more limited target specificity. CD4"’ T cells also provide help for B cell activation and class switching to enable antibody production. They thus have an important role in antiviral immune responses, where antibodies are the main effector arm. CD4* T cells also provide help for CD8^ I cell responses, by stimulating dendritic cells which activate and induce expansion of naïve antigen-specific CD8^ T cell precursors (Bennett et al., 1998; Lanzavecchia, 1998; Ridge et al., 1998). Although
CD4'' T cells are not essential for initial CD8* T cell priming and activation, they are important for the generation and maintenance of memory (Bourgeois et al., 2002;
Janssen et al., 2003). In the absence of CD4" T cell help, CDO"" T cells are more likely to become exhausted and non-functional (Zajac et al., 1998), CD4"^ responses may thus be dispensable for the control of some acute infections that are contained by CD8'’
T cells, for example acute LCMV infection (Christensen et al., 1994) but they are essential for control in chronic infections such as CMV (cytomegalovirus) (Gamadia et al., 2003), HIV (Jin et al., 1999; Rosenberg et al., 1997) and LCMV (Planz et al., 1997).
72 Little is known about the liver specific CD4* T cell responses in acute infections as liver biopsies are rarely taken in both humans and chimpanzees, but in the peripheral blood the CD4* response has been seen to correlate with the clinical course of infection, with a strong correlation between a sustained, vigorous and multi-specific response and virai clearance, even after the loss of antibody responses (Diepolder et al., 1995;
Gerlach et al., 1999; Harcourt et al., 2001; Missale et al., 1996; Rosen et al., 1999).
CD4‘" responses against the protease and helicase domains are stronger and more frequent in patients who resolve acute HCV than those who develop chronic infections
(Diepolder et al., 1995). In some acutely infected patients recurrent viraemia is associated with the loss of CD4* responses (Gerlach et al., 1999). Further evidence for the importance of CD4'' T cell responses in the control of acute HCV infection comes from the association between class II genotypes (DRB*1101 & DRB1*0301) and control of acute infection (Thursz et al., 1999), however this allele is not protective in all populations. This protective MHC class II molecule may present well processed or highly conserved peptides from the non-structural proteins (Diepolder et al., 1999).
In patients who develop chronic infections weaker CD4* responses are detected and these responses are not sustained (Gerlach et al., 1999). The CD4^ response is frequently directed against the core protein in the peripheral blood, and this same preference has also been seen in the liver (Penna et al., 2002). A correlation has also been found between CD4'’ responses to the core protein and a healthy carrier status of patients where viral elimination did not occur (Botarelli et al., 1993; Lechner et al.,
2000b). There is no apparent relationship between the intrahepatic CD4" response and clinical parameters, although the vigour of the peripheral blood CD4"’ response has correlated with low ALT levels and less fibrosis in a small number of patients (Chang et al., 2001; Hoffmann et al., 1995). In general, the impaired CD4^ response is associated with increased liver disease (Puoti et al., 2001) and a sustained therapy
response is associated with stronger CD4'" responses (Cramp et al., 2000; Missale et
73 al., 1997). This suggests that the HCV-specific CD4'' T cell response may contribute to the control of the infection. However, if a deficient CD4* response is detected then
CD8'’ and B cell responses may also be inadequate, so this is difficult to prove.
1.7.3.3 CD8'’ T Cell Responses
CD8^ T cells are key effector cells in the control of many established virus infections
For example, CD8* T cells are important in LCMV (Christensen et al., 2001; Oxenius et
al., 1998), HIV (Wordarz, 2001; Wordarz and Jansen, 2001) and CMV (Reddehase et
al., 1988) infections. They can be highly expanded and are MHC class I restricted, which allows them to recognise most cells when virally infected. Efficient control of
viral infections occurs if broad and highly specific CD8'’ T cell responses are mounted.
If a narrow CD8* T cell response is mounted, it may be easier for viruses to evade
immune control by the generation of escape viral variants (discussed later). CD8‘" T
cells have several effector mechanisms for combating infected cells. They produce
soluble factors such as IFNy and TNFa which contribute to viral clearance in a non-
cytolytic manner (Guidotti and Chisari, 2000). This non-lytic control by cytokines
“cures” cells of virus infection. In addition, they clear virus infections via their ability to
lyse infected cells. There are several pathways by which CTLs can induce apoptosis in
the target cells; these are shown in figure 1.4.
CTL lysis can be in a calcium-dependent, contact-dependent manner, by perforin and
granzymes (Russel and Ley, 2002). Once the CTL has identified the target cell by cell
cell contact, signals are induced which lead to the release of granules containing
perforin and granzymes. Perforin is polymerised in a calcium-dependant manner and
enters the target cell membrane, creating a passage for granzymes to enter the target
cell. The granzymes, particularly granzyme B, cleaves proteins within the target cell
resulting in the activation
74 NK CTL LTa1p2
Fas-L TRAIL perforin & granzymes TNFa LTa3
Fas IN F R1 Lip R DR5 DD DD --J Lfl DD FADD FADD FADD Grb
Granzyme B
Procaspases
Caspase Cascade
APOPTOSIS Figure 1.4. A schematic outline of apoptosis pathways. Lysis of target cells can be induced via several pathways. TRAIL binds to the receptor DR5, Fas-Ligand binds to Fas and TNFa, LTa1p2 or LTa2(31 bind to the TNF-R1. These all recruit an adapter protein FADD, which binds to death domains on these receptors. This leads to the activation of procaspases that initiate a caspase cascade that results in apoptosis of the target cell. LTa3 binds to the LTpR which recruits adapter molecules belonging to the TRAF family which initiates apoptosis within the cell. NK cells and CTL release perforin which forms a pore for granzymes to enter the target cell and activate an adapter called Grb. This activates granzyme B which results in DNA fragmentation and apoptosis of the target cell.
76 of caspases involved in the apoptotic pathways, ultimately resulting in lysis of the target cell.
The Fas-Fas Ligand pathway of apoptosis induced by CTL is a calcium-independent, contact-dependant process (Rouvier et al., 1993). Fas expressed by the target cell, has death domain (DD) regions intracellulariy and upon ligation with Fas-ligand expressed by the CTL, a reformation of a death-inducing signalling complex (DISC) occurs. This complex contains adapter proteins such as FADD (Fas-associated death domain protein) which activates caspases (in particular, caspase-8) in a cascade which
results in the induction of apoptosis within the target cell (details of the pathway are in
(Wajant, 2002).
Apoptosis pathways may also be induced within target cells via pathways involving other members of the TNF receptor superfamily, such as TRAIL (TNF related apoptosis inducing ligand), lymphotoxin (LTa3, LTaip2 or LTa2pi) and TNFa. These all act in a similar way to induce apoptosis within target cells. Ligation of the ligand with an
appropriate receptor (for example, TRAIL binding to DR5, LTa3 binding to the LTpR,
LTa1p2, LTa2p1 and TNFa binding to the TNFR-1) results in the recruitment of
adapter proteins which recognise death domain regions of the receptors. This results in
the activation of caspase cascades which induce apoptosis within the target cell. A
schematic overview of the pathways is shown in figure 1.4 (further details can be found
in Abe et al., 2000; Schulze-Osthoff et al., 1998).
The relative importance of the effector pathways involved in controlling viral replication
depends on several factors, including the virus, the stage of infection and the host cell
type or tissue (Guidotti and Chisari, 2000). For example, in acute LCMV infections,
CD8* T cells mediate control via lysis in a perforin dependant manner, whereas in
77 acute vaccinia virus infection, CD8* T cells mediate control by the release of cytokines.
In chronic infections, “curing” of cells by cytokine release (IFNy and TNFa) from CD8* T cells is very important, as seen in hepatitis B virus infection in transgenic mice (Guidotti et al., 2002; Wieland et al., 2003) (reviewed by (Guidotti and Chisari, 1996); however there is some active lysis of infected cells as well.
CD8* T cell responses can be detected in HCV-infected individuals in acute and chronic infections (Chang et al., 1997; Koziel et al., 1993; Koziel et al., 1992; Koziel et al., 1997).
In acute HCV infection the virus-specific CD8^ T cell response can be very strong: up to 7% of circulating CD8* cells were targeted to the dominant epitope, with 1% against the second dominant epitope (Lechner et al., 2000a). During acute infection the CD8* cells have a highly activated phenotype (CD38"^ CD69"^) but have been found to be unable to produce IFNy (Lechner et al., 2000a; Thimme et al., 2001). These cells have been called “stunned”. The emergence of these “stunned” cells correlated with the onset of liver inflammation, suggesting that these cells may have a pathological role in the liver. It is unknown whether these cells can produce other cytokines. Resolution of viraemia and liver inflammation correlates with the emergence of less activated CD8* cells with high IFNy production. The frequency of CD8* cells decreases after the acute
phase, with only 0.01-1.2% of circulating HCV-specific CD8* cells being reported in
HCV seropositive patients (He et al., 1999).
In acute HCV infection the CD8* response is detectable in the liver and blood and has
been shown to correlate with the virological outcome. An early, multi-specific
intrahepatic CD8* CTL response is associated with viral clearance in chimpanzees
(Cooper et al., 1999; Lechner et al., 2000b) and the nature of the peripheral blood
78 CD8* response is also correlated with clearance of infection (Gruner et al., 2000;
Lechner et al., 2000b; Thimme et al., 2001): broad, vigorous responses against multiple epitopes are associated with good control of virus replication. This may be important in preventing CD8* escape and in controlling viral replication. The proportion of activated CD8* cells has also correlated with the degree of liver inflammation (ALT levels) in acute disease (Lechner et al., 2000a).
In chronically infected HCV patients CD8* CTL responses are not sustained (Chang et al., 2001; Lechner et al., 2000a; Lechner et al., 2000b; Takaki et al., 2000). In one study, recovered patients had 0.09% of circulating HCV-specific CD8* cells that had a greater IFNy production than those in chronically infected patients who had 0.07% of circulating HCV-specific CD8'' cells (He and Greenberg, 2002). The poor CD8* response is only HCV-specific as responses to other viruses including EBV (Epstein
Barr virus), influenza and HIV are not affected (Chang et al., 2001; He et al., 1999;
Lauer et al., 2002); therefore the immunosuppression seen in HCV is not a generalised immunosuppression. The relationship between HCV-specific CD4* and CD8* T cell responses could be important. The absence of a sufficient CD4* T cell response may compromise the CD8* T cell response, either by the poor induction of responses or by the lack of maintenance of responses. Therefore it is likely that the weak CD8* response in HCV patients may be due to the ineffective CD4* response.
During chronic HCV infection CD8* CTL epitopes in all viral proteins have been identified in both human (Battegay et al., 1995; Cerny et al., 1995; Koziel et al., 1993;
Koziel et al., 1995; Koziel et al., 1992) and chimpanzee studies (Erikson et al., 1993).
The magnitude of the HCV-specific CD8* response can be heterogeneous, but this does not correlate with viraemia (Wong et al., 1998). However, it has been found to correlate with an increase in ALT levels and IFNy (Nelson et al., 1998; Wong et al..
79 1998), which may relate to liver damage. During chronic infection, HCV-specific CTL responses may be too weak to achieve viral clearance but may be strong enough to mediate some control over the viral load, which could influence the outcome of infection. This occurs at the expense of liver disease resulting ultimately in the development of cirrhosis and hepatocellular carcinoma.
1.7.4 Immunopathological Role of the Immune Response in HCV-associated Liver
Damage
T cells are likely to have an immunopathological role during HCV infection, promoting inflammation within and damage to the liver. HCV-specific T cells are found within the liver with compartmentalisation of CD4* cells to the portal areas (Wejstal et al., 1992) and CD8* cells in the hepatic lobules (Yuk et al., 1986). These cells have an activated or memory phenotype. B cells are also found in germinal centres of lymphoid follicles in the portal tracts (Gonzalez-Peralta et al., 1994; Onji et al., 1992).
The liver of HCV infected patients has a 30x greater frequency of CD8* cells than the blood (He et al., 1999). However, most of the intrahepatic T cells are not HCV-specific
(Bertoletti et al., 1997; Carding et al., 1993; He et al., 1999). HCV-specific CTL are also present in the liver, at a higher frequency than in the peripheral blood (Koziel et al., 1993; Koziel et al., 1995; Koziel et al., 1992; Nelson et al., 1998) but are at lower frequencies in the liver than is seen in for other viral infections such as LCMV (Byrne and Oldstone, 1984). Aggressive macrophage and monocyte responses are also present in the liver, which will enhance antigen presentation and the inflammatory process (Marrogi et al., 1995; Mosnier et al., 1994). The presence of pro-inflammatory cytokines, particularly IFNy, in the liver will also enhance activation of non-specific inflammatory responses. IFNy enhances the ability of TNFa to mediate hepatic
80 damage (Morita et al., 1995), and initiate recruitment of T cells (Cook et al., 1995;
Shields et al., 1999; Taub et al., 1993), NK and NKT cells (Kaneko et al., 2000;
Salazar-Mather et al., 1998) :o the liver, all of which are capable of mediating hepatic damage in a non-antigen-specific manner. The presence of innate cells such as NK,
NKT and yô T cells in HCV liver may also be contributing to immunopathogenesis as they create and sustain an inflammatory environment activating non-specific T cells. If this response is not down-regulated damage to uninfected hepatocytes could occur, thus enhancing inflammation and damage to the liver.
HCV-infected hepatocytes are destroyed by CD8* CTL cells via Fas-FasL (Hiramatsu et al., 1994; Kondo et al., 1997; Lehman et al., 1996; Mita et al., 1994), TNFa
(Kinkhabwala et al., 1990; Vassalli, 1992) and perforin (Ando et al., 1997; Kondo et al.,
1997). However, uninfected hepatocytes may also be damaged by cells which are non- specifically activated within the liver or that have been recruited by the expression of chemokines from activated cells as part of the inflammatory process. Another
mechanism that can amplify hepatocyte death is that of CD40-CD40L binding between activated lymphocytes and macrophages with hepatocytes, which amplifies Fas-
mediated death of hepatocytes (Afford et al., 1999). This can also cause liver damage
irrespective of the antigen-specificity of the cells.
A study by Napoli et al (Napoli et al., 1996) found a correlation between Thi cytokine
responses and hepatic fibrosis and portal inflammation. The generation of a pro-
inflammatory environment in the liver enhances necroinflammatory and fibrotic liver
disease such as cirrhosis and hepatocellular carcinoma. In contrast, Cacciarelli at a!
(Cacciarelli et al., 1996) suggested that an elevated Th2 response was present which
may downregulate the HCV-specific immune response.
81 Antibodies may also contribute to immunopathology in HCV infection. Over half of chronically infected patients have increased expansions of CDS* B cells in the peripheral blood (Pozzato and Moretti, 1994). Activation of these cells has been associated with autoimmune diseases such as rheumatoid arthritis (Jarvis et al., 1992) and may be linked with autoimmunity and B cell lymphomas in HCV patients. HCV- specific antibodies are also implicated in the pathophysiology of mixed cryoglobulinemia, where cold precipitable serum monoclonal and polyclonal IgG and
IgM immunoglobulins cause a systemic vasculitis with a range of clinical manifestations
(Agnello, 1997). Approximately 80% of mixed cryoglobulinemia cases are secondary to HCV infection (Agnello, 1997). In addition, a high HCV seroprevalence has also been reported in patients with other diseases, including B-cell-non-Hodgkin's lymphoma (reviewed by Haufater et al., 2000) and Sjogren’s syndrome (Almasio et al.,
1992; Haddad et al., 1992).
1.7.5 Implications for Vaccine Development
The preceding information gives some insight into what kind of immune response vaccines may need to elicit to combat HCV infection. In order to prevent infection, a vaccine would have to induce high titre neutralising antibodies presumably directed against the envelope glycoproteins (El & E2). To control acute infection, a strong, sustained, vigorous and multi-specific antiviral CD4* and CDS* response would be required; therefore a vaccine would need to induce broad spectrum T cell responses targeting multiple conserved regions of the virus stimulating CD4* and CDS* responses to provide protection against re-infection. Antibodies may also be important in reducing spread and possibly aiding elimination of the virus. A therapeutic vaccine could aim to elicit or restore a similar type of response to eliminate infection, or would at minimum need to reduce infection in the liver in order to prevent the development of further liver
82 damage. Therefore the inducticn of liver specific CD4* and CD8* T cell responses and antibody responses would be lequired, without causing enhancement of non-specific immunopathological damage. To aid the development of vaccines stimulating the correct immune responses without enhancing immunopathology, a better understanding of the immune responses occurring in HCV infection and why they frequently fail to control viral repication is required.
1.8 Viral Immune Evasion Strategies
1.8.1 General Immune Evasion Strategies used by Viruses
Viruses have evolved many different strategies for evading the host immune response to allow them to establish persistent infections. There are three main types of viral immune evasion strategies. Firstly, viruses may interfere with the induction or maintenance of the host immune response. They may achieve this by inducing a generalised immunosuppression, or cause tolerisation of virus-specific T cells or cause
CD8* cytotoxic T cells to become exhausted. Secondly, viruses may avoid recognition by the host immune response by mechanisms such as latency, infecting
immunoprivileged sites, downregulation of MHO and adhesion molecule expression or antigenic variation. Finally, viruses may avoid host control by possessing resistance to effector mechanisms, for example, by down-regulating Fas expression on infected cells or possessing resistance to control by antiviral cytokines. Most viruses use a
combination of these evasion mechanisms to establish persistent infections within the
host. Viruses develop these evasion mechanisms under pressure from the host
immune response in order to survive and replicate, thus providing them an evolutionary
advantage.
83 1.8.1.1 Interference with the induction or maintenance of host immune responses
Viruses may downregulate the host immune responses by inducing a generalised immunosuppression within the host in order to establish a persistent infection. There are several ways viruses can induce immunosuppression. They may encode proteins that have immunosuppressive effects, for example, some human and animal retroviruses encode the p15E protein (Haraguchi et al., 1997) which has effects on monocytes, NK cells and CTL activity (Cianciolo et al., 1988; Gottlieb et al., 1990).
Alternatively viruses can infect cells and tissues of the immune system and impair their functions, or simply target them for destruction. A good example of a virus that takes this approach is measles virus, which infects T cells, B cells, DCs and monocytes, destroying them by syncytium formation (Joseph et al., 1975). Measles virus also inhibits the immune response in vitro by downregulating IL-12 production (Borrow and
Oldstone, 1994). These are non-antigen-specific mechanisms of impairing the host immune response. Sometimes the host response is only transiently impaired in a localised area; however in extreme cases the host response may become severely impaired, for example, in measles virus infection or end-stage HIV infection, which may render the host susceptible to opportunistic infections.
Viruses may also induce specific immunosuppression by several mechanisms. A virus may induce neonatal tolerance, if infection occurs during early stages in life when the T cell repertoire is being shaped by thymic selection, resulting in the deletion of virus- specific clones as they are recognised as self. An example of this occurs when neonatal mice are infected with LCMV: they become persistently infected and tolerant to the virus (Pircher et al., 1989). An alternative strategy for generating a specific immunosuppression is to cause exhaustion of antigen-specific T cells. Again, LCMV uses this strategy. Infection of mice with an aggressive strain of LCMV generates a rapid expansion of CTL, which become exhausted and cannot control the virus,
84 resulting in a persistent infection (Moskophidis et al., 1993). Exhaustion may involve deletion of virus-specific T cells, or the persistence of T cells with impaired effector function (Zajac et al., 1998).
1.8.1.2 Avoidance of recognition by the host immune response
The second approach viruses may use is to avoid recognition by the host immune response, in order to gain extra time for replication and transmission to a new host.
Latency is a strategy a virus may use to reside in the host undetected. Herpes simplex virus type 1 (HSV-1) is a good example of a virus which uses latency to avoid host recognition. It infects via the skin and undergoes lytic replication; it then infects local sensory neurones but in these cells it does not express any gene products, hence avoiding MHC class I recognition by CTL. However, this strategy is not ideal for transmission; therefore HSV periodically reactivates and undergoes lytic replication in the skin in order to generate viral particles (Sawtell and Thompson, 1992).
Another strategy viruses use to avoid recognition by the host immune response is to infect immunoprivileged sites. These are sites within the body which the immune system has limited access to, and which therefore provide a safe haven for viruses.
Immunoprivileged sites include the kidney, the salivary glands and the central nervous system (CNS). Lymphocyte trafficking to the CNS is limited due to the blood brain barrier, and there is an immunosuppressive environment there with little MHC class I expression. Several viruses persist in the central nervous system, including HSV-1 and varicella zoster virus (VZV).
Viruses may also avoid recognition by the host immune system by interfering with MHC or adhesion molecule expression on infected cells. There are several ways viruses can
interfere with MHC expression; virtually every part of the process involved in generating
85 MHC expressing viral peptides is targeted by a virus in order to allow enhanced virus production before detection. EBV targets the proteolysis of proteins. The viral protein
EBNA-1 which is the only protein produced in latently-infected cells is resistant to proteolysis by the proteasome because it contains glycine-alanine repeats (Levitskaya et al., 1995). Other viruses prevent the transport of peptides into the ER. Herpes simplex virus encodes a protein called ICP47, which blocks TAP (transporter associated with antigen processing) from the cytoplasmic side (Fruh et al., 1995; Hill et al., 1995; York et al., 1994), whilst the human cytomegalovirus (HCMV) US6 protein interferes with peptide translocation from the luminal side of the ER (Ahn et al., 1997).
HCMV also impairs MHC class I expression using the US11 protein which disassociates MHC chains resulting in their degradation (Wiertz et al., 1996). The transport of MHC may also be targeted by viruses; for example, the adenoviral encoded E3/19K protein retains MHC class I molecules in the ER (Andersson et al.,
1985; Burgert and Kvist, 1985). The HIV-1 nef protein causes endocytosis of surface
MHC class I (Schwartz et al., 1996). Viruses may also downregulate adhesion molecules in order to reduce their susceptibility to CTL recognition, for example, EBV can downregulate expression of ICAM-1 (intracellular adhesion molecule-1) and LFA-3
(leukocyte function antigen-3) on the surface of infected cells (Gregory et al., 1988).
Another strategy viruses can take in order to avoid recognition by the host is to undergo antigenic variation. This can be achieved by various mechanisms including reassortment of the viral genome, recombination between genomes or by mutation.
RNA viruses or viruses which have an RNA intermediate step have extremely high mutation rates, generating diversity which can cause escape from both antibody and
CTL control. CTL escape viral variants are generated when CD8* cells have a major role in controlling the infection. This has been seen in HIV infection (reviewed by
Borrow and Shaw, 1998).
86 1.8.1.3 Resistance to host effector mechanisms
Viruses can also possess resistance to host effector mechanisms: they can interfere with the activity of antiviral cytokines (such as type 1 IFNs and IFNy) or render cells resistant to lysis via apoptotic pathways. Antiviral cytokines are an important part of the host immune response as they can “cure” cells of a viral infection (as described in the immune responses section), therefore these cytokines are the most likely to be targeted by viruses, which may encode cytokine receptor homologues or chemokine agonists or antagonists or possess resistance to cytokine control mechanisms. Many viruses target interferons: for example, pox viruses such as vaccinia virus produce an interferon receptor homologue to mop up host interferons (Alcami and Smith, 1996); and adenoviruses encode a protein (ElA) which inhibits the JAK/STAT signalling pathways of interferon (Mahr and Gooding, 1999). Viruses may also target antiviral mechanisms induced by type 1 IFNs, such as the RNA-dependent protein kinase
(PKR). Poliovirus can cause the degradation of PKR and other viruses such as adenoviruses and EBV encode RNA inhibitors which compete with dsRNA for binding to PKR (Mahr and Gooding, 1999).
Viruses may render host cells resistant to lysis by targeting apoptotic pathways in order to provide more time for replication and transmission. Some viruses encode viral homologues of FLIP (FLICE inhibitory proteins) which blocks the formation of the DISC
(death inducing signalling complex) and prevents apoptosis (Thome et al., 1997). Pox viruses target caspases, which are an integral part of many apoptotic pathways (see figure 1.4), by encoding a protein called crmA. Other viruses encode homologues of anti-apoptotic molecules such as Bcl-2; examples of this are used by HSV-1 (Kroemer,
1997) and EBV (Henderson et al., 1993).
87 1.8.2 HCV Immune Evasion Strategies
Approximately 20% of individuals acutely infected with HCV contain viral replication efficiently. As discussed in the previous section, and by analogy with other virus infections it would be expected that this would require efficient early control of virus replication by innate responses (especially type 1 IFNs), and efficient adaptive responses (especially CD4* and CD8* T cell responses). The available evidence from
HCV infection supports this, for example, associations between strong multi-specific
CD4* and CD8* T cell responses, “NOB” antibody responses and the control of acute
HCV infection have all been reported. However, approximately 80% of individuals don’t control acute HCV infection efficiently and go on to become chronically infected.
This suggests that HCV has strategies for impairing, avoiding and/or resisting control by immune responses mounted by these individuals. As discussed below, many mechanisms have been proposed to be involved.
It is unclear why HCV does not establish a persistent infection in all infected individuals. This could be due to host or viral factors. Host factors playing a role in the establishment of a persistent infection could include genetics, for example, the HLA type may affect the magnitude and breadth of HCV-specific CD4* and CD8* T cell responses. Polymorphisms in the CD81 gene may alter the level of viral replication, as
CD81 is a proposed as a putative receptor for HCV. Alternatively CD81 polymorphisms may alter the efficiency of antiviral host responses. Prior exposure of the host to HCV infection would affect memory T cell responses, which again could alter the magnitude and breadth of HCV-specific T cell responses. Viral factors influencing the outcome of
HCV infection may include the infectious dose and the route of infection. The virus
genotype has also been implicated; as discussed previously some genotypes are more
88 responsive to therapy and immune control than other genotypes. This is also discussed in more detail below.
1.8.2.1 Type 1 Interferons
Interferons play an important role in the host antiviral response, so it is not surprising that viruses have evolved strategies to avoid or counteract interferons. HCV may be impairing the host type 1 IFN response, through the action of the NS5A protein. This protein has been observed to impair IRF-1 (interferon regulatory factor) and IRF-3 activation (see figure 1.2 in previous section for interferon induction pathways) (Foy et al., 2003; Pfiugheber et al., 2002). NS5A has also been found capable of binding to another molecule involved in the interferon signalling pathway called Grb2 (growth factor receptor bound protein 2). Binding to Grb2 results in a decrease in the phosphorylation of ERK-1/2 (extracellular signal-related protein kinase 1 and 2) which leads to a reduction in interferon induction (Tan et al., 1999). NS5A has also been shown to inhibit the activity of the transcription factor AP-1 (MacDonald et al., 2003) also involved in the interferon signalling pathways. Conversely, NS5A may also activate an interferon promoter which leads to an increase in the expression of IRF-3
(Ghosh et al., 2003) which may augment virus mediated interferon activity.
HCV also possesses resistance to interferons. This is probably very effective as it is
clear that interferon therapy fails in the majority of patients. HCV may be resisting the
host interferon response in several ways; NS5A has been shown to induce IL-8, a
cytokine which can counteract the antiviral activity of interferons (Polyak et al., 2001).
HCV also has effects on the interferon signalling pathways, for example, the core
protein has been implicated in the inhibition of ST ATI expression, a component of the
interferon signalling pathway (Basu et al., 2001). In addition, HCV has mechanisms for
impairing IFN-induced effector mechanisms. Enomoto et al (Enomoto et al., 1996)
89 originally identified a region within NS5A in which mutations affected the response to interferon therapy in Japanese patients with a type 1b infection. This region was called the interferon sensitivity determining region (ISDR). A correlation between the ISDR and interferon response has also been found in Indonesian HCV patients infected with genotypes 1b, 1c and 2 (Lusida et al., 2001). The mechanism of interferon evasion via the ISDR has since been identified to involve the NS5A gene product binding to the dsRNA-dependant protein kinase (PKR) and preventing its dimérisation and the subsequent phosphorylation of the protein synthesis initiation factor-2a (elF-2a), thus blocking the antiviral activity of interferons (Gale et al., 1997). Other viral proteins may also be involved in HCV resistance to the host interferon response. Taylor et al (Taylor et al., 1999) found the E2 protein contains a domain with high homology to the autophosphorylation domain of PKR and the phosphorylation target region of elF2a, called PePHD (PKR- elF2a phosphorylation homology domain). This region could block PKR activity, more efficiently with genotype 1 E2 protein than genotypes 2 and 3
E2 protein. This may also help to explain the difference in the sensitivity to interferon of different HCV genotypes.
In summary, HCV appears to be capable of interfering with multiple different components of the pathways involved in interferon induction, signalling pathways and activity in host cells, all of which result in impairment of the control of viral replication by interferons.
1.8.2.2 NK Cells
If HCV impairs type 1 IFN induction, this may in turn affect the activation of NK cells in early HCV infection. HCV may also impair NK responses in other ways, for example, it has been observed that NK cells from HCV patients have a decreased cytotoxic
90 capacity compared to those from uninfected individuals (Corado et al., 1997).
However, it is unclear when in infection this is occurring, and it may be a consequence of a persistent infection. There may be a role for interaction between the HCV E2 protein and CD81 in impairment of NK responses, as it has been shown that E2 can cross-link CD81 expressed on NK cells and block NK cell activation, cytokine production (IFNy), proliferation and cytotoxic granule release (Crotta et al., 2002; Tseng and Klimpel, 2002).
Alternatively, HCV could avoid NK cell recognition by inducing an upregulation of MHC class I (Herzer et al., 2003). In addition, HCV could induce resistance to NK effector mechanisms, by effects on the antiviral actions of interferons (described above) or by affecting the susceptibility of cells to NK lysis (discussed below). As the role of NK cells in HCV infection is unclear, the physiological role of these effects of HCV infection on NK cells remains to be clarified. However, they could conceivably limit the antiviral activity of NK cells early in the infection process, allowing a persistent infection to be established.
1.8.2.3 Antibody Response
Humoral responses are also potential targets for HCV immune evasion. There seems to be a delay in the induction of the antibody response during HCV infection (Chen et al., 1999) which could contribute to HCV persistence. Ray et al (Ray et al., 2000), found the antibody response in experimentally infected chimpanzees to be of a low titre, short-lived and did not provide protection against re-infection with a homologous strain of virus (Farci et al., 1992), therefore these observations suggest that the magnitude and duration of the antibody response is impaired by HCV. However, HCV persistence and progression of hepatitis are seen even in the face of circulating antibodies, which would suggest the virus also avoids control by the host antibody
91 responses. One strategy HCV may take to avoid the antibody response is that of antigenic variation in the hypervariable region 1 (HVR1) of the E2 protein (Farci et al.,
1996; Higashi et al., 1993; Taniguchi et al., 1993) which is a major target for neutralising antibodies. Mutations in this region account for a large degree of sequence variation between isolates from infected patients (Farci et al., 1996). The emergence of sequence variants will allow escape from neutralising antibodies which may lead to persistent infection (Farci and Purcell, 2000; Farci et al., 2000). However, the generation of viral escape variants may be a consequence of a persistent infection and immune pressure rather than the cause of persistent infection, which would imply that HCV has other strategies for evading host immune responses in order to establish a persistent infection.
1.8.2.4 T Cell Responses
HCV may impair the induction and/or maintenance of T cell responses via effects on
DCs. HCV RNA has been detected in cells of the immune system (Bain et al., 1999;
Lerat et al., 1998) suggesting that HCV is capable of infecting these cells which could impair the immune responses generated by these cells. There is mounting evidence that there is an alteration in the ability of antigen presenting cells to induce and maintain immune responses during infection with HCV. It has been shown that dendritic cells from chronically infected individuals have an impaired stimulatory capacity in vitro and a reduced ability to produce IL-12 (Auffermann-Gretzinger et al.,
2001; Bain et al., 2001; Kakumu et al., 2000; Kanto et al., 1999). This has also been shown in murine model systems where bone marrow derived DCs expressing HCV proteins have a reduced allostimulatory capacity (Kim et al., 2002a). Similarly Hiasa et al (Hiasa et al., 1998) found murine lymphoid DCs expressing HCV structural proteins also had a decreased allostimulatory capacity and a decrease in IL-12 production.
Sarobe et al (Sarobe et al., 2002) have also seen a decrease in T cell allostimulatory
92 responses and IL-2 production stimulated by monocyte-derived DCs expressing HCV core and E1 proteins. Macrophages are other antigen presenting cells that may also be affected by HCV as it has been seen that the HCV core protein can inhibit macrophage IL-12 and nitric oxide production and these macrophages also had a decreased ability to stimulate allogenic T cell responses (Lee et al., 2001). These defects may contribute to deficits in the antiviral T cell responses in vivo.
In acute HCV infection, strong CD8* T cell responses are induced but impairments in their effector functions have been reported. It has been observed that in patients with acute HCV infection highly activated CTL populations were detected but they temporarily failed to secrete IFNy. These cells have been described to have a
“stunned” phenotype as they recovered the ability to secrete IFNy as the levels of viraemia decreased (Lechner et al., 2000b). Potentially these stunned CTL may secrete other cytokines such as IL-4 or IL-10, as HCV-specific CTL clones have been found to secrete these cytokines (Koziel et al., 1995). The perforin content, lytic ability and proliferative capacity has also been found to be reduced in patients with acute
HCV infection (Urban! et al., 2002). These “stunned” cells may result from a lack of the appropriate cytokines produced by DCs and macrophages (Bain et al., 2001; Lee et al.,
2001; Sarobe et al., 2002). However, it is more likely that this phenotype may be a consequence of a high viral load in vivo, as a similar phenomenon has been observed in other viral infections, such as LCMV infection (Moskophidis et al., 1993).
Alternatively, the impaired effector functions of HCV-specific CTL may be due to an effect of the core protein interacting with the complement receptor gClqR. This has been proposed as a mechanism for inhibition of antiviral CTL responses and pro- inflammatory cytokine production (IL-2 and IFNy) by Yao et al (Yao et al., 2001a; Yao et al., 2001b).
93 Although strong HCV-specific 008"^ T cell responses are frequently observed in acute
HCV infection, loss of CD4* T cell responses and low magnitude CDS* T cell responses frequently accompany chronic HCV infection (Gerlach et al., 1999; Lechner et al.,
2000a; Wong et al., 2001). Again, the mechanisms responsible for this are not clear; but impairment of DC function may be of importance here.
HCV may also avoid recognition by the host T cell response via its high mutation rate which could result in the generation of escape viral variants. CTL escape has been described in a chimpanzee with a chronic HCV infection (Weiner et al., 1995a; Weiner et al., 1995b). If the initial CTL response is polyclonal and multispecific it is less likely to be escaped, but an initially narrow response could be evaded by the virus by this mechanism.
HCV may also possess resistance to T cell effector mechanisms. Several studies have found the core protein to reduce the susceptibility of cells to apoptosis by interactions with the TNFR-1 and Fas (Ruggieri et al., 1997; Zhu et al., 1998). Altering the susceptibility of cells to apoptosis would prevent their destruction by CTLs, thus providing longer for viral replication.
1.8.3 Potential Involvement of the HCV Core Protein in HCV Evasion Strategies
The HCV core protein has properties that suggest it may contribute to HCV persistence via several different mechanisms. First, it may act extracellularly: it has been shown that the core protein can bind to the complement receptor (gClqR), leading to impaired
T cell responses (Kittlesen et al., 2000; Yao et al., 2001a; Yao et al., 2001b). Second, the presence of the core protein within the cytoplasm of cells has been shown to bind to the cytoplasmic tail of members of the TNFR family (Chen et al., 1997; Matsumoto et
94 al., 1997; Ruggieri et al., 1997; Zhu et al., 1998) and may modulate signalling through these molecules (Hahn et al., 2000; Machida et al., 2001; Otsuka et al., 2002; Zhu et al., 2001). This family includes members which are involved in DC activation; therefore core could decrease DC activation and functions and hence impair the host immune response. The TNFR family also contains members involved in NK cell and CTL effector functions, therefore the core protein could increase the susceptibility of DCs to apoptosis leasing to impaired immune responses, or it may decrease the susceptibility of hepatocytes to apoptosis to increase viral production. Third, the core protein could also affect all these pathways by effects on downstream signalling pathways, for example, NFkB (Yoshida et al., 2001). Fourth, the core protein has been suggested to impair JAK/STAT signalling pathways involved in cellular responses to IFNs (Basu et al., 2001). Finally, the core protein has been shown to have effects on the regulation of gene expression in the nucleus, for example, it has been shown to have effects on p53
(Herzer et al., 2003; Ray et al., 1997), which again could increase or decrease the susceptibility of cells to apoptosis and have effects on the activation of responses.
Given the reports that HCV was able to infect DCs (Goutagny et al., 2003; Navas et al.,
2002) and that DCs from HCV patients had impaired functions, including a reduced stimulatory capacity and IL-12 production (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Kakumu et al., 2000; Kanto et al., 1999) and the variety of mechanisms by which core could potentially effect these cells, I set out to explore the effects of the core protein on the activation and functions and the sensitivity of cells to apoptosis. Two studies that provided further rationale for this work were those by Hiasa et al (Hiasa et al., 1998), who found murine DCs expressing HCV structural proteins had a decreased stimulatory capacity and reduced IL-12 production and Lee at a! (Lee et al., 2001), who found that the core protein decreased IL-12 and nitric oxide production by macrophage cell lines. I aimed to address effects of the core protein on activation and interaction of
DCs with T cells and also effects core may have on the susceptibility of DCs to
95 apoptosis by means of adenoviral vectors, which would be more physiologically
relevant than previous studies.
96 Chapter 2 - Materials & Methods
2.1 Materiats
2.1.1 Biochemical Reagents
Reagent Supplier Catalogue Number lOObp DNA Ladder Invitrogen Life 15628-019 Technologies, Paisley, UK 1 Kb"" DNA Ladder Invitrogen Life 10787-018 Technologies, Paisley, UK 2-Mercaptoethanol Sigma Aldrich Company M7522 Ltd, Poole, UK ^H-Thymidine Amersham Pharmacia TRA120 Biotech UK Ltd, Little Chalfont, UK Agarose Promega UK, V-3121 Southampton, UK Alexa Fluor 647 Dextran Molecular Probes Europe D22914 (10,000 MW, anionic) BV, Leiden, Netherlands Ampicillin Sigma Aldrich Company A-9518 Ltd, Poole, UK Aldrich Beta Plate Scint Wallac, Turku, Finland SC/9200 BO-PRO-3 iodide Molecular Probes Europe B-3587 BV, Leiden, Netherlands Bovine Serum Albumin Sigma Aldrich Company A4503 (BSA) Ltd, Poole, UK Butan-1-ol BDH Lab Supplies, Poole, 100615-Y UK Caesium Chloride Sigma Aldrich Company C3139 Ltd, Poole, UK Chloroform BDH Lab Supplies, Poole, 100776B UK Chromogenic Alkaline BioRad Laboratories Ltd, 170-6432 Phosphatase Substrate Hamel Hempstead, UK (ELISPOTS) Deoxy nucleotide Promega UK, U1240 Triphosphates (DNTPs) Southampton, UK Dimethylsulphoxide Sigma Aldrich Company D2650 (DMSO) Ltd, Poole, UK DNA Polymerase 1 Invitrogen Life 18012-021 Technologies, Paisley, UK DNA Polymerase Mg Free Promega UK, H190G Buffer (lOx) Southampton, UK
97 E.CO//BJ5183 Stratagene, Cambridge, UK 200154 Electroporation Competent Cells E.coli Max Efficiency Invitrogen Life 18297-010 DH10B Competent Cells Technologies, Paisley, UK E.coli Subcloning Efficiency Invitrogen Life 18265-017 DH5a Competent Cells Technologies, Paisley, UK Effectene Transfection Qiagen, Crawley, UK 301307 Reagent Ethanol lAH Stores, Compton, UK Ethidium Bromide Sigma Aldrich Company E1510 Ltd, Poole, UK FcR Blocking Reagent Miltenyi Biotech, Bisley, UK 130-059-901 Fluorescent Mounting Dako Corporation, Ely, UK 53023 Medium Fugin Autogen Bioclear, Caine, Ant-fn UK Gel Loading Solution Type 1 Sigma Aldrich Company G7654 (6x) Ltd, Poole, UK Gelatin (type A) Sigma Aldrich Company (32500 Ltd, Poole, UK GeneJuice Transfection CN Biosciences, Beeston, 70967-5 Reagent UK Glacial Acetic Acid BDH Lab Supplies, Poole, 1022466 UK Glycerol BDH Lab Supplies, Poole, 101186M UK Glycine Invitrogen Life 15709-017 Technologies, Paisley, UK Goat serum Invitrogen Life 16210-064 Technologies, Paisley, UK Heparin (Pump-Hep) Leo Laboratories Ltd, Princes Risborough, UK Isopropanol BDH Lab Supplies, Poole, 1022466 UK Kanamycin Sigma Aldrich Company K0879 Ltd, Poole, UK Lipopolysaccharide (LPS) Sigma Aldrich Company L2880 E.co//055.135 Ltd, Poole, UK Low Endotoxin FCS (Batch Harlan Sera Labs, 009501 91607) Loughborough, UK Low Range Marker BioRad Laboratories Ltd, 161-0306 Hemel Hempstead, UK Luria Broth (LB) lAH Media Supplies, Compton, UK Luria Broth (LB) Agar lAH Media Supplies, Plates Compton, UK Magnesium Chloride 6- BDH Lab Supplies, Poole, 101494V Hydrate UK Methanol BDH Lab Supplies, Poole, 10158BG
98 UK Mineral Oil Sigma Aldrich Company M5904 Ltd, Poole, UK Na“'Cr04 Amersham Pharmacia CJS11 ^ ’Chromium) Biotech UK Ltd, Little Chalfont, UK Opti-Phase Supermix Wallac, Turku, Finland SC/9235/21 Scintillant Paraformaldehyde Sigma Aldrich Company P6148 Ltd, Poole, UK Phalloidin-Alexa-Fluor-568 Molecular Probes Europe A12380 BV, Leiden, Netherlands Phenol Sigma Aldrich Company P4557 Ltd, Poole, UK Phosphate Buffered Saline Invitrogen Life 14200-067 (PBS) (1 OX) Technologies, Paisley, UK Potassium Chloride Sigma Aldrich Company P4504 Ltd, Poole, UK Potassium Hydroxide Sigma Aldrich Company P5958 Pellets Ltd, Poole, UK Recombinant Lymphotoxin R&D Systems, Abingdon, 678-LY a1(32 UK Recombinant Lymphotoxin R&D Systems, Abingdon, 679-TX a231 UK Recombinant TRAIL CN Biosciences, Beeston, 616376 UK Sodium Azide Sigma Aldrich Company S8032 Ltd, Poole, UK Sodium Carbonate BDH Lab Supplies, Poole, 102405Y Anhydrous UK Sodium Chloride BDH Lab Supplies, Poole, 102415K UK Sodium Citrate BDH Lab Supplies, Poole, 102425L UK Sodium Hydrogen BDH Lab Supplies, Poole, 102475W Carbonate UK Sodium Hydroxide Pellets BDH Lab Supplies, Poole, 102524X UK Sucrose BDH Lab Supplies, Poole, 102744B UK Sulphuric Acid BDH Lab Supplies, Poole, 102761C UK T4 DNA ligase Promega UK, Ml 801 Southampton, UK TAE (25x) Anachem Ltd, Luton, UK 0796-1.6R Tag Polymerase Promega UK, Ml 865 Southampton, UK Terrific Broth Invitrogen Life 22711-022 Technologies, Paisley, UK TMB Substrate R&D Systems, Abingdon, DY999
99 UK Tris-HCi Sigma Aldrich Company T3253 Ltd, Poole, UK Triton-X-100 Sigma Aldrich Company T9284 Ltd, Poole, UK Tween-20 Sigma Aldrich Company P7949 Ltd, Poole, UK
2.1.2 Plastic ware
Product Supplier Catalogue Number 12ml Polyclear Centrifuge Beckman Instruments UK 362305 Tubes Ltd, High Wycombe, UK 13mm Coverslips Western Lab Supplies, ML-579-13 Aldershot, UK 15ml Falcon Tubes Becton Dickinson Labwear, 352096 NJ, USA 19g Needles Sherwood Medical, 1100-252012 Gosport, UK 24 Well Plates Becton Dickinson Labwear, 353047 NJ, USA 3.2ml Polycarbonate Thick Beckman Instruments UK 362305 Wall Tubes Ltd, High Wycombe, UK 50ml Falcon Tubes Becton Dickinson Labwear, 352070 NJ, USA 6 Well Plates Becton Dickinson Labwear, 353046 NJ, USA 6mm Dishes Corning, NY, USA 430166 96 Flat Bottom Plates Corning Costar, High 3595 Wycombe, UK 96 V Bottom Plates Corning Costar, High 3894 Wycombe, UK 96 Well Sample Plate (CTL Wallac, Turku, Finland 1450-401 Counting Plate) Bijou Tubes BDH Lab Supplies, Poole, 275/0460/24 UK Cell Scrapers 18cm Becton Dickinson Labwear, 3085 25cm NJ, USA 3036 Cell Strainers (40pm) Becton Dickinson Labwear, 352340 NJ, USA Chamber Slides Invitrogen Life 154461K Technologies, Paisley, UK Cluster Tubes (FACS) Abgene, Surrey, UK AB-0672 ELI SPOT plates Millipore Corp, Bedford, MAHA S45 USA Eppendorf Tubes 0.5ml Tref Ag, Degersheim, 96.4625.9.01 1.5ml Switzerland 96.8160.9.02
100 FACS Tubes (5ml Round Becton Dickinson Labwear, 352054 Bottom Polystyrene) NJ, USA Flasks T150 Helena Biosciences, 90151 T75 Sunderland, UK 9076 125 9026 Gene Puiser Cuvette 0.2cm BioRad Laboratories Ltd, 165-2086 gap Hemel Hempstead, UK Immune 96 Well Nunc Life Technologies, Paisley, 439454A Plates (Maxisorp) UK Internal Thread 1.8ml Nunc Life Technologies, Paisley, 377267 Cryovials UK LS^ MACS Columns Miltenyi Biotech, Bisely, UK 130-042-401 Microscope Slides BDH Lab Supplies, Poole, 406/0184/04 UK Pasteur Pipettes Jencons Scientific, Leighton 475-068 Buzzard, UK PCR reaction Tubes Perkin Elmer Applied N801-0580 (tubes) Biosystems, Warrington, N801-053 (caps) UK Pipettes 5ml Bibby Sterilin, Stone, UK 40105 10ml 47110 25ml 18327 50ml Becton Dickinson Labwear, 357550 NJ. USA Plate Sealers Camlab Ltd, Cambridge, EC/005.SPN UK Reagent Reservoirs Corning Inc, NY, USA 4873 (100ml) Slide-A-Lyzer (1000 Pierce & Warriner UK Ltd, 6638122 MWCO) Dialysis Cassettes Chester, UK Syringes 2ml lAH Stores, Compton, UK 50ml Tips 20ul Rainin Instruments Co Ltd, GPS25 200ul Woburn, USA GPS250 lOOOul GPS1000 Universal T ubes BDH Lab Supplies, Poole, 275/0460/04 UK
2.1.3 Restriction Enzymes
Restriction Enzyme Supplier Catalogue Number BglW Invitrogen Life 15213-028 Technologies, Paisley, UK C/al Invitrogen Life 15416-068 Technologies, Paisley, UK EcoRI invitrogen Life 15202-013 Technologies, Paisley, UK
101 HindlW Invitrogen Life 15207-020 Technologies, Paisley, UK Pad New England Biolabs (UK) R0547L Inc, Hitchen, UK Pme\ New England Biolabs (UK) RO5605 Inc Hitchen, UK Sma\ Invitrogen Life 15228-034 Technologies, Paisley, UK Spe\ Invitrogen Life 15443-013 Technologies, Paisley, UK Sph\ Invitrogen Life 15413-040 Technologies, Paisley, UK SSp\ Invitrogen Life 15458-011 Technologies, Paisley, UK
2.1.4 Plasmids
Plasmid Supplier Catalogue Number pGEM-1-195 Dr J. McLauchlan, MRG pGEM-1-169 Virology Unit, Glasgow, UK pGEM-1-124. 145-169 PGDNA3-EGFP Dr M. Flint pCA13 Microbix Biosytems Inc. Kite pBHGIO Toronto, Canada pABS.4 pFG140 pAEIsplA pAdTrack-CMV Drs He & Vogelstein, pAdEasy-1 Howard Hughes Medical Institute Research Laboratories, Baltimore, USA
2.1.5 Tissue Culture Reagents
Reagent Supplier Catalogue Number Agar-Agar Merck, Darmstadt, 1.01613 Germany DMEM with Glutamax Invitrogen Life 31955-021 Medium Technologies, Paisley,UK Dulbecco’s MOD Eagle Invitrogen Life 41966-029 Medium Technologies, Paisley, UK Foetal Bovine Serum Invitrogen Life 10106-169 (GFGS) Technologies, Paisley, UK (Lot: 40Q1282F) Foetal Calf Serum (FGS) PAA Laboratories, Linz, A15-002
102 Austria (Lot: 083) Gentamlcln Sigma Aldrich Company G-1272 Ltd, Poole, UK Histopaque-1077 Sigma Aldrich Company H8889 Ltd, Poole, UK Horse Serum Invitrogen Life 26050-088 Technologies, Paisley, UK Human Male AB Serum Sigma Aldrich Company H-4522 Ltd, Poole, UK L-Glutamine Sigma Aldrich Company G-7513 Ltd, Poole, UK Low Endotoxin Foetal Calf Harlan Sera Labs, 009501 Serum (LE PCS) Loughborough, UK (Lot: 91607) Male AB Serum Sigma Aldrich Company H4522 Ltd, Poole, UK Ml 99 Medium Invitrogen Life 21157-029 Technologies, Paisley, UK MEM with Glutamax Invitrogen Life 41090-028 Medium Technologies, Paisley, UK Neutral Red Invitrogen Life 15330-046 Technologies, Paisley, UK Penicillin & Streptomycin lAH Media Supplies, Compton, UK RPMI (Phenol-Red Free) Invitrogen Life 1835-030 Medium Technologies, Paisley, UK RPMI 1640 with Glutamax Invitrogen Life 72400-021 Medium Technologies, Paisley, UK Saline Baxter Healthcare, F7124 Compton, UK Trypan Blue Invitrogen Life 15250-061 Technologies, Paisley, UK Trypsin-EDTA Invitrogen Life 45300-019 Technologies, Paisley, UK
2.1.6 Cytokines
Cytokine Supplier Catalogue Number GM-CSF GlaxoSmithKline, Gift From Dr John Tite Stevenage, UK IL-2 GlaxoSmithKline, Gift From Dr John Tite Stevenage, UK IL-4 R&D Systems, Abingdon, 204-IL-025 UK M-CSF R&D Systems, Abingdon, 216-MC-025 UK TNFa Upstate Biotechnologies, 01-164 Boltoph Claydon, UK
103 2.1.7 Antigens & Peptides
Antigen Supplier Catalogue Number Purified Protein Derivative Statens Seruminstitut, 2390 (PPD) Copenhagen, Denmark CMV Peptide Mr L. Hunt, lAH, Compton (NLVPMVATV)
2.1.8 Cell Lines
Cell Line Supplier Catalogue Number 293 Microbix, Toronto, Canada PD-02-02 293T Dr M. Stevenson, Worcester, USA Hep16 Dr A. Patel, Glasgow, UK Hep3B European Collection of Cell 86062703 Cultures HLA-A2' EBV-BLCL (EJ4) Dr N. Jones, Oxford, UK HLA-A2^ EBV-BLCL (BORI) Dr P. Borrow, Compton, UK Jurkat European Collection of Cell 88042803 Cultures K562 European Collection of Cell 89121407 Cultures
2.1.9 Kits
Kit Supplier Catalogue Number Annexin V (Cy3) Apoptosis Biovision Research K102-100 Kit Products, CA, USA Gen-Probe Mycoplasma Gen-Probe Inc, San Diego, 1591 Testing Kit USA Human IL-12 p40 ELISA Kit R&D Systems, Abingdon, DY1240 UK Human IL-2 ELISA Kit R&D Systems, Abingdon, DY202 UK NK Cell Isolation Kit Miltenyi Biotech, Bisley, UK 130-046-502 Qiagen, Crawley, UK 27104
2.1.10 Antibodies
Antibody Supplier Catalogue Clone Isotype Number Agonistic Anti- Upstate Ltd, 05-201 CH11 igM Fas Ab Milton Keynes,
104 UK Anti- IFNy Mablech AB, 3420-6 7-B6-1 (ELISPOT Nacka, Sweden detection Ab) Anti-biotin ALP Vector Sp-3020 Laboratories Inc, Peterborough, UK Anti-CD3 Harlan Sera Custom 0KT3 Lab UK Ltd, produced Biscester, UK Anti-IFNy Mablech AB, 3420-3 1-D1K (ELISPOT Nacka, Sweden coating Ab) CD11cPE BD Biosciences 5555392 B-Ly6 igGi Pharmingen, Oxford, UK CD 14 MACS Miltenyi 130-050-201 lgG2a Beads Biotech, Bisley, UK CD3 MACS Miltenyi 130-050-101 lgG2a Beads Biotech, Bisley, UK CD4 MACS Miltenyi 130-045-101 SK3 IgGi Beads Biotech, Bisley, UK CD40 BD Biosciences 33070D SC3 IgGi Pharmingen, Oxford, UK CD40 PE BD Biosciences 33075X 5C3 IgGi Pharmingen, Oxford, UK CDS MACS Miltenyi 130-045-201 BW135/80 lgG2a Beads Biotech, Bisley, UK CD80 PE BD Biosciences 557227 L307.4 IgGi Pharmingen, Oxford, UK CD83 PE BD Biosciences 36935X HB15E IgGi Pharmingen, Oxford, UK CD86 PE BD Biosciences 555658 2331 (FUN-1) IgGi Pharmingen, Oxford, UK CD95 BD Biosciences 33452X DX2 IgGi Biotinylated Pharmingen, Oxford, UK CD95L BD Biosciences 65322X NOK-1 IgGi Biotinylated Pharmingen,
105 Oxford, UK Goat Anti Jackson 115-176-068 mouse F(ab ’)2 Immuno Fragment - Research Labs Gy5 Inc, West Grove, PA, USA HOV Core Dr J. JM122 McLauchlan, Glasgow, UK HLA-A,B,C PE BD Biosciences 32295X G46-2.6 IgGi Pharmingen, Oxford, UK HLA-DR PE BD Biosciences 32415X TU36 lgG2b Pharmingen, Oxford, UK Mouse lgG2b BD Biosciences 33804X 27-35 lgG2b FITC Pharmingen, Oxford, UK Purified Mouse BD Biosciences 555746 MOPC-21 lgG1 Pharmingen, Oxford, UK Streptavidin PE BD Biosciences 13025D Pharmingen, Oxford, UK TRAIL Ri R&D Systems AF347 (DR4) Europe Ltd, Abingdon, UK TRAIL Rll R&D Systems AF631 IgG (DR3) Europe Ltd, Abingdon, UK
106 2.2 Methods
2.2.1 Preparation & Quantification of Plasmid DNA
Competent Escherichia coil (E. coil) DH5a or DH10B cells were transformed with 0.2-1 pg of plasmid DNA using the heat shock method (Sambrook et al., 1989). Briefly, 1-5pl of plasmid DNA was added to lOOpI of competent cells in Eppendorf tubes. Cells were incubated on ice for 30 minutes before heat shocking at 37°C for 20 seconds. They were then incubated on ice for 2 minutes before addition of 1 ml of Luna broth (LB) media. They were incubated for 1 hour at 37°C in an InnOva 4230 shaking incubator (New Brunswick
Scientific, Edison, NJ, USA) at 200rpm to allow expression of the antibiotic resistance genes. Cells were plated onto LB agar plates containing an appropriate antibiotic
(ampicillin at lOOpg/ml or kanamycin at 25pg/ml) and incubated overnight at 37°C.
Individual colonies were picked and inoculated into 10ml of LB containing the appropriate antibiotic and grown overnight at 37°C in an InnOva 4230 shaking incubator at 200rpm.
DNA was extracted using the standard alkaline lysis method (Sambrook et al., 1989) or the
Qiagen Qiaprep Spin Miniprep kit (following the manufacturer's protocol). In the later case, bacterial cells were pelleted at top speed in a microfuge (Eppendorf centrifuge 5415C,
Eppendorf, Hamburg, Germany) and resuspended in 250pl of buffer PI. Cells were lysed with an equal volume of buffer P2 with gently mixing, then this solution was neutralised with 350pl of buffer N3. Cellular debris was pelleted at top speed in a microfuge and the supernatants added to the QIAprep spin columns placed in clean Eppendorf tubes.
Columns were spun at top speed in a microfuge and the flow-through discarded. The columns were then washed with buffer PE and the flow-through discarded again. Finally,
107 the columns were placed into clean Eppendorf tubes and plasmid DNA was eluted in 50pl of buffer EB. Extracted plasmid DNA was quantified using a GeneQuant Pro UV spectrophotometer (Amersham Pharmacia Biotech, Little Chalfont, UK). Samples of plasmid DNA were digested with appropriate restriction enzymes following standard methods (Sambrook et al, 1989) and were run on a 0.8% agarose mini-gel to confirm the plasmid identity. DNA bands were visualised by ethidium bromide, viewed on a UV light box (Jencons, Forest Row, UK) and photographed with a Polaroid GelCam (Jencons,
Forest Row, UK).
2.2.2 Preparation & Gel Purification of Plasmid Fragments
Plasmids (2-20pg) were linearised or gene fragments were excised from the plasmids by restriction enzyme digestion following standard methods (Sambrook et al., 1989).
Fragments were separated by running the digest mix on a 0.8% low melting point agarose gel, and the fragment band(s) were excised from the gel. The gel fragments were weighed and 5 volumes of 20mM Tris-HCI/ImM EDTA (pH8) (TE buffer) were added. The fragments were then incubated in a 56°C waterbath until the gel had melted. Phenol, phenol: chloroform (1:1) and chloroform extractions and ethanol precipitations were carried out following standard methods (Sambrook et al., 1989). Linearised plasmids or purified gel fragments were resuspended in an appropriate volume of distilled water or Tris-EDTA
(TE) buffer.
108 2.2.3 Subcloning Techniques
Gene fragments were subcloned into the adenoviral vector plasmids pABS.4, pCA13 and pBHGIO (Microbix method) or pAdTrack-CMV (AdEasy method). T4 DNA ligase was used to ligate fragments into the appropriate vector following standard techniques
(Sambrook et al, 1989). Briefly, a range of vector: insert ratios were set up (for example,
1:1, 1:2, 1:4 and 2:1), using 50-1 OOng of plasmid with 1 unit of 14 DNA ligase in the appropriate buffer. The final volume per reaction was 10 or 20pl. Ligation mixes were incubated at 16°C overnight. To identify the desired recombinants, the ligation mixes were transformed into competent E. coil DH5a or DH10B cells by the heat shock method.
Colonies were picked and grown up in the presence of the appropriate antibiotic. Plasmid minipreps were prepared as described in section 2.2.1. These were then screened by restriction enzyme digestion. Glycerol stocks of plasmids were prepared by adding 500^1 of the overnight bacterial culture to SOOpI of glycerol in cryovials. The glycerol stocks were stored at -80°C.
2.2.4 Plasmid DNA Maxi preps
500ml of LB containing an appropriate antibiotic was inoculated with a small scraping of the glycerol stock of plasmid-containing bacteria. This was cultured overnight at 37°C in an InnOva 4230 shaking incubator at 200rpm. Bacterial cells were pelleted at ZOOOrpm in an Avanti J-301 centrifuge (Beckman, High Wycombe, UK) in a JA-14 rotor. The pelleted cells were resuspended in 40ml of 50mM Tris-HCI/IOmM EDTA (pH8) solution. Cells were lysed with 80ml of freshly prepared 0.2M NaOH/1% SDS solution and incubated on ice for
109 5 minutes with gentle mixing. Neutralisation was carried out with 40ml KAc (pH4.8) with complete mixing. The samples were incubated on ice for 15 minutes. Cell debris was pelleted at SOOOrpm for 15 minutes in an Avanti J-301 centrifuge in a JA-14 rotor. The supernatant was passed through a 40pm cell strainer to remove any further clumps of debris before precipitation with 0.6 volumes of isopropanol. The precipitated DNA was pelleted at SOOOrpm for 15 minutes in an Avanti J-301 centrifuge in a JA-14 rotor, and resuspended in exactly 10ml of TE buffer. lOg of caesium chloride were added to the
DNA solution with 1ml of ethidium bromide (lOmg/ml stock). This DNA solution was dispensed into centrifuge tubes (3.2ml polycarbonate thick walled tubes) and balance tubes prepared. These were spun in a Beckman Optima TLX ultracentrifuge (Beckman,
High Wycombe, UK) overnight at 80,000rpm in the TLA100.4 rotor at 20°C. After the overnight spin, the plasmid bands were visualised under UV light and the lower (plasmid
DNA as opposed to genomic DNA) of the two bands carefully removed into fresh tubes.
The tubes were topped up with freshly made caesium chloride/ethidium bromide solution and spun for 4-6 hours at 80,000rpm in the ultracentrifuge. The lowest band was removed and diluted with 3 volumes of water in universal tubes. One volume of TE-saturated butan-1-ol was added, and the tubes were shaken and allowed to settle. The upper pink organic phase containing the ethidium bromide was removed. These steps were repeated until the upper phase was colourless with all ethidium bromide removed. Plasmid DNA was precipitated with 2.5 volumes of 100% ethanol at -20°C overnight. Precipitated DNA was pelleted at 10,000rpm in a Beckman Avanti J301 centrifuge in a JA-30.50 rotor for 15 minutes before washing with 70% ethanol. The plasmid DNA was allowed to air dry before resuspension in an appropriate volume of TE buffer or distilled water. The concentration of the plasmid DNA was measured by UV spectrophotometry and the plasmid identity was checked by restriction digest analysis.
110 2.2.5 Plasmid Recombination in E.coli BJ5183 Competent Cells
As part of the Ad Easy system for generation recombinant adenoviruses recombination took place in E. coli BJ5183 electroporation competent cells. 2pg of pAdTrack-CMV-195, pAdTrack-CMV-169 or pAdTrack-CMV-124 were digested with Pme I to linearise the plasmids. Digested plasmid was separated from undigested plasmid by gel purification as described in section 2.2.2 and the linearised plasmids were resuspended in 6pi of distilled water. Electroporation competent E. coli BJ5183 cells were thawed on ice and 40pl aliquots placed into pre-chilled Eppendorf tubes. 1pl of pAdEasy-1 (100ng) and 6pl of pAdTrack-CMV-195, pAdTrack-CMV-169 or pAdTrack-CMV-124 were added to the competent cells and gently mixed. This mixture was transferred into a pre-chilled electroporation cuvette and the cuvette was inserted into the Gene Puiser II electroporation chamber (BioRad, Hemel Hempstead, UK). Cells were pulsed with 2.5KV,
200 Ohms, 25mFD and 1 ml of sterile LB was added after electroporation. The cells were then transferred into 15ml Falcon tubes and incubated at 37°C in a shaker incubator
(200rpm) for 1 hour to allow expression of antibiotic resistance genes. Cells were plated onto LB plates containing kanamycin (25pg/ml) and incubated overnight at 37°C.
2.2.6 Polymerase Chain Reaction (PCR)
Primers were designed using the Primer 3 programme available on the internet (www- genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and synthesised by MWG Biotech,
(Ebersberg, Germany) on a 0.2pM scale, HPSF (high purity, salt free) purified. Primers were resuspended to 1 pg/pl in distilled water. A PCR reaction mix was prepared using
111 75)il of PCR buffer, 45^1 of 25mM MgCb, 6)il of a 1:1:1:1 mix of DNTPs, 75^1 of 0.1 % (w/v) gelatin and 549^1 of distilled water. For each PCR reaction 14.6^1 of the reaction mix,
0.2)liI of each primer, O.SpI of Taq polymerase, l^il (50-1 OOng) of template DNA and 3.5pl of distilled water was added to PCR reaction tubes. The final volume for each reaction was 20pl. Two to three drops of mineral oil were dropped on top of the PCR mix to prevent evaporation and caps sealed on the tubes. The PCR tubes were placed into a
GeneAmp PCR System 9700 (Applied Biosystems, Warrington, UK) and the following
PCR conditions used:
94°C - 45 seconds 94°C - 30 seconds 55°C -1 minute 25 cycles 72°C - 1 minute 72°C -1 0 minutes 4°C - stop
Control reactions with no template, no primers, each primer alone and no Taq polymerase were set up as well as the test samples. After completion of the PCR cycles, IOp.1 of the product mix was diluted with 4|il of loading buffer and 4pl of distilled water and run on a
2% agarose gel at 110V for 90 minutes and product bands were visualised under UV light.
2.2.7 293 & 293T Cells
Low passage 293 cells were maintained in culture in MEM (minimum essential medium) supplemented with 10% PCS (foetal calf serum) and penicillin/streptomycin (lug/ml).
Cells were passaged every 3-5 days, sub-culturing at 1:10, using citric saline to detach cells from the flask surface. 293T cells were maintained in culture in DMEM (Dulbecco’s minimum essential medium) supplemented with 7% PCS and penicillin/streptomycin
(lug/ml). Cells were passaged every 3-5 days, sub-culturing at 1:10, using 1x trypsin-
112 EDTA (0.05% trypsin, 0.53mM EDTA) to detach cells from the flask surface. Cells were shown to be mycoplasma negative by testing with the Gen-Probe mycoplasma testing kit, following the manufacturer’s instructions.
2.2.8 Generation of Recombinant Adenoviruses
One day prior to transfection 293 cells were seeded at 10® cells in 5ml of MEM supplemented with 10% GFGS (good foetal calf serum) into 6mm dishes (Microbix method) or T25 flasks (AdEasy method). Cells were incubated overnight at 37°C in a humidified CO2 incubator (Forma Scientific, Ohio, USA). Cells were 70-90% confluent on the day of transfection.
The Qiagen Effectene transfection reagent was used for co-transfection of the adenoviral genome plasmids from the Microbix method, following the manufacturer’s protocol for transfection of adherent cells. 2^g of plasmid DNA (pAEIsplA & pBHGIO, pCA13 & pBHGIO or pFG140) was dissolved into buffer EC (DNA condensation buffer) using various ratios of plasmids (shuttle plasmid: genome plasmid) (1:1, 2:1, 1:2). Enhancer was added at a constant ratio of 1:8 (DNA:Enhancer) and mixed by vortexing before incubating at room temperature for 2-5 minutes. Effectene reagent was added at different ratios (1:10, 1:25, 1:50, DNA:Effectene). Samples were vortexed and incubated at room temperature for 5-10 minutes to allow transfection complexes to form. During this time, old culture medium was aspirated from cells in 6mm dishes. Cells were washed with PBS and
4ml of fresh MEM supplemented with 10% GFCS added per dish. 1ml of MEM supplemented with 10% GFCS was added to the Eppendorf tubes containing the transfection complexes and these were briefly mixed before addition drop-wise to the cells.
113 Dishes were gently rocked to disperse transfection complexes and incubated overnight at
37°C in a humidified incubator to allow gene expression. The following day culture medium was removed from the dishes and cells were overlayed with 5ml of 1% agar- agar/M199 medium supplemented with 10% horse serum. Cells were incubated for 10,15 or 20 days to allow adenoviral plaque formation. On days 9, 14 or 19 cells were overlayed with 5ml of 1% agar-agar/M199 supplemented with 10% horse serum supplemented with
1 % neutral red to allow easier identification of adenoviral plaques. Potential plaques were picked one day after neutral red overlays were added with a Pasteur pipette and eluted into SOOpI of MEM (without serum) in 24 well plates by incubation overnight at 37°C. 200pl of the eluted virus was used to infect 293 cells (seeded at 10® cells/well in 24 well plates one day prior to infection). The remaining virus was frozen at -80°C (seed stocks). Virus was added to cells for 30 minutes at room temperature before addition of 1.5ml of MEM supplemented with 10% GFCS. Infected cells were incubated until complete cytopathic effect (CPE) was observed. These cells were harvested and lysed by three freeze/thaw cycles at -80°C/37°C to generate pi virus stocks.
The GeneJuice transfection reagent was used to transfect T25 flasks of 293 cells with the adenoviral vector pAdEasy-HCV core genome plasmids generated using the Ad Easy system. 4pg of Pac I digested pAdEasy-195, pAdEasy-169 or pAdEasy-124 was added to
300pl of MEM supplemented with 10% GFCS pre-incubated with 12pl of GeneJuice.
Transfection complexes were allowed to form for 5-10 minutes at room temperature before addition drop-wise to the cells in T25 flasks. Old culture medium was removed from each flask and 6ml of MEM supplemented with 10% GFCS added per flask before addition of transfection complexes. Transfected cells were incubated at 37°C in a humidified incubator to allow gene expression. Cells were incubated for 7-10 days and CPE due to
114 the production of recombinant adenovirus was observed with an inverted light microscope
(Leica Microsystems (UK) Ltd, Milton Keynes, UK). When maximal CPE was observed cells were harvested into 50ml falcon tubes using a cell scraper to remove adherent cells.
Cells from multiple flasks of viruses transfected with the same adenoviral genome plasmid were pooled and pelleted by spinning at 1300rpm in a Sorvall RT7 benchtop centrifuge
(DuPont (UK) Ltd, Stevenage, UK) for 15 minutes. Pellets were resuspended in 5ml of
PBS and lysed with 3 freeze/thaw cycles at -80°C/37°C to generate the seed stocks.
These stocks were amplified by sequential infection of 293 cells in T25, T75 and I I 50 flasks using 30-50% of the viral stock from the previous round of amplification for each new infection step. Briefly, 1-2.5ml of viral lysate was added to 293T cells and incubated for 30 minutes at 37°C before addition of DMEM supplemented with 7% PCS to the flasks.
Cells were then incubated for 3-5 days until maximal CPE was observed, after which they were pelleted and resuspended in a small volume of serum-free DMEM (the volume used was appropriate to the number and size of flasks harvested, for example, 1ml for a T25 flask or 2ml for a T75 flask). Three freeze/thaw cycles were then carried out to release viral particles from the cells. The amount of each virus in stocks generated in this way was determined using a CPE assay described in section 2.2.9.
2.2.9 CPE Assay
293 cells were seeded into 24 well plates at 10® cells/well in 2ml of MEM supplemented with 10% GFCS one day prior to infection, so that they were 80-90% confluent at the time of infection. Samples to be titrated serially diluted from 10’^ to 10'^^ in MEM (without serum). Cells were washed once with serum-free media and 200pl of each virus dilution
115 was added to individual wells. MEM supplemented with 10% GFCS was added to control wells. Cells were incubated for 30 minutes at room temperature before addition of 1.5ml of
MEM supplemented with 10% GFCS per well. Cells were incubated for 5 days and CPE was observed with an inverted light microscope. The viral titre was calculated from the last well showing CPE. The dilution factor here was multiplied by 5 (as only 200pl of virus was added per well) to give the viral titre in infectious units/ml.
2.2.10 Generation of Ad-GFP & Ad-CEI E2 Stocks
Small quantities of high titre Ad-GFP and Ad-CEI E2 were provided by other investigators.
These were amplified by sequential infection of 293 cells with increasing MOIs (starting with the highest MCI possible from the initial stock) in T150 flasks. High titre master stocks were generated from thirty T150 flasks infected at a MCI of 50. Flasks were infected in a small volume (approximately 3ml per flask) for 90 minutes at 37°C in a humidified incubator and then 27ml of DMEM supplemented with 7% FCS and Ipg/ml of penicillin/streptomycin was added. The cells were incubated for 3 days until complete
CPE was observed. Cells and supernatant were harvested from the flasks, pooled and frozen in 50ml aliquots at -80°C. Three freeze/thaw cycles were carried out and the titre of each virus determined by CPE assays.
Working stocks of each virus were prepared from the master stocks. Thirty T150 flasks were infected at a MOI of 50 for 90 minutes in a small volume (approximately 3ml per flask) at 37°C in a humidified incubator and then 27ml of DMEM supplemented with 7%
FCS and Ipg/ml of penicillin/streptomycin was added to each flask. The cells were incubated for 3 days until complete CPE was observed at 37°C in a humidified incubator
116 and then the cells and supernatants were harvested. The cells were pelleted and combined together. An equal volume of supernantant was added to the cell pellets and this was frozen at -80°C. Three freeze/thaw cycles were carried out. After the final freeze/thaw cycle, the lysate was pelleted at 4000rpm in a Sorvall RT7 benchtop centrifuge for 20 minutes and the supernatant carefully removed into a fresh Falcon tube.
This supernantant was purified on caesium chloride gradients as described in section
2.2.11.
2.2.11 Adenovirus Purification
Caesium chloride gradients were prepared in 12ml polyclear centrifuge tubes. Firstly 2ml of a high density caesium chloride solution (1.4g/ml) was placed into a tube, and then 3ml of low density caesium chloride solution (1.2g/ml) was layered on top. The virus supernatant was layered onto the low density caesium chloride solution. Gradients were spun at 100,000g for 90 minutes in a Sorvall Discovery 10008 ultracentrifuge (DuPont
(UK) Ltd, Stevenage, UK) in the TH-641 rotor. Purified virus appeared as an opalescent band at the interface of the high and low density caesium chloride solutions. It was harvested by puncturing the tube with a 19g needle and gently pulling the virus band into a
2ml syringe. Purified virus was dialysed against ImM MgCl 2, lOmM Tris-HCI (ph7.8) plus
10% glycerol in Slide-A-Lyzer dialysis cassettes with several changes of buffer at 4°C overnight. Virus was aliquoted and stored at -80°C and titrated in the CPE assay described in section 2.2.9.
117 2.2.12 Peripheral Blood Mononuclear Cell (PBMC) Isolation
Buffy coat cells from normal human peripheral blood obtained from the National Blood
Service (Colindale, London, UK) or fresh blood obtained from EJIVR donors was diluted
1:2 with saline. 15ml of diluted blood was layered onto 10ml of Histopaque-1077 and centrifuged at 1600rpm for 30 minutes in a Sorvall RT7 benchtop centrifuge with the brake switched off. Peripheral blood mononuclear cells (PBMC) were harvested from the gradient interface with a Pasteur pipette into fresh 50ml Falcon tubes. Cells were washed three times in 50ml of saline. Following the final wash cells were resuspended in RPMI supplemented with 10% LE FCS (low endotoxin foetal calf serum). An aliquot of cells was diluted in trypan blue and viable cells were counted in a haemocytometer (Weber Scientific
International, Lancing, UK).
2.2.13 Human Monocyte-Derived Dendritic Cell (DC) Production
PBMCs were pelleted by spinning at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge and the supernatant was discarded. Cells were resuspended in human anti-
C D # MACS beads at a concentration of lOpI of beads per 10^ cells. Cells were incubated on ice for 10 minutes and then washed twice in 50ml of MACS buffer (RPMI, 2%
FCS, 2mM EDTA). Cells were then resuspended to 5x10^cells/ml in MACS buffer and passed through a 40pm cell strainer to remove any clumps of cells. The cells were then added to LS"^ MACS columns which had been previously equilibrated with 3ml of MACS buffer, using 3ml of cells per column. Positive selection of CD14* monocytes occurred within the columns; other cells passed through. Each column was washed twice with 3ml
118 of MACS buffer to remove all unbound cells. The CD14^ cells were then eluted from the columns in 3ml of DC culture medium (RPMI supplemented with 10% LE FCS and Ipg/ml of penicillin/streptomycin) per column. Cells were eluted by insertion of the plunger, which forced the cells out into a 15ml Falcon tube. Eluted CD 14^ monocytes were counted in trypan blue using a haemocytometer and resuspended to 10® cells/ml in DC culture medium supplemented with IL-4 (50ng/ml) and GM-CSF (200ng/ml). Cells were plated into
6 well plates (5ml per well) and incubated for 6-7 days at 37°C in a humidified CO 2 incubator to allow differentiation of monocytes into dendritic cells. Three days after their initial isolation, cells were fed by removing 1ml of the old culture medium and replacing it with 1.5ml of fresh DC culture medium containing IL-4 (interleukin-4) and GM-CSF
(granulocyte-macrophage-colony stimulating factor). The cells obtained were routinely greater than 98% CDIIc positive, as assessed by antibody staining and analysis by flow cytometry (described in section 2.2.20).
2.2.14 Infection of Monocyte-derived Dendritic Cells with Adenoviruses
Immature or mature monocyte-derived DCs were infected at an MOI of 200 in a small volume of media (1-3ml, depending upon cell numbers) in 15ml Falcon tubes for 2 hours at
37°C in a humidified CO2 incubator. Cells were resuspended to 10® cells/ml and plated into 24 well plates at 1 ml/well and incubated at 37°C until use in a humidified CO 2 incubator. In most experiments DCs were used 24 hours post infection.
119 2.2.15 Assessment of GFP Expression by Flow Cytometry
293 cells or DCs were Infected with Ad-GFP, Ad-Core-195, Ad-Core-169, Ad-Core-124 or
Ad-Empty at a range of multiplicity of infections (MOIs) (0, 1, 10, 100, and 1000) and incubated for 24 hours (to determine the optimal MOI) or at a MOI of 75 for 4-32 hours (to determine the optimal length of infection). They were then harvested into FACS tubes and pelleted at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge. 293 cells were resuspended in 200pl of 1% paraformaldehyde, whilst DCs were resuspended in 50pl of
Facstain (PBS, 2% FCS, 0.1% azide) and lOpI of PE-conjugated anti-CDIIc was added per sample. Cells were stained for 30 minutes on ice in darkness, washed with 1ml of
Facstain and resuspended in 200pl of 1% paraformaldehyde. Cells were analysed on a
Becton Dickinson (Oxford, UK) FacsCalibur for GFP expression (FL-1) and CDIIc expression (FL-2) by DCs. Data was analysed with the WinMDI program version 2.7 (Joe
Trotter, TSRI, USA).
2.2.16 Investigation of HCV Core Protein Expression by Confocal Microscopy
Monocyte-derived DCs were infected with adenoviruses at a MOI of 200 and plated into 24 well plates containing coverslips at 10® cells/well, in a 1 ml volume. They were incubated at
37°C/5%C02 in a humidified incubator for 24 hours. Coverslips were removed into fresh plates and gently washed with PBS/0.5% BSA four times before fixing in 1:1 acetoneiethanol for 15 minutes at room temperature. Coverslips were allowed to air dry prior to rehydration in PBS/0.5% BSA for 15 minutes. Cells were blocked with FcR
(Fragment crystallisable receptor) blocking reagent (2pl/10® cells, diluted in PBS/0.5%
120 BSA) for 15 minutes, on ice. They were then washed as before and stained for core protein expression with 400|jl of anti-core antibody diluted in PBS/0.5%BSA (JM122, a mouse monoclonal antibody, diluted 1:200) or an isotype control antibody (purified mouse
IgGi) at the same dilution as the core antibody . Cells were stained for 30 minutes on ice and washed as before. Cells were stained with a secondary Cy-5-conjugated F(ab ’)2 fragment (goat anti-mouse F(ab ’)2 fragment, diluted 1:250) for 30 minutes on ice, in darkness. Cells were washed four times in PBS/0.5%BSA and co-stained with either phalloidin-alexa-fluor-568 (diluted 1:100) to reveal the cytoskeleton or with BO-PRO-3
(1:400) to reveal the nuclei, in a 400pl volume for 20 minutes, on ice, in darkness. The cells were then washed as before and the coverslips were mounted onto microscope slides in Dako fluorescent mounting medium. The edges of the coverslips were sealed with clear nail varnish and the slides were examined with a Leica confocal microscope
(Leica Microscope Systems (UK) Ltd, Milton Keynes, UK) and digital images were captured and analysed with the Leica confocal software (Leica Microscope Systems (UK)
Ltd, Milton Keynes, UK).
2.2.17 Stimulation of Monocyte-derived Dendritic Cell Maturation
Immature dendritic cells (10® cells/ml) were matured with a range of LPS
(lipopolysaccharide) concentrations (0-100ng/ml), recombinant TNFa (tumour necrosis factor-alpha) concentrations (0-500ng/ml) or by stimulation with an anti-CD40 monoclonal antibody at a range of concentrations (0-10pg/ml) in a 1 ml volume in 24 well plates for 24 or 48 hours at 37°C in a humidified incubator.
121 2.2.18 Production of Monocyte-derived Macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated on Histopaque-1077 gradients as described in section 2.2.12. monocytes were isolated by MACS positive selection, as described in section 2.2.13.
Monocytes were resuspended in macrophage medium (Dulbecco’s modified Eagle medium supplemented with 10% heat inactivated human serum, glutamine (2mM/ml) and gentamicin (Ipl/ml) plus with lOOOU/ml of M-CSF (macrophage-colony stimulating factor).
5x10® cells in 2ml media were plated per well in 24 well plates or 2x10'^ cells in 200pl per well in 96 well flat bottom plates. Cells were incubated at 37°C in a humidified CO 2 incubator for 7-12 days to allow differentiation into macrophages, replacing half of the media in each well with fresh macrophage medium without M-CSF every 2-3 days.
2.2.19 Infection of Monocyte-derived Macrophages with Adenoviruses
Culture medium was removed from macrophages and cells were washed with 1 ml of PBS.
Adenoviruses were diluted in macrophage medium (without M-CSF) to allow infection at a
MOI of 1000 in a 200pl volume per well. Cells were incubated at 37°C in a humidified CO 2 incubator for 2 hours before addition of 1.5ml of macrophage medium (without M-CSF) per well. Macrophages were incubated for 24 hours at 37°C in a humidified CO 2 incubator before use.
122 2.2.20 Analysis of Cell Surface Marker Expression by Flow Cytometry
The level of expression of a variety of maturation/activation markers (MHC class I, MHC class II, CD40, CD80, CD86, CD83, CD95 and CD95L) or surface markers (CD11c and
CD14) on DCs or surface marker expression on purified I cells (CD3, CD4 and CD8) was determined by flow cytometry. Cells were pelleted by spinning at 1300rpm for 5 minutes and resuspended in an appropriate volume of Facstain (PBS with 2% FCS) to give 10® cells/ml, then 50pl of cells was added per well to 96 well V bottomed plates. Cells were stained with PE-conjugated or biotinylated antibodies against a range of markers, or with appropriate isotype matched controls, using 5-1 OpI of antibody, for 30 minutes on ice in darkness. Cells were then washed with 150pl of Facstain, followed by spinning the plates at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge and flicking out the supernatant. Cells stained with directly-conjugated antibodies were resuspended in 200pl of 1% paraformaldehyde solution and transferred into cluster tubes. Cells stained with biotinylated antibodies were resuspended in 50pl of a 1:200 dilution of streptavidin-PE and incubated on ice for 20 minutes in darkness. The cells were washed in the same way as before and resuspended in 200pl of 1% paraformaldehyde solution and transferred into cluster tubes.
The level of expression of T cell activation markers was determined by three colour staining and analysis by flow cytometry. Cells from DC-T cell co-cultures were resuspended and pelleted by spinning at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge. Cells were resuspended in an appropriate volume of Facstain to give 10® cells per well in 50pl. Cells were stained with 7 or 8pl of different cocktails of antibodies for the various combinations of T cell surface and activation markers, as detailed below:
123 Cocktail 1 = GD3-APC, CD4-PerCP, CD69-PE Cocktail 2 = CD3-APC, CD8-PerCP, CD69-PE Cocktail 3 = CD3-APC, CD4-PerCP, CD25-PE Cocktail 4 = CD3-APC, CD8-PerCP, CD25-PE
Single stains with each antibody were also carried out to allow compensation settings to be made on the FacsCalibur. The cells were incubated for 30 minutes on ice, in darkness.
Cells were then washed with 150pl of Facstain, followed by spinning the plates at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge and flicking out the supernatant. Cells were resuspended in 200pl of 1% paraformaldehyde solution and transferred into cluster tubes.
Samples were analysed on a Becton Dickinson FacsCalibur immediately or stored at 4° until analysed. Data was analysed with WinMDi version 2.7 or CellQuest software (Becton
Dickinson, Oxford, UK).
2.2.21 Dextran Uptake by Dendritic Cells
The antigen uptake capacity of DCs was measured by analysis of their ability to take up fluorescently-labelled dextran,, based on the description in Kanto et al., 1999. Briefly, 10® dendritic cells were resuspended in 200pl of dendritic cell medium in FACS tubes. Alexa- fluor-647-dextran (10,000MW)) was added at 20pg/ml (1:100 dilution of stock) and cells were incubated at 37°C for a range of times (0-30 minutes) or at 4°C for the same time range (negative controls). (Cells were then washed four times in 5ml of Facstain, centrifuging between washes at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge, to remove any external dextran from the cell surface. Cells were fixed in 250pl
124 of 1% paraformaldehyde and analysed for dextran uptake by flow cytometry using a
Becton Dickinson FacsCalibur. Data was analysed with WinMDi version 2.7 software.
2.2.22 Cytokine ELISAs
IL-12 production by DCs and macrophages, and IL-2 production by T cells was assessed by measurement of cytokine levels in cell supernatants using capture ELISA kits, following the manufacturers’ instructions. The capture antibody was diluted to the recommended working concentration in PBS (IL-12 p40; 4pg/ml, IL-2; 4pg/ml) and lOOpI was added per well in Immune 96 well Nunc plates (Maxisorp). Plates were sealed and incubated overnight at room temperature. Unbound antibody was removed by inverting plates and washing three times with 400pl of PBS/0.05% Tween per well. Plates were blotted dry after the final wash and blocked with 300pl of blocking solution (1% BSA (bovine serum albumin), 5% sucrose in PBS). Plates were incubated at room temperature for at least one hour. The washing step was repeated prior to addition of standards and samples to the plates. Serial dilutions of an appropriate range were made for the standards (IL-12 p40; 2000-0pg/ml, IL-2; 1000-0pg/ml) in reagent diluent (0.1% BSA, 0.05% Tween in Tris- buffered saline, pH7.2-7.4, 0.2pm filtered) and lOOpI added per well. Samples were added neat (200pl/well) and doubling dilutions were made down the plate in reagent diluent giving a final volume of lOOpl/well. Duplicate wells for each standard and sample were prepared. Plates were sealed and incubated for 2 hours at room temperature. The washing step was repeated prior to addition of the biotinylated detection antibody which was diluted to an appropriate concentration in reagent diluent (IL-12 p40; 175ng/ml, IL-2;
100ng/ml). lOOpI of detection antibody was added per well, and plates were sealed and incubated at room temperature for 2 hours. The washing step was repeated before
125 addition of 100pl of streptavidin-HRP (diluted 1:200 in reagent diluent) per well. Plates were sealed and incubated at room temperature in darkness for 20 minutes. The washing step was repeated again and lOOpI of substrate was added per well. The substrate was prepared by mixing equal volumes of colour development buffer A and colour development buffer B. Plates were sealed and incubated at room temperature in darkness for 20 minutes to allow colour development. The reaction was stopped by addition of 50pl of stop solution (2N H 2SO4) per well. Plates were gently tapped to ensure thorough mixing and the absorbancy at 450nm was read on a Spectramax 340 plate reader (Molecular Devices
Corp, Winnersh, UK). Data was analysed with the Softmax Pro software (version 3.1.2)
(Molecular Devices Corp, Winnersh, UK).
2.2.23 Mixed Lymphocyte Reactions (MLR)
The ability of DCs to stimulate allogenic T cells was assessed using several readouts: T cell proliferation (James, 2000), IL-2 production, and activation marker expression (based on the description in Sarobe et al., 2002.
2.2.23.1 Proliferation
PBMCs were isolated on Histopaque-1077 gradients as described in section 2.2.12 and
CD3"^ cells were purified by positive selection using anti-CD3 MACS beads and LS^ MACS columns (following the same protocol for CD14 cell isolation, described in section 2.2.13 but substituting anti-CD3 MACS beads for anti-CD14 MACS beads). The purity of the populations obtained 90-95% as determined by flow cytometry (described in section
2.2.20). CD3* cells were resuspended to 10® cells/ml in RPMI supplemented with 7% PCS
126 and penicillin/streptomycin (1pg/ml) and plated into 96 well round bottom plates at 10® cells per well (lOOpI volume).
Uninfected or adenovirus infected DCs were resuspended to 10® cells/ml and an appropriate range of DC dilutions were made to enable 10® to 10^ DCs to be added per well in a lOOpI volume. Control wells with T cells alone and DCs alone were included in the assay set up. All variable were assayed in triplicate.
Cells were incubated at 37°C in a humidified CO2 incubator in a damp box. The plates were pulsed with 0.8pCi/well of ®H-thymidine (added in a 20pl volume) on either day 3, 4 or 5 and incubated overnight prior to harvesting on a Tomtec 96 well plate harvester
(Perkin Elmer, Cambridge, UK). Filters were counted on a Trilux 1450 Microbeta counter
(Wallac, Turku, Finland) using version 2 of the Microbeta software. Mean counts per minute (CRM) were calculated for each set of triplicates.
2.2.23.2 IL-2 Production & T Cell Surface Marker expression
Uninfected or adenovirus infected DCs (10®, 10"* or 10®cells per well) were co-cultured with
10® CD3* cells in 24 well plates (prepared as described above). Each well had a final volume of 1.5ml. Cells and supernatants were harvested after 1,2 or 3 days incubation at
37°C in a humidified CO2 incubator. Supernatants were frozen at -20°C and later assayed for IL-2 levels by ELISA as described in section 2.2.22. The cells were triple-stained with antibodies against CD3 with CD4 or CD8 and the activation markers CD69 or CD25 following the method described in section 2.2.20. Cells were analysed on a Becton
Dickinson FacsCalibur using CellQuest software.
127 2.2.24 Stimulation of antigen-specific T cell responses
The ability of DCs to stimulate CD4* and 008"^ I cell responses to antigen was assessed using IFNy ELISPOT assays (Kilinman and Nutman, 2000).
2.2.24.1 IFNy ELISPOT Assay
Millipore MAHA S45 plates were coated with 100pl per well of anti-IFNy mAh (clone 1-
D1K) diluted to 10pg/ml in 0.1M sodium bicarbonate buffer pH9.6. Plates were incubated at 4°C overnight. Unbound antibody was removed by inverting plates and washing with
200pl of PBS/well six times. Plates were blotted dry after the final wash and blocked with lOOpI of blocking solution (RPMI with 10% heat-inactivated pooled human serum) per well.
Plates were incubated at 37°G for at least 1 hour. Prior to addition of cells to the wells the blocking solution was removed by inverting the plate.
Dendritic cells were counted and diluted in RPMI with 10% pooled human serum so that between 1x10^ and 1x10® cells could be added in lOOpI per well. T cells were purified from PBMCs isolated on Histopaque-1077 gradients and CD4"' or CD8^ cells were positively selected using MACS beads, as previously described. The purity of cells was
80-90% determined by flow cytometry. CD4"^ or CD8^ cells were resuspended to 1 x10® cells/ml in RPMI supplemented with 10% pooled human serum and 50pl were added per well, giving a final cell number of 5x10* cells per well.
CD4^ cells were stimulated with purified protein derivative (PPD) at 5pg/ml final concentration; this was added in a volume of 20pl per well. Previous studies carried out by
128 Dr Simone Gloster (EJIVR) showed that 5|jg/ml of PPD was an optimal concentration that
CD4* cells from BCG vaccinated individuals respond to. CDS'" cells were stimulated with a
HLA-A2 restricted CMV peptide (NLVPMVATV) from the pp65 protein at a final concentration of 10'®M, again added in a 20pl volume per well. Controls without antigen or peptide, and no DCs were also set up. All variables were assayed in duplicate.
The plates were incubated for 1 hour at 37°C in a damp box in a humidified CO 2 incubator.
30pl of filtered PCS was then added per well and the plates were incubated overnight in a damp box at 37°C.
After stimulation the plates were inverted to remove cells and wells were washed with
200pl of filtered PBS/0.05% Tween six times. After the final wash the plates were patted dry. 100pl of biotinylated anti-IFNy detection mAb (clone 7-B6-1 ) diluted to 1pg/ml in PBS was added per well and the plates were incubated at room temperature in a damp box for
2 hours. The plates were then inverted and washed with 200pl of PBS/0.05% Tween per well as before to remove unbound antibody. 100pl of anti-biotin alkaline phosphatase diluted 1:1000 in PBS/Tween was added per well and the plates incubated at room temperature in a damp box for a further 1-2 hours. The plates were then washed as before and after the sixth was h the bottom of the plate was removed and the underneath of the plate washed in PBS/Tween to remove all unbound antibody from the wells. The plates were carefully re-assembled, ensuring the wells were firmly sealed, and blotted dry.
10ml of chromogenic alkalinie phosphatase substrate was prepared by diluting the development buffer 1:25 in distilled water and addition of lOOpI of colour reagents A and B. lOOpI of substrate was added per well and the plates were watched for spot development.
After 30 minutes the plates weire inverted to remove the substrate, dismantled and washed
129 with tap water. The plates were allowed to dry at room temperature and spots with a fuzzy border were counted on an AID automated image analysis system with AID ELISPOT version 2.5 software (Autoimmun Diagnostika GmbH, Stassberg, Germany). The results were calculated as the mean (of duplicate wells) number of spot-forming cells per 5x10*
CD4"^ or CDS'" cells.
2.2.24.2 Antigen-Specific Proliferation Assays
Another method used to evaluate the ability of DCs to stimulate antigen-specific CD4* T cell responses was the measurement of T cell proliferation by ^H-thymidine incorporation assays (James, 2000).
PBMCs were isolated on Histopaque-1077 gradients as described in section 2.2.12 and
CD4* cells isolated by positive selection using anti-CD4 MACS beads and LS"’ MACS columns (following the same protocol as used for CD14 cell isolation, described in section
2.2.13, but substituting anti-CD4 MACS beads for anti-CD14 MACS beads). CD4* cells were resuspended to 1.4x10® cells/ml in RPMI supplemented with 10% LE PCS and penicillin/streptomycin (Ipg/ml) and plated into 96 well plates, giving 1.4x10® cells per well
(in a lOOpI volume).
Uninfected or adenovirus infected (MCI 200) DCs were resuspended to 10® cells/ml and an appropriate range of DC dilutions were made in RPMI supplemented with 10% LE PCS and penicillin/streptomycin (Ipg/ml) to enable between 10^ and 2x16* DCs to be added per well in a lOOpI volume.
130 PPD was added in a 10|j| volume per well to give a final concentration of 5pg/ml. Control wells with no PPD and no DCs were set up and all variables were assayed in triplicate.
Plates were incubated at 37°C in a humidified CO 2 incubator in a damp box and pulsed with 0.8pCi/well of ^H-thymidine on day 5. After a further overnight incubation, they were harvested and counted as described in section 2.2.23.1.
2.2.25 Induction of Apoptosis
The extent to which different cell types underwent apoptosis in response to various stimuli was assessed by staining with Annexin V. This is a fluorescently labelled protein which has a high affinity for phosphatidyl-serine of the plasma membrane which becomes exposed as cells undergo apoptosis (Zhang et al., 1997).
Uninfected or adenovirus infected Jurkat cells (cultured in RPMI supplemented with 7%
PCS and penicillin/streptomycin (Ipg/ml)), Hep16 or Hep3B cells (cultured in DMEM supplemented with 7% PCS and penicillin/streptomycin), macrophages or DCs were stimulated with different inducers of apoptosis; each used at a range of concentrations.
1x10® cells were incubated in 24 well plates in a 1ml volume of the appropriate media for
24 hours at 37°C in a humidified incubator together with: an agonistic anti-Pas antibody
(clone CH-11 ) at 0-1000ng/ml, recombinant TNPa at 0-500ng/ml, TRAIL receptor 1 (TRAIL
R1) antibody at 0-2pg/ml, TRAIL receptor 2 (TRAIL R2) antibody at 0-10pg/ml, recombinant TRAIL at 0-500ng/ml, recombinant lymphotoxin aip2 (LTa1(32) at 0-500ng/ml or recombinant lymphotoxin a2p1 (LTa2pi) at 0-500ng/ml. The cells were then resuspended and transferred at 10® cells per well into 96 well V-bottomed plates for annexin V-cy3 staining.
131 Annexin V staining was perforned using the annexin V (Cy3) apoptosis kit. Cells were pelleted in a 96 V-bottomed piste by spinning at 1300rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge. The plate was flicked to remove the supernatant and the cells were resuspended in 50pl of annexin V-Cy3, diluted in annexin V binding buffer (2.5pl annexin per sample) from the annexin V binding kit. Cells were incubated for 15 minutes on ice, in darkness, and then washed with 150pl of annexin V binding buffer per well and pelleted at
1300 rpm for 5 minutes in a Sorvall RT7 benchtop centrifuge. The cells were finally resuspended in 200pl of annexin V binding buffer and transferred into cluster tubes.
Samples were immediately run on a Becton Dickinson FacsCalibur. Data was analysed with WinMDi version 2.7 software.
2.2.26 Natural Killer (NK) Cell Assay
The cytotoxic activity of NK cells against different target cell types was assessed using
^^Chromium release assays (Wunderlich and Shearer, 2000).
Target cells were uninfected or adenovirus infected (MOI 200) K562 cells (cultured in
RPMI supplemented with 7% PCS and penicillin/streptomycin (Ipg/ml)), DCs, macrophages, Hep16 or Hep3B cells. K562 and DCs were labelled by pelleting 10® cells in a 15ml Falcon tube, resuspending the pellet with lOOpCi of Na®^Cr 04, and then incubating for 1-2 hours at 37°C in a humidified incubator. Cells were washed four times with 5ml of RPMI (phenol-red free) supplemented with 10% GFCS and resuspended at 10® cells/ml after the final wash, so that they could be added to assay plates at lO'^ cells per well in a lOOpI volume. Macrophages, Hep16 and Hep3B cells were plated at 2x10* cells
132 per well into flat bottom 96 well plates. Macrophages were allowed to differentiate for IQ-
12 days and Hepi 6 and HepSB cells allowed to adhere overnight. They were then labelled in the plates by addition of 3.3uCi of Na^^Cr 04 per well in a 40pl volume, for 1-2 hours at
37°C in a humidified incubator. These cells were washed four times with 200pl per well of
RPMI (phenol-red free) supplemented with 10% GFCS and lOOpI of medium was added to each well after the final wash.
In some experiments, NK cells were purified from blood bag PBMCs (isolated as described in section 2.2.12) using an NK cell isolation kit, following the manufacturers instructions.
Briefly, PBMCs were incubated with 20pl per 10^ total cells of the hapten-antibody cocktail
(a cocktail of hapten-conjugated monoclonal CD3, CD14, CD19, CD36 and anti-lgE antibodies) for 10 minutes on ice. Cells were washed twice in MACS buffer and resuspended in 20pl per 10^ total cells of anti-hapten microbeads. They were then incubated on ice for 10 minutes and washed as before. Cells were resuspended in 500pl per 10^ total cells of MACS buffer and added to LS* MACS columns. The unlabelled flow through was collected as this contained the purified NK cells. The purity of the cells was
70% as determined by flow cytometry. The cells were then counted and resuspended in
RPMI (phenol-red free) supplemented with 10% GFCS at concentrations appropriate for addition of cells in a lOOpI volume to achieve the desired effectortarget (E:T) ratios.
Alternatively, blood was drawn from an EJIVR donor who has a high frequency of NK cells and PBMCs were isolated on histopaque-1077 gradients, following the method described in section 2.2.12. Whole PBMCs (containing the effector NK cells) were resuspended in
RPMI (phenol-red free) supplemented with 10% GFCS at concentrations appropriate for addition of cells in a lOOpI volume to achieve the desired E:T ratios.
133 Assays were set up in flat-bottomed 96 well plates. Effector and target cells were each added in a 100pl volume per well. Target cells were used at 10"* or 2x10^ cells per well and effectors were added to give a range of E:T ratios (for example, ranging from 100:1 to
5:1 with whole PBMCs or 40:1 to 5:1 with purified NK cells). Controls were included to enable the measurement of the spontaneous and maximum chromium release from each target cell type. These wells had lOOpI of target cells with either lOOpI of media
(spontaneous release) or lOOpI 2% (v/v) Triton-X-100 (maximum release). All variables were assayed in triplicate.
Plates containing K562, DCs, Hepi 6 and HepSB target cells were incubated for 5 hours at
37°C in a humidified incubator before harvesting, whilst plates containing macrophages were incubated for 5 hours or overnight before harvesting. After this incubation period,
40pl of supernatant was harvested from each well into counting plates. lOOpI of Opti- phase scintillant was added per well and plates were sealed and left overnight to allow the scintillant and sample to become homogenously mixed before counting in a Trilux 1450
Microbeta counter. The percentage specific chromium release was calculated using the equation below. mean test counts - mean spontaneous counts x 100 = % specific ®^Cr release mean maximum counts - mean spontaneous counts
2.2.27 Generation of antigen-specific CD8"^ cytotoxic T cells
50ml of blood was taken from a HI_A-A2^ EJIVR donor with a known response to a HLA-A2 restricted CMV peptide (NLVPMVATV). PBMCs were isolated on Histopaque-1077 gradients as described in section 2.2.12 and CDS'" cells isolated by positive selection using anti-CDS MACS beads and LS^ MACS columns (following the same protocol as for CD14
134 cell isolation, described in section 2.2.13, but substituting anti-CD8 MACS beads for anti-
CD 14 MACS beads). CD8"^ cells were plated at 2x10® cells per well into 96 well plates with 10® allogenic feeder cells (PBMCs irradiated at 3000 rads) per well in T cell media
(RPMI supplemented with 10% GFCS, 25mM 2-mercaptoethanol and Ipg/ml of penicillin/streptomycin) together with the HLA-A2 CMV peptide at a final concentration of
IpM. After 3 days IL-2 was added to the medium to give a final concentration of lOU/ml.
Every 3 days half of the medium in each well was replaced with fresh medium supplemented with IL-2. As the cells proliferated and increased in numbers they were expanded into 24 well plates. Fresh feeder cells were added every 2 weeks (5x10® cells per well in 24 well plates) and fresh media supplemented with IL-2 was added every 3 days, replacing half of the medium per well.
2.2.28 Culture of Epstein-Barr virus transformed B cell lines
Epstein-Barr virus transformed B lymphoblastoid cell lines (EBV-BLCL) generated from
HLA-A2"^ BLCL (BORI) and HLA-A2' BLCL (EJ4) donors were cultured in RPMI supplemented with 20% GFCS and Ipg/ml of penicillin/streptomycin in T75 flasks, sub- culturing at 1:10 approximately every 7 days.
2.2.29 CDO"" CTL Cytotoxicity Assays
The cytotoxic activity of MHC-restricted, antigen-specific CD8^ CTL against different target cell types was assessed using ®^Cr release assays as described in section 2.2.26 for assessment of NK cell cytotoxicity.
135 The target cells used here were uninfected, or adenovirus infected (MOI 200), HLA-A2‘^ or
HLA-A2' EBV-BLCLs or DCs, which were labelled in suspension with lOOpCi of Na^^Cr 04 for 1 hour at 37°C in a humidified incubator. The cells were washed four times with 5ml of
RPMI (phenol-red free) supplemented with 10% GFCS and resuspended in an appropriate volume to give 2x10* target cells per well in a volume of 50pl.
CDS'" CMV-specific CTL generated as described in section 2.2.27 were resuspended to
2x10® cells/ml in phenol-red free RPMI supplemented with 10% GFCS and lOOpI of cells added per well to give 2x10® cells per well, an effectortarget ratio of 10:1. Cells were incubated in the presence of a range of CMV peptide concentrations (10'®M to 10'®M final concentration, and no peptide). Peptides were diluted from the stock (0.04M) to 4 times the desired final concentration (e.g. 4x10'®M for a final concentration of 10'®M) in 1:10 serial dilutions, and 50pl added per well. Controls were included to allow measurement of the spontaneous and maximum ®^chromium release from each target cell type. All variables were assayed in triplicate.
Plates were incubated for 5 hours at 37°C in a humidified incubator, then harvested and counted as described in section 2.2.26. The percentage specific ®^chromium release was calculated using the equation described in section 2.2.26.
2.2.30 Statistical Analysis
Statistical analysis was performed with the Mintab 13.1 software. Statistical analysis of variance was performed by two-way ANOVA tests and by one-way ANOVA and Tukey’s pairwise comparison.
136 Chapter 3 - Generation of Recombinant Adenoviruses Expressing the HCV Core Protein
3.1 Introduction
The aim of this project was to gain a better understanding of mechanisms that may contribute to HCV’s ability to evade control by the host immune response, focusing particularly on effects of core expression within dendritic cells. As it is not possible to assess the effects of infection of DCs with HCV directly, the approach taken was to express the core protein within DCs by the means of adenoviral vectors, so that its effect on their immune functions could be addressed.
Adenoviral vectors were chosen to transduce dendritic cells with the HCV core protein.
They represent an efficient gene delivery system, with high levels of gene expression being achieved in many cell types, including both cell lines and primary cells (Graham,
2000). The advantage of adenoviral vectors over many other viral vectors (e.g. vaccinia or herpesvirus vectors) is they induce minimal damage to or alteration to the target cell function (Graham, 2000; Wilkinson and Akrigg, 1992; Zhong et al., 1999) making them an ideal vector to look at effects of expression of individual genes of interest within the transduced cells.
Replication-deficient adenoviral vectors are capable of infecting many cell types and expressing genes of interest within them, however they cannot undergo productive
replication as they lack regions of the adenoviral genome essential for replication.
These replication-deficient adenoviruses are generated and expanded in a helper cell
line (e.g. 293 cells), which provides the essential functions required for replication that
are lacking in the adenoviral vector genome. Most replication-deficient adenoviral
137 vectors (including the ones used here) have deletions within the early region 1 (E1) and early region 3 (E3) of the adenovirus type 5 genome (Bett et al., 1994; McGory et al.,
1988). Inserts between 4.7-9.4kb can be placed into the El region and inserts
between 3.7-3.9kb can be placed into the E3 region (Bett et al., 1994). El is essential for adenovirus replication within cells therefore deletions in this region render the vectors replication-deficient. The El deletions also include genes which are needed for oncogenic transformation of cells in culture and genes which are essential in regulating the adenoviral lytic cycle. However, the deletion in El does not affect the packaging signal sequences within the virus which are essential for packaging of the full length genome into functional virions. The deletion in E3 does not affect viral replication but disrupts viral immunomodulatory genes including genes that can modulate immune mediated apoptotic mechanisms. This helps to ensure that any immunomodulatory effects seen within the target cell are due to the gene of interest expressed in the vector rather than the immunomodulatory genes encoded by the adenovirus vector.
This chapter describes the generation of replication-deficient adenoviral recombinant vectors co-expressing green fluorescent protein (GFP) and the HCV core protein or fragments thereof, and analysis of core protein expression in dendritic cells (DCs) transduced with these viruses.
3.2 Choice of HCV Core Protein and Fragments for Expression In Adenoviral
Vectors.
A series of pGEM plasmids encoding a full length version (amino acid residues 1-195) and fragments of the HCV core protein derived from the HCV type la laboratory strain
Glasgow (Hope and McLauchlan, 2000) were provided by Dr J. McLauchlan (MRC
Virology Unit, Glasgow, UK). The full length version of core in plasmid pGEM-1-195, a
138 truncated version of the core protein in plasmid pGEM-1-169 and a mutated version of core in plasmid pGEM-1-124, 145-169 were chosen to be inserted into adenoviral vectors, for reasons explained below. Figure 3.1 illustrates the full-length core protein and these fragments.
Previous studies have suggested that the full-length core protein has a cytoplasmic cellular localisation (Lo et al., 1995; Lo et al., 1994). However it is thought that truncated versions of the core protein, which may be generated by proteolytic processing, may have a different cellular localisation to the full-length version.
Versions of the core protein truncated at residues 182, 178 (Hussy et al., 1996), 171
(Liu et al., 1997) or 153 (Lo et al., 1994) that lack the COOH domain may be translocated into the cell nucleus, where core may have different functions within the cell. For example, truncated versions of the core protein may be capable of activating or suppressing cellular promoters, which may be important in the development of hepatocellular carcinoma in some chronically infected individuals (Chang et al., 1998;
Ray et al., 1997; Ray et al., 1998b; Shih et al., 1993). By generating an adenoviral vector expressing a version of the core protein truncated at residue 169, it was hoped to achieve localisation of core to the nucleus, so that functions there could be addressed.
The third version of core chosen for use in this project was a mutated version of the core protein lacking residues 125-144. Loss of this sequence results in the core protein not being cleaved at a maturation site at residue 172. This leads to over expression of the mutated core protein compared to the wild type protein in BHK cells transfected with the core encoding plasmids (Hope and McLauchlan, 2000). An adenoviral vector expressing this version of the core protein was generated so that if the level of expression of core from the vector containing the full length core protein (1-
139 5' E2 NS2 NS3 NS4 NS5A N85B HCV Genome
iEncodes Full length Core
195
Truncated Core 169
Core - lacking residues 125-144
124 145 195
Figure 3.1. HCV core fragments derived from the pGEM plasmids provided by Dr J. McLauchlan. The numbers underneath each core protein fragment represent amino acid residue numbers in the HCV genotype 1a polyprotein.
140 195) proved to be exitremely bw; it may be possible to achieve higher levels of core expression by means of this alemative vector.
3.3 Microbix Method for Construction of Adenoviral Vectors
Dr Frank Graham and his co-workers simplified the construction of recombinant adenoviral vectors by developing a series of bacterial plasmids containing circular forms of the adenovirus type 5 genome with deletions in the El and E3 regions (Bett et al., 1994; Bett et al., 1993; Graham, 1984; Hanke et al., 1990; McGrory et al., 1988).
As outlined in figure 3.2, recombinant adenoviral vectors are created using two
plasmids, a shuttle plasmid that contains the gene of interest flanked by El sequences, and a large genome plasmid that encodes the remainder of the adenoviral genome, with a large insert in the El region which renders the genome too large for packaging, and a deletion in the E3 region. To generate recombinant adenovirus, a helper cell line
(293) that constitutively provides El functions is co-transfected with both plasmids.
Homologous recombination occurs between these plasmids and during recombination the large insert in the El region of the genome plasmid is reduced in size as the gene of interest is incorporated into the adenoviral genome. The reduction in size of the genome allows packaging of the recombinant adenoviral genome into virions. These virions are released and infect neighbouring 293 cells, ultimately giving rise to a plaque in the helper cell monolayer which can be visualised by neutral red staining 10-20 days post-transfection. Recombinant virus can be expanded from the plaque and amplified
in 293 cells. Recombinant viruses generated in this way can infect multiple cell types
but can only replicate in helper cell lines, as they lack the El region.
141 Large insert in E1
E1 E3 Homologous recombination Plasmid containing Transfer plasmid Ad genome & gene containing genes of of interest in E3 interest in E1
Replication deficient Ad genome that can be packaged into virions, which are then released & infect neighbouring cells
Figure 3.2. Outline of the Microbix method for generating recombinant adenoviruses. Following transfection of the transfer plasmid and adenoviral genome plasmid into the helper cell line (293), homologous recombination occurs and the size of the genome is reduced, allowing the recombinant replication-deficient adenoviral genome to be packaged into virions. These virions are released and infect neighbouring cells, ultimately giving rise to a plaque in the helper cell monolayer which can be visualised by neutral red staining 10-20 days post transfection. Recombinant virus can be expanded from the plaque by growth in 293 cells. The recombinant viruses generated can also infect other cell types, but cannot replicate within them as they lack the El region.
142 This system was Initially used to try and construct recombinant adenoviruses co- expresslng the marker GFP with the different versions of the hepatitis C virus core
protein described In section 3.2. The GFP gene was cloned Into the E3 region of the adenoviral genome plasmid pBHGIO and the core protein and fragments thereof were
cloned Into the transfer plasmid pCA13 within the E1 region of the adenoviral genome.
As Illustrated In figure 3.3, homologous recombination between these two plasmids would result In generation of a vector co-expressIng GFP and the HCV core protein or fragments thereof.
3.3.1 Sub-cloning core protein fragments Into the El region within the transfer plasmid
pCA13
Figure 3.4 outlines the strategy taken for subcloning the core fragments from the pGEM vectors provided by Dr J. McLauchlan Into the transfer plasmid pCA13.
The core protein fragments were excised from lOpg of the pGEM vectors (pGEM-1-
195, pGEM-1-169 and pGEM-1 -124,145-195) with the restriction enzymes Hind\\\ and
EcoRI. Hind\\\ digestion was carried out first, and then the plasmids were ethanol
precipitated before digestion with EcoRI. After EcoRI digestion the fragments (0.59kb for full length core, 0.50kb for the truncated core and 0.53kb for the mutated core) were
separated on 1% low melting point agarose gels, as shown In figure 3.5. Isolated fragments were purified by phenol, phenol/chloroform and chloroform extraction and
ethanol precipitation. Fragments were resuspended In lOpI of TE buffer; yields ranged
between 1.5-1.7pg as determined by UV spectrophotometry.
143 pCA13 Kan pBHG10-GFP Core -HCV Amp'' Ad5 genome Ad5 enome
GFP CMV CO TRANSFECT 293 CELLS
Figure 3.3. Plasmids used for generation of recombinant adenoviruses by the Microbix method. GFP was cloned into the E3 region of the adenoviral genome plasmid pBGHIO, to generate pBHG10-GFP. The HCV core protein or fragments thereof were cloned into the shuttle plasmid pCA13. These two plasmids and the replication-deficient adenoviral genome generated by homologous recombination between then in 293 cells. This is illustrated diagrammatically in this figure. The adenoviral genome is represented by the purple lines, the core protein by the red box, GFP by the green box, the turquoise box represents the CMV IE promoter, the blue checked box represents the ampicillin resistance gene and the yellow box represents the kanamycin resistance gene.
144 core HindWl EcoRI
pGEM-HCV core
CMV HindWl
EcoRI Amp"
pCA13-core pCA13
Figure 3.4. Strategy for cloning the HCV core fragments into the transfer plasmid pCA13. Core fragments were excised from the pGEM plasmids (provided by Dr J. McLauchlan) by digestion with Hind\\\ & EcoRI, and cloned into pCA13 within the Hind\\\ & EcoRI restriction enzyme sites, to generate the transfer plasmid pCA13-HCV-core for each version of the core protein. This is illustrated diagrammatically in this figure. The plasmids and sequences within them are not drawn to scale. The red box represents the HCV core protein fragment, the turquoise box represents the CMV IE promoter, the blue checked box represents an amplicillin resistance gene and the purple lines represent the adenoviral genome sequences.
145 s
Remainder of pGEM plasmid
Core protein 0.5kb fragments
Figure 3.5. Isolation of HCV core protein encoding fragments by restriction enzyme digestion & agarose gel electrophoresis. Plasmids pGEM/1-124,145-169, pGEM/1-169 and pGEM/1-195 were digested with HindWl and EcoRI. The digestion products were separated by electrophoresis on a 1% low melting point agarose gel. Core protein-containing fragments of the expected sizes (0.53kb for the mutated core fragment, O.SOkb for the truncated fragment and 0.59kb for the full- length core fragment) were generated from each plasmid; the positions of these and the remainder of the plasmid are indicated.
146 15|jg of the E1 shuttle plasnid pCA13 was cut at the Hind\\\ restriction site within the
multiple cloning site. It was then ethanol precipitated and digested with EcoRI. The digested plasmid was isolated on a 1% low melting point agarose gel and the 7kb fragment cut from the gel was purified by phenol, phenol/chloroform, and chloroform
extraction and ethanol precipitation. The HCV core protein fragments were ligated into the digested shuttle plasmid using T4 DMA ligase at a 1:2 vector:insert ratio (25ng of
pCA13 and 50ng of core fragment). The directional subcloning ensured the correct
orientation of the fragments with respect to the CMV promoter in pCA13. Ligation
mixes (3 or 6pl) were transformed into E.coli DH5a competent cells using the heat
shock method. Bacteria were cultured on LB agar plates containing ampicillin, then were expanded and miniprep quantities of DNA were prepared and screened for the
presence of the core protein fragment by digestion with the restriction enzyme C/al.
The digestion site for this enzyme is unique to the HCV core fragments, therefore digestion results in linearisation of only core-containing recombinant plasmids, giving a
band of 7-8kb depending on the size of the core protein insert, as shown in figure 3.6.
Plasmids containing the core fragments were selected and large quantities produced
by growing large scale cultures of bacteria and carrying out maxi preps.
3.3.2 Subcloning GFP into the E3 region of the Adenoviral Plasmid pBHGIO
Figure 3.7 outlines the strategy taken to clone the GFP gene into the adenoviral genome plasmid pBHGIO.
The GFP gene was excised the plasmid pCDNA3-EGFP by digestion with the
restriction enzymes BglW and Xba\. 20pg of plasmid pCDNA3-EGFP were digested with
6g/ll. The overhanging ends resulting from BglW digestion were filled by incubation with
30 units of the Klenow fragment and O.SmM DNTPs for 15 minutes at room
147 O)IT)
ID
pCA13-HCV-core recombinants
Figure 3.6. Screening for pCA13-HCV-core recombinants by C/al restriction enzyme digestion. Core fragments were ligated into the shuttle plasmid pCA13 within the Hind\\\ and EcoRI restriction sites and ligation mixes were transformed into E.coli DH5a competent cells. Miniprep quantities of DNA were prepared and digested with C/al, an enzyme that has a unique restriction site within the core fragments. This results in linearisation of pCA13-HCV-core recombinant, which we see as a 7-8kb band (dependant upon the size of the core fragment insert) when separated on an agarose gel as indicated above.
148 Xbal GFP
CMV pCDNAS-EGFP BglW
Amp'^
VO Pad pABS.4 PABS.4-GFP
AE3 AE3 pBHGIO pBHG10-GFP Adenoviral genome Figure 3.7. Strategy for cloning GFP into the Adenoviral genome plasmid pBHGIO. The GFP fragment was excised from pCDNA3-EGFP with BglW & Xba\ prior to cloning into the shuttle vector pABS.4 within the Spel & Xba\ sites. The GFP fragment was then isolated by Pad digestion and cloned into pBHGIO within the E3 region of the adenoviral genome. This is illustrated diagrammatically and the plasmids and sequences are not drawn to scale. The green box represents GFP, the turquoise box represents the CMV IE promoter, the blue checked box represents an ampicillin resistance gene, the pink box represents the T7 promoter, the pale purple box represents the Sp6 promoter, the yellow box represents a kanamycin resistance gene and the purple lines represent the adenoviral genome sequences.
150 temperature. The Klenow fragment was inactivated and phenol, phenol/chloroform and chloroform purifications and ethanol precipitation carried out prior to digestion of the plasmid with Xba\ to release the GFP-containing fragment. The GFP-containing fragment (~1.7kb) was isolated on a 1% low melting point agarose gel, excised, purified and precipitated in the same way as described for the HCV core protein fragments. It was finally resuspended in 20pl of TE buffer. The yield was shown by UV spectrophotometry to be 4.8pg.
As the pBHGIO adenoviral genome plasmid did not contain the correct restriction sites for direct cloning of GFP into the E3 region, the shuttle plasmid pABS.4 was used to provide a means of inserting GFP into pBHGIO. The pABS.4 shuttle vector contains two Pad restriction sites. These sites allow the cloning of a gene of interest (GFP here) into the plasmid pBHGIO. To enable directional cloning of the GFP fragment into pABS.4, pABS.4 (20pg) was first digested at the Sph\ restriction site and the overhanging ends were filled by incubation with the Klenow fragment and dNTPs, before digestion of the plasmid at the Xba\ site. The GFP fragment was then ligated into pABS.4. Ligations (1:1, 1:2, 1:3, 1:4 and 2:1 vector:insert ratios with lOOng of vector) were carried out using T4 DMA ligase and 3pl of each ligation mix were transformed into E.coli DH5a competent cells using the heat shock method. Bacterial colonies with kanamycin resistance were selected and expanded and screened to identify those carrying plasmids containing GFP inserted into pABS.4. The restriction enzyme EcoRI was used to screen for the presence of GFP in miniprep quantities of plasmid DMA, as this enzyme should cut out a 0.8kb fragment from the GFP gene.
Large scale cultures of bacteria containing pABS.4-GFP were grown and plasmid DMA purified by the maxiprep method.
The GFP fragment was excised from 20pg of pABS.4-GFP by digestion with Pad and the fragment (2.8kb) was isolated on a 1% low melting point agarose gel, as shown in
151 figure 3.8. This fragment was purified by phenol, phenol/chloroform, and chloroform extraction and ethanol precipitated. The fragment was resuspended in 10pl of TE buffer. 10pg of the adenoviral genome plasmid pBHGIO was linearised with Pad, and the GFP fragment was ligated into the Pad site of pBHGIO with T4 DMA ligase. This was not a directional cloning step; however the direction of the insertion of the fragment which contains the GFP gene (together with its own transcriptional control elements) in the E3 region of the adenoviral genome plasmid was not of importance. Again a range of vectorinsert ratios were used (1:1, 1:2,1:3 and 1:4). 6pl of each ligation mix were transformed into E.coli DH5a competent cells and recombinants selected on the basis of ampicillin and kanamycin resistance. Individual colonies were picked and cultured further before isolation of DMA by minipreps. These were checked for the presence of pBHGIO containing the GFP fragment by several restriction digests. Xba\ digestion should linearise pBHGIO-GFP, whilst EcoRI digestion should give three fragments of
BOObp, 1.6kb and 7kb, and Pad digestion should release the GFP fragment (2.8kb) from the plasmid, as shown in figure 3.9. Large scale cultures of bacteria containing pBHGIO-GFP were grown and DMA isolated by maxiprep ready for transfection into
293 cells.
3.3.3 Generation of Recombinant Adenoviruses by Co-Transfection of 293 cells
Large quantities of two other plasmids, pFG140 and pAEIsplA were also grown up and purified by the caesium chloride maxiprep method prior to transfections being carried out. pFG140 is a plasmid containing a circular form of the Ad5 genome that can generate infectious virus when transfected alone into 293 cells. This can be used as a positive control for the transfection process and generation of adenoviral plaques. pAEIsplA is an “empty” shuttle plasmid that can be used for the generation of control adenoviruses that only express GFP, with no insert in El (i.e. no core protein insert).
152 Q) S
3
2.8kb GFP fragment Remainder of pABS.4
Figure 3.8. Isolation of the GFP-containing fragment from pABS.4 recombinants by Pad digestion. The GFP fragment was ligated into pABS.4 within the Sph\ and EcoRI restriction sites, to provide the Rad restriction sites for subcloning the GFP fragment into pBHGIO. pABS.4 was digested with Rad to release the GFP-containing fragment (2.8kb) which was isolated on a low melting point agarose gel as indicated. The lower band corresponds to the remainder of the pABS.4 plasmid (-2.5kb).
153 (a) b)
U) 0)
' 1
28kb
0.8kb
Figure 3.9. Confirrmation of the presence of GFP inpBHG10-GFP. The GFP fragment was ligated into the adenoviral geiome plasmid pBHGIO within the Pad site. Ligation mixe;s were transformed into E.coli Dh5a competent cells and recombinants with kanamycin and! ampicillin resistance were selected and amplified. Mhiprep quantities of DMA were digested with (a) Xba\ or EcoRI and (b Pad. Xba\ digestion linearises the plasmids whilst EcoiRI digestion yields three fragmentsof 0.8kb, 1.6kb andZkb as indicated in (a). Pad digestion rreleases the 2.8kb GFP fragment as indicated in (b).
154 This plasmid was used to generate the control viruses as problems growing large stocks of pCA13 occurred and a purified stock of this plasmid had been produced for another project. This allowed the generation of the control viruses to begin whilst the core protein and fragments thereof were being cloned into pCA13.
Plasmids were transfected or co-transfected into low passage 293 cells seeded in
60mm dishes using the Effectene transfection reagent. The transfection process was
initially optimised using control plasmids. First, the efficiency within which 293 cells could be transfected using Effectene was determined using a plasmid that only expressed GFP. A range of plasmid DNA concentrations were transfected into 293 cells and the transfection efficiency was determined by assessing the percentage of cells expressing GFP 24 hours later by flow cytometry. At DNA concentrations
between 2pg and 4pg, 98-99% of 293 cells were found to be GFP positive (data not shown), indicating the Effectene method was efficient for transfection of 293 cells. The ratio of DNA to Enhancer was also optimised, this time using the (much larger) adenoviral control plasmid pFG140. Ratios of 1:10, 1:25 and 1:50 were tried simultaneously; however no differences in the number of plaques produced was observed, so all future transfections were carried out at a DNA:Enhancer ratio of 1:25.
The ratio of DNA to Effectene was consistently kept at 1:8 as recommended by the
manufacturer.
To generate recombinant adenoviruses, 293 cells were co-transfected with plasmids
pAEsplA and pBHGIO or pBHGIO-GFP, or each of the pCA13-HCV core plasmids and pBGHIO-GFP. As a positive control, cells were transfected in parallel with the
adenoviral genome plasmid pFG140. The amount of DNA transfected into cells ranged from 0.5pg to 8pg, with different amounts of each plasmid and different ratios of each
plasmid being used (1:1 and 2:1). Transfected 293 cells were incubated for 7, 10, 15
or 20 days and then examined for the presence of plaques by addition of neutral red
155 overlays. The control plasmid pFG140 consistently produced adenoviral plaques, with the number of plaques ranging from 20-30 per plate, as shown in figure 3.10. These plaques started to appear at day 10. At this time no plaques were seen on cells transfected with the different shuttle plasmids and pBHGIO-GFP; however it might be expected that the recombinant viruses would take longer to form plaques as they have to undergo homologous recombination before infectious virus is generated, whereas the control plasmid pFG140 already contains all the components required for viral replication within 293 cells. By day 20, small numbers of potential plaques (1 or 2) were visible on cell monolayers transfected with the shuttle plasmids and pBHGIO-
GFP. These plaques were picked and virus eluted, however upon infection of more 293 cells no cytopathic effect was observed suggesting that these “plaques” were not due to adenoviral replication. To control for the possibility that mutations had been introduced into the adenoviral genome plasmid pBHGIO-GFP when GFP was subcloned into this, that made it difficult or impossible to recover infectious virus from this, 293 cells were also co-transfected with pAEIsplA and the parental plasmid pBHGIO; but this also did not result in the generation of adenoviral plaques.
This led me to believe that the homologous recombination event between the two plasmids is a very low efficiency event. In an attempt to improve the probability that recombination would occur, further transfections were carried out using linearised plasmids. Each was digested with a restriction enzyme that cut it at a single unique site. The enzymes used were SspI for pAEIsplA and the pCA13-HCV-core plasmids and C/al for the adenoviral genome plasmids pBHGIO and pBHGIO-GFP.
Combinations of linearised shuttle and genome plasmids were transfected at a ratio of
2:1 (genome plasmidishuttle plasmid) into 293 cells using a total of 6pg of linear plasmid DNA. However, once again plaques were generated only in cells transfected
156 y
Figure 3.10. Adenoviral plaques produced from transfection of 293 cells with the plasmid pFG140 (positive control plasmid containing a circular form of the AdS genome) revealed by neutral red staining.
157 with the control plasmid pFG140, indicating that homologous recombination and expansion of recombinant virus had failed to take place.
The continued problems encountered with generating adenoviral recombinants with this method led me to look for another method of generating adenoviral vectors. The method I chose to try instead was the Ad Easy system designed by Dr He and Dr
Vogelstein (He et al., 1998).
3.4 AdEasy Method for Constructing Adenoviral Vectors
The AdEasy system developed by Drs He and Vogelstein (He et al., 1998) relies upon homologous recombination occurring between plasmids containing the adenoviral genome and a transfer plasmid containing the gene of interest in El, as did the
Microbix method. However, in the AdEasy system the recombination step takes place within bacterial cells that are highly efficient at performing recombination instead of mammalian cells where recombination is a lower efficiency event. This method has the additional advantage that once recombinant adenoviral genomes have been generated, they can be expanded in bacteria and transfected into large numbers of cells, meaning that production of homogenous adenovirus stocks can be rapidly achieved without the need for plaque purification. Figure 3.11 outlines the AdEasy method for generating recombinant adenoviruses.
This system was used to generate replication-deficient adenoviral vectors co expressing HCV core protein or a fragment thereof and GFP. An advantage of this system was that GFP was already present in the shuttle vector pAdTrack-
158 Pad HCV Core AMP'
Left Arm Right Arm
pAdTrack-CMV | pAdEasy-1
Adenoviral genome
Pad Linearise with Pmel Co transform into E.coli BJ5183 cells
HCV Core
CMV CMV
Right Arm Recombinats are Recombination occurs Left Arm between pAdTrack-CMV selected with Kanamycin & pAdEasy 1 arms of homology
pAdEasy-HCV-core
Linearise adenoviral genome with Pad
GFP HCV Core ^^1 AE3
Transfect 293 Cells Pad Pad
Large quantities of virus produced and purified Figure 3.11. The AdEasy method for generation of recombinant adenoviruses. HCV core fragments were cloned into the shuttle plasmid pAdlrack-CMV. This was linearised & co-transformed with the genome plasmid pAdEasy-1 into E.coli BJ5183 cells where homologous recombination occurs between the arms of homology. Recombinant genomes are linearised with Pad prior to transfection into the helper cell line (293) where infectious virus is generated over 7 days. This process is illustrated in diagrammatic form in this figure. The plasmids are not drawn to scale and the red box represents the core protein fragment, the green box represents GFP, the turquoise box represents the CMV IE promoter, the blue checked box represents an ampicillin resistance gene, the yellow box represents a kanamycin resistance gene and the purple lines represent adenoviral genome sequences.
160 CMV so the only subcloning that needed to be carried out was subcloning the core protein encoding DNA fragments into the shuttle plasmid pAdTrack-CMV.
3.4.1 Subcloning the HCV Core Fragments into pAdTrack-CMV
The HCV core protein fragments described in section 3.2 were excised from the pGEM vectors (10pg) in which they were supplied using BglW and were isolated by separation on 1% low melting point agarose gels, as shown in figure 3.12, and purified by phenol, phenol/chloroform and chloroform extraction and ethanol precipitation. Figure 3.13 outlines the strategy taken for subcloning the core fragments into the shuttle vector pAdTrack-CMV. The shuttle vector pAdTrack-CMV (20pg) was also digested with BglW.
The vector ends were dephosphorylated using calf intestinal alkaline phosphatase
(CIP) to prevent them re-ligating, then the digested plasmid was purified by phenol, phenol/chloroform and chloroform extractions and ethanol precipitation and resuspended in lOpI of distilled water. The core fragments were ligated into the linear shuttle vector using T4 DNA ligase, setting up ligations at vector:insert ratios of 1:1 and
1:2 or vector alone. 3pl of each ligation mix were transformed into E.coli DH10B competent cells using the heat shock method. Bacterial cells were plated onto LB agar plates containing kanamycin. The 1:2 vectorinsert ratio proved to be most efficient, with hundreds of colonies growing on these plates. Individual colonies were screened for the presence of plasmids carrying the core protein fragments. Miniprep quantities of
DNA were prepared and digested with the restriction enzyme Smal. This enzyme cuts uniquely within the core protein DNA sequence and also allows the orientation of the core fragments within the shuttle vector to be determined, as shown in figure 3.14.
BglW digestion was also used to confirm the presence of the core fragments within pAdTrack-CMV as this enzyme removes the core fragment cloned into the vector.
161 $ C i S (D 0) § s CD JD LU LU LU O O O Q. a. Q.
ore ~Q-5ktj , .Core ~0.5kb fragments fragment
Figure 3.12. Isolation of the HCV core protein encoding fragments from plasmids pGEM/1-169, pGEM/1-195 and pGEM/1-124,145-195 by restriction enzyme digestion & agarose gel electrophoresis. The pGEM plasmids were digested with BglW to release the HCV core protein fragments, which were isolated on a low melting point agarose gel, as shown above. The full length core protein fragment from pGEM/1-195 is 0.58kb, the truncated core protein fragment from pGEM/1-169 is 0.51 kb and the mutated core protein fragment from pGEM/1-124,145-195 is 0.53kb.
162 core BglW BglW
pGEM-HCV core
BglW CMV CMV GFP
Kan pAdTrack-CMV pAdT rack-CMV core
Figure 3.13. Strategy for cloning HCV core fragments into the shuttle plasmid pAdTrack-CMV. Core fragments were excised from the pGEM plasmids (supplied by Dr J. McLauchlan) by digestion with BglW and subcloned into the BglW restriction enzyme site in pAdTrack-CMV, to generate pAdTrack-CMV-core for each version of the core protein. This is illustrated diagrammatically in this figure. The plasmids and sequences within them are not drawn to scale. The red box represents the HCV core protein fragment, the green box represents GFP, the turquoise box represents the CMV IE promoter and the yellow box represents a kanamycin resistance gene.
163 Potential recombinants Potential recombinants A ______
O) $ i > 6.8kb 2.1kb 1.5kb 1kb 0.8kb Core fragments in the correct orientation Figure 3.14. Screening pAdTrack-CMV-HCV-core plasmids for the presence of HCV core encoding protein fragments by Smal digestion. Core protein fragments were ligated into pAdTrack-CMV within the BglW restriction site. Ligation mixes were transformed into E.coli DH5a competent cells and miniprep quantities of DNA were prepared and digested with Smal, which cuts uniquely within the core fragment sequence and allows determination of the orientation of the core fragment within pAdTrack-CMV. Separation of a Ikb fragment indicates that core is in the correct orientation (highlighted by the blue arrows) whilst a 1.5kb fragment indicates that core is in the wrong orientation. 164 Once pAdTrack-CMV-core plasmids were identified large quantities of plasmid DNA were produced and purified on caesium chloride gradients. Large quantities of the adenoviral genome plasmid p Ad Easy-1 were also produced and purified on caesium chloride gradients, ready for co-transformation with the pAdTrack- CMV-core plasmids into E.coli BJ5183 electroporation competent cells. 3.4.2 Co-transformation of E.coli BJ5183 Cells with pAdTrack-CMV-Core & p Ad Easy-1 The pAdTrack-CMV-core plasmids (pAdTrack-CMV-195, pAdTrack-CMV-169 and pAdTrack-CMV-124) were linearised by digestion with Pmel (4pg of plasmid) and undigested plasmid was removed by running the digest mixes on 1 % low melting point agarose gels and excising the linearised DNA. This was purified by phenol, phenol/chloroform and chloroform extractions and was ethanol precipitated and resuspended in 6pl of distilled water. The linear pAdTrack-CMV-core plasmids and the circular adenoviral genome plasmid pAdEasy-1 were co-transformed into electroporation competent E.coli BJ5183 cells by electroporation as described in chapter 2. Following transformation, bacterial colonies with kanamycin resistance were selected and DNA prepared by minipreps. This was then checked for recombinant adenoviral genomes by Pad digestion, which yields fragments of 4.5kb and ~30kb when a recombinant is present, as shown in figure 3.15. Bacteria could also have been co-transformed with pAdTrack-CMV (without core) and pAd Easy-1, to generate a recombinant adenoviral genome plasmid from which a control adenovirus expressing GFP only could be generated (Ad-Empty). However this was not carried out, as I had been given a small amount of Ad-Empty by another investigator (Dr S. Smith, EJIVR), so I did not need to generate this control virus. 165 a> V)‘ Potential Recombinants 1 m I A. I I A Recombinant Figure 3.15. Screening Ad-Easy-HCV-core recombinants with Pad digestion. E.coli BJ5183 competent cells were co-transformed with pAdEasy-1 and linearised pAdTrack-CMV core plasmids by electroporation. Colonies with kanamycin resistance were selected and miniprep quantities of DNA prepared. The presence of recombinants was checked by Pad digestion which yields a 4.5kb fragment and the remainder of the genome (~30kb) as indicated above. The red arrow shows the presence of an adenoviral recombinant genome. 166 The presence of the core protein-encoding sequences in the recombinant adenoviral genomes was confirmed by PCR as described in chapter 2. Briefly, primers specific to a sequence present in all three versions of the core protein were designed. If core sequences were present in the genomes a 177bp PCR product was amplified, as shown in figure 3.16. Once the identity of the recombinant genome plasmids was confirmed, large quantities of DNA were produced and purified on caesium chloride gradients. The genome plasmids (20pg) were then linearised by Pad digestion prior to transfection into 293 cells using the GeneJuice transfection reagent as described in chapter 2. The GeneJuice transfection reagent was used rather than Effectene as it was a simpler and quicker method of transfection, requiring only one reagent. The efficiency of transfection with GeneJuice was checked as for Effectene, and high levels of transfection were achieved. Transfected cells were incubated for 7-10 days at 37°C in a humidified incubator until CPE was visible. The cells were then harvested by scraping them off the flask surface and transferring them into tubes. The cells were pelleted and resuspended in 2ml of sterile PBS, and viral lysates were prepared by performing three freeze/thaw cycles at -80°C/37°C. 3.4.3 Expansion of Ad-Core-195, Ad-Core-169 & Ad-Core-124 The virus in the initial lysates obtained after the transfections was amplified by sequential rounds of growth in 293 cells. Ad-Empty (a control virus created using the Ad-Easy system, small quantities of which were provided by Dr S. Smith, EJIVR) was amplified in parallel with the Ad-Core series of viruses. At first successive rounds of infection were carried out in flasks of cells of increasing size (T25, T75, T150), but the level of viral amplification at each passage was too low for this to be successful - there 167 I I 0 > u 1 (/) o € ■o a> e (0 s u 1 (D ! O Ia 177bp Figure 3.16. Checking for the presence of the HCV core sequences in recombinant pAdEasy-HCV-core genomes by PCR. Primers specific for a sequence common to all three versions of the core protein were designed, PCR ampification with which would yield a 177bp band if core sequences were present. The pAdTrack-CMV-core plasmids were used as positive controls and the empty plasmids pAdEasy-1 and pAdTrack-CMV were used as negative controls in the PCR. The PCR reaction generated 177bp fragments for all three pAdEasy-core plasmids, as indicated on the gel above, confirming the presence of core sequences in the recombinant adenoviral genomes. 168 was a decline in the titre of virus obtained in successive lysates. The transfections were thus repeated, and virus was amplified from the initial lysates more gradually, performing successive rounds of growth in the smallest flasks (T25) until complete CPE was observed before moving on to infect larger flasks. 1 ml of each of the initial lysates was used to infect a T25 flask of 293 cells for 1 hour at 37°C before addition of 9ml of medium. The cells were incubated for 5-7 days, until CPE was observed. Lysates were generated in the same way as after the initial transfection and put into a 2ml volume. This cycle of infection and harvesting was repeated in T25 flasks until complete CPE was observed by 2-3 days after infection, indicating that the amount of virus present in the lysates had increased. The volume of the lysates was kept at 2ml throughout to prevent the virus becoming too diluted. Once this stage was achieved the infection and harvesting process was repeated in T75 flasks in order to increase titre of lysates further. Initial rounds of infection gave low levels of CPE, but upon sequential amplification the amount of CPE increased. Once full CPE was observed by 2-3 days post-infection, the infection process was repeated in T150 flasks, again until complete CPE was observed. At this stage the adenoviral titre in each lysate was measured using a CPE assay, as described in chapter 2. If the titre was at least 5x10^ infectious units, T150 flasks of 293 cells were infected at a MCI of about 5 and a further round of viral amplification was performed. The viral titres of the resulting lysates were measured by CPE assay and a further round of infection was performed, this time using a higher MCI of 25. Once a titre sufficient to allow infection of T250 flasks at an MOI of 50 was achieved the volume of high titre virus increased by infecting multiple flasks, as described in chapter 2, in order to generate a master stock. These master stocks could then be used to generate working stocks that could be purified on caesium chloride gradients as described in chapter 2. 169 Although the problems initially encountered with amplification of the viral stocks were overcome and cell lysates containing high viral titres of each virus were generated, attempts to generate master stocks and working stocks of these viruses was thwarted by fungal infection. When fungal infection became apparent in the first set of high titre cell lysates produced, these were discarded and cells were re-transfected in an attempt to generate new clean stocks. Initially this looked promising but again as the amplification process proceeded the fungal infection returned. The plasmids used in the transfection were a possible source of the fungal contamination; attempts were made to generate “clean” plasmids by subjecting the digested plasmids to phenol, phenol/chloroform and chloroform extraction and ethanol precipitation, after agarose gel purification. Cells were then re-transfected with the “clean” plasmids and the amplification process was repeated, however fungal infection still occurred. Time did not permit further attempts to generate large scale master stocks and working stocks of the series of core-GFP co-expressing viruses. As described in the following section, the small amounts of reasonably high titre virus that were generated from each recombinant plasmid did enable preliminary investigation of the GFP and core expression in cells infected with the different adenoviral recombinants. However, all further studies in this thesis were carried out using two recombinant adenoviruses provided by other investigators, Ad-GFP and Ad-CE1E2. 3.5 Ad-GFP & Ad-CE1E2 I was given a small amount of two recombinant adenoviruses, Ad-GFP (provided by Dr M, Stevenson, Worcester, USA) and Ad-CE1E2 (provided by Dr B. Clarke, GlaxoSmithkline, UK). I generated high titre master stocks in 293 cells of each of these viruses, and caesium chloride purified working stocks as described in chapter 2. 170 Ad-GFP, contains GFP inserted in the E1 region of the adenoviral genome, and is used as a control adenovirus. Ad-CE1E2 contains the HCV type 1a core, El and E2 glycoproteins inserted into the El region of the adenoviral genome. However, unlike the recombinant viruses I generated, this virus does not co-express GFP. Ad-CE1E2 was used to assess effects of the core protein on cell functions, both activation and susceptibility to death by different apoptotic pathways. 3.6 Analysis of GFP & Core Protein Expression in Ceiis infected with Different Adenoviral Recombinants The expression of GFP by Ad-GFP was confirmed in 293 cells by flow cytometry. Uninfected, Ad-GFP or Ad-CE1E2 infected 293 cells remaining from CPE assays were scraped from the wells showing complete CPE, pelleted and fixed in 1% paraformaldehyde and analysed by flow cytometry for GFP expression, measuring GFP fluorescence in the FL-1 channel. As can be seen in figure 3.17, cells infected with Ad-GFP did indeed express high levels of GFP, whilst the uninfected cells and cells infected with Ad-CE1E2 did not have an increase in FL-1 fluorescence, confirming that Ad-GFP did express GFP and that fluorescence detected in the FL-1 channel was not due to infection-associated damage of the cells. GFP expression in 293 cells infected with Ad-Empty and each Ad-Core virus was determined by flow cytometric analysis of 293 cells remaining from CPE assays. Once the titres of the viruses had been determined, the 293 cells were scraped from the wells showing complete CPE, pelleted and fixed in 1% paraformaldehyde and analysed by flow cytometry for GFP expression. As shown in figure 3.18, a high level of fluorescence in the FL-1 channel, indicative of GFP expression, was detected in the cells infected with Ad-Empty (the control virus with no core insert in El), Ad-Core-195 171 B c g LU M1 10° 10' 10- 10^ 10* GFP FL1-H Uninfected 10° 1 0 ' 10- 10 ^ 10 * 10° 10 ' 10- 10^ 10* FL1-H FL1 -H Ad-GFP Ad-CE1E2 Figure 3.17. Determination of GFP expression in 293 cells infected with Ad-GFP & Ad-CE1E2. Uninfected 293 cells and cells infected with Ad-GFP and Ad-CE1E2 from CPE assays were harvested, fixed and analysed by flow cytometry for GFP expression in the FL-1 channel. 172 B c M1 g LU 10° 10 ' 10^ 10^ 10 * GFP FL1 -H Uninfected M l I Ml LJJ 10° 10 ' 10^ 10^ 10 * 10° 10 ' 10^ 10^ 10 * FL1 -H FL1-H Ad-Core-195 Ad-Core-169 Ml 10° 1 0 ' 10^ 10^ 10 * 10° 1 0 ' 10^ 10^ 10 * FL1 -H FL1 -H Ad-Core-124 Ad-Empty Figure 3.18. Determination of GFP expression in 293 cells infected with Ad- Core-195, Ad-Core-169, Ad-Core-124 & Ad-Empty. Uninfected 293 cells and cells infected with Ad-core-195, Ad-Core-169, Ad-Core-124 and Ad-Empty from CPE assays were harvested, fixed and analysed by flow cytometry for GFP expression in the FL-1 channel. 173 (full length core), Ad-Core-169 (truncated core) and Ad-Core-124 (mutated core), but not in uninfected cells. This result showed that GFP was expressed, as would be expected, from Ad-Empty and each of the Ad-Core viruses. Expression of the HCV core protein in cells infected with Ad-CE1E2 and the Ad-Core viruses was analysed by immunofluorescent staining and confocal microscopy, as this not only enabled verification of core protein expression, but also allowed simultaneous analysis of the intracellular location of the expressed protein. The cells used for this analysis were monocyte-derived DCs, as future work would focus on these cells, making it important to verify core protein expression within cells infected with in particular Ad-CEI E2. Monocyte-derived DCs were infected at a high MOI (200) with purified Ad-GFP and Ad- CEI E2 or at the highest MOI achievable using the Ad-Core containing lysates of each Ad-Core virus. These were MOI's of approximately 9 with Ad-Core-195 and Ad-Core- 124, and a MOI less than 1 with Ad-Core-169. HCV core expression was assessed 24 hours post-infection by staining the cells (and as a control, uninfected DCs) with an anti-core mouse monoclonal antibody (JM122) provided by Dr J. McLauchlan, binding of which was detected using a fluorescently labelled (Cy5) anti-mouse F(ab ’)2 fragment. The cells were also co-stained with either phalloidin alexa-fluor 568 to detect the cell cytoskeleton, or with a nuclear stain (BO-PRO-3), to enable analysis of the intracellular localisation of the core protein. Using three-colour confocal microscopy, expression of GFP, the HCV core protein and either cytoplasmic or nuclear markers could thus be simultaneously assessed in individual cells. Representative results from this analysis are shown in figures 3.19 -3.25. Figure 3.19 Shows analysis of GFP expression in DCs infected at a high MOI with Ad- GFP, and illustrates the use of co-staining with cytoplasmic (phalloidin) or nuclear (BO- 174 (a) Phalloidin Overlay LA (b) BO-PRO-3 Overlay Figure 3.19. Expression of GFP in Ad-GFP infected DCs. DCs infected with Ad-GFP (MCI 200) were stained with (a) phalloidin to detect the cytoskeleton or (b) BO- PRO-3 to detect cell nuclei and examined by confocal microscopy. The yellow arrows indicate cells where GFP expression can be seen in the nucleus and cytoplasm. Pha bidin (a) Overlay (b) BO-PRO-3 Overlay Figure 3.20. Uninfected DCs stained for HCV core protein. Uninfected DC were co-stained with anti-core antibody and (a) phalloidin to detect the cytoskeleton or (b) BO-PRO-3 to detect cell nuclei and examined by confocal microscopy. Phalloidin Overlay -J Figure 3.21. Staining Ad-CE1E2 infected DCs with an isotype control antibody. Ad-CE1E2 infected DCs were co-stained with an lgG1 isotype control antibody and phalloidin to detect the cytoskeleton. (a) Phalloidin Overlay (b) BO-PRO-3 Overlay Figure 3.22 Expression of HCV core protein in Ad-CE1E2 infected DCs. DCs infected with Ad-CE1E2 (MCI 200) were stained with (a) phalloidin to detect the cytoskeleton or (b) BO- PRO-3 to detect cell nuclei and examined by confocal microscopy. The yellow arrows indicate cells where expression of core can be seen in the nucleus and cytoplasm. (a) Phalloidin Core Overlay (b) BO-PRO-3 Core Overlay Figure 3.23. Expression of HCV core protein in Ad-Core-195 infected DCs. DCs infected with Ad-Core-195 were stained with (a) phalloidin to detect the cytoskeleton or (b) BO-PRO-3 to detect cell nuclei and examined by confocal microscopy. The yellow arrows indicate cells where core expression can be detected in the nucleus and cytoplasm. 179 (a) Phalloidin Core Overlay (b) BO-PRO-3 Overlay Figure 3.24 Expression of HCV core protein in Ad-Core-124 infected DCs. DCs infected with Ad-Core-124 were stained with (a) phalloidin to detect the cytoskeleton or (b) BO-PRO-3 to detect cell nuclei and examined by confocal microscopy. The white arrows indicate cells where core expression can only be detected in the nucleus and the yellow arrows indicate cells where core expression is seen in both the nucleus and cytoplasm, (a) Phalloidin Overlay Core (b) BO-PRO-3 Overlay Figure 3.25 Expression of HCV core protein in Ad-Core-169 infected DCs. DCs infected with Ad-Core-169 were stained with (a) phalloidin to detect the cytoskeleton or (b) BO-PRO-3 to detect cell nuclei and examined by confocal microscopy. The white arrows indicate cells where core expression can be detected in the nucleus. 181 PRO-3) markers to determine the intracellular location of the protein. High levels of GFP expression were seen in Ad-GFP infected DCs, and this protein was found in both the cytoplasm and nucleus. Figures 3.20-3.22 show analysis of the core protein expression in DCs infected with Ad-CE1E2. Figure 3.20 shows uninfected DCs stained with the anti-core antibody; no core expression was detected as expected, showing the antibody was not binding non- specifically to the cells. Similarly, when DCs infected with Ad-CE1E2 were stained with an isotype control antibody and the secondary Cy5-F(ab’)2 fragment, no background staining that may be mistaken for core expression was seen. This is shown in figure 3.21. Figure 3.22 Shows core protein staining of DCs infected with Ad-CE1E2. Expression of the core protein could be detected in almost all cells (consistent with the Ad-CE1E2 infection being carried out at a high MOI). Much of the core protein within individual cells appeared to be located in the nucleus (figure 3.22b); but some of the core staining was also seen in the cytoplasm (figure 3.22a). Analysis of expression of both GFP and the HCV core in DCs infected with the Ad-Core viruses is shown in figures 3.23-3.25. Although some GFP expression was detected in DCs infected with each of these viruses, the level of GFP expression was much lower than that seen in cells infected with Ad-GFP (compare figure 3.23 with figure 3.19). As discussed further at the end of this chapter, this may have been due to the relatively low MOI used for infection of DCs with the Ad-Core series of viruses. Alternatively, it may reflect differing efficiencies in GFP production from Ad-GFP versus the Ad-Core viruses. Importantly, not only GFP, but also the HCV core protein could be detected in cells infected with all three Ad-Core viruses. The level and cellular sites of core protein expression in cells infected with Ad-Core-195 (figure 3.23) appeared very similar to that 182 in cells infected with Ad-CE1E2, with core protein expression being seen in both the nucleus and, at lower levels in the cytoplasm. It is harder to detect the core staining in cells infected with Ad-Core-124, as can be seen in figure 3.24. This may be due to the low MOI used for infection. The core appears to be expressed in both the nucleus and cytoplasm of infected cells. To determine whether there are really differences in the level of core expressed from this virus compared to Ad-Core-195, infections would need to be carried out at a range of MO Is with both viruses and the levels of expression looked at over time (the core protein expressed by Ad-Core-124 may accumulate more at later time points) by a more qualitative means, for example, intracellular staining and flow cytometry. The core protein expression in DCs infected with Ad-Core-169 is the hardest to see, as shown in figure 3.25, which is consistent with the lowest MOI. This version of the core protein appears to be localised in the nucleus of the cells, but this needs to be confirmed with infection at a higher MOI. In conclusion, the Ad-Core viruses I constructed co-expressed GFP and the HCV core protein. The intracellular location of the core protein was nuclear and cytoplasmic for the full length and mutated core protein but expression of the truncated core protein may have been restricted to the nucleus. Ad-CE1E2 also expressed the core protein within DCs; in this case the core protein had a nuclear and cytoplasmic location. 3.6 Discussion I successfully constructed adenoviral vectors co-expressing a full-length version of the HCV core protein (amino acids 1-195), a truncated core protein (amino acids 1-169) or a mutated core protein (amino acids 1-124, 145-195), together with the marker green fluorescent protein, using the Ad-Easy system. However I encountered many problems whist making and expanding these viruses. 183 The initial method I used to try and construct the adenoviral vectors was the Microbix method. The cloning of the core protein fragments and GFP fragments into the respective plasmids in order to generate the recombinant adenoviral genome was relatively straightforward, however this method consistently failed to produce viral plaques following co-transfection into the helper cell line (293) which was required to generate infectious virus. The transfection efficiency using the Effectene method was high when a control GFP expressing plasmid was transfected into the 293 cells. The control plasmid pFG140 which contained the entire infectious adenovirus genome also consistently produced plaques, which again made it unlikely that poor transfection efficiencies were responsible for virus not being generated. This also provided a control for the process of virus generation from adenoviral genomes. To exclude the possibility that mutations had been introduced into the adenoviral genome plasmid during the subcloning of GFP, which may have made it impossible for the recovery of infectious virus when co-transfected with the shuttle plasmids, the parental plasmid pBHGIO and the pAE1Sp1A shuttle plasmid were also co-transfected into 293 cells; but this also did not result in the generation of recombinant adenoviral plaques. The Microbix method relies upon homologous recombination taking place between the two plasmids containing the adenoviral genome in mammalian cells; this was most likely the limiting step in generating the recombinant viruses. Homologous recombination in mammalian cells is of a low efficiency (He et al, 1998), and recombination efficiencies may have been further reduced if not all transfected cells contain both plasmids. The size of the plasmids may also have affected recombination. As pBHG10-GFP was very large (~35kb) compared to the pCAl 3-core containing plasmids (~7kb) the areas of homology may be too distant for recombination to occur. I made attempts to enhance the likelihood of recombination occurring by, changing the ratios of the two plasmids, increasing the amount of each plasmid transfected into the 184 cells, and by linearising the plasmids prior to transfection. However, recombination still failed to occur and no recombinant adenoviruses were generated. The continued failing of the Microbix method led me to try a different approach. The second approach I took to generate the recombinant adenoviral vectors was using the Ad-Easy method, which was successful. The main reason this method was successful compared to the Microbix method is likely to be due to the recombination step taking place in bacterial cells rather than mammalian cells, as bacterial cells are more efficient at carrying out recombination (Chartier et al., 1996; Crouzet et al., 1997). The Ad-Easy method also had other advantages over the Microbix method, mainly that there was no need to plaque purify virus as large amounts of homogenous virus were generated at the initial transfection step, making the method quicker than the Microbix method. Another advantage of the Ad-Easy system was that the adenoviral genome already contained the marker GFP, so there was no need to clone a marker into the genome as there was with the Microbix method. This also made the Ad-Easy method quicker as there were fewer cloning steps required as only the HCV core fragments had to be inserted into the adenoviral genome. Although the Ad-Easy method did successfully generate recombinant adenoviral vectors, I had problems expanding these viruses for experimental use. Once I had generated the initial stocks of each virus, I followed the suggested amplification protocol provided with the Ad-Easy system; however I did not get expansion of the viruses occurring. This was probably to be due to the initial viruses being expanded too quickly resulting in a low MOI and a resulting decrease in output viral titres. To overcome this problem I started again and took more time to expand the viruses, waiting for complete CPE after each step before proceeding to the next round of amplification. This was successful and the titre and volume of each virus did increase. However, during the amplification process a problem with fungal contamination 185 occurred. I tried adding an anti-fungal reagent (Fugin) to the cell cultures, however this proved to be toxic to the cells. Therefore to prevent the spread of the fungal contamination to other cell lines and between the viruses I generated new viral stocks by re-transfecting cells and following the slow amplification process. This initially appeared to have overcome the problem of fungal contamination; however, as the amplification process proceeded the fungal contamination reappeared. The fact that contamination occurred only during amplification of this series of viruses and not during passage of uninfected 293 cells or preparation of stocks of Ad-GFP or Ad-CE1E2 (which were expanded from virus provided by other investigators) suggested that the plasmids transfected into the cells may be the source of contamination. So I decided to carry out phenol, phenol/chloroform and chloroform purification of the linearised plasmids before transfection as this treatment should destroy any contaminating fungal spores. Unfortunately, this step did not resolve the problem of fungal contamination in the recombinant adenoviruses. As it was going to take more time than I had available to solve this problem of contamination, the decision to use adenoviral vectors constructed by other investigators (Ad-GFP and Ad-CE1E2) for the remaining studies was taken. I did have enough of the un purified Ad-Core viruses to determine whether they expressed GFP and the HCV core protein and to look for any differences in the pattern of core expression within infected cells. Ad-Empty and the Ad-Core viruses expressed GFP. In the confocal experiments, the level of GFP expression seen with Ad-GFP is higher than with the Ad-Core viruses, however, you wouldn’t expect there to be differences in the levels of expression as both contain enhanced GFP (He et al., 1998; Swingler et al., 1999) under control of the CMV promoter. The differences are more likely to be due to the differences in the MOI of infections, as the MOIs used for the Ad-Core viruses were lower than the MOI used 186 with the purified Ad-GFP. Experiments in chapter 4 also show that the level of GFP expression in Ad-GFP infected cells at any given time point depends on the MOI. It may be conceivable that at higher MOIs the cells may be infected with multiple virions, leading to higher GFP expression. In support of this, when GFP expression was analysed in 293 cells from CPE assays, high levels of GFP expression were seen. These cells would have been infected at a higher MOI and 293 cells are also more susceptible to adenoviral infection than DCs. The viruses productively replicate within 293 cells, but cannot in DCs; therefore more viral particles are present in these cells. The HCV core protein was expressed in DCs infected with Ad-CE1E2 and the three Ad-Core viruses. It is easiest to see core expression in the DCs infected with Ad- CE1E2 and Ad-Core-195. This may relate to these being infected at the highest MOIs. To properly compare levels, infections at a range of MOIs with these viruses needs to be carried out, to look at levels of core expression over time, by a more qualitative means, such as intracellular staining and flow cytometric analysis. This would allow you to see whether, as might have been expected, that you get higher levels of core expression in cells infected with the adenovirus expressing the mutated version of the core protein (Ad-Core-124). The mutated version of the core protein expressed by Ad- Core-124 is not cleaved at a maturation site, which results in over-expression of the protein in BHK transfected cells (Hope and McLauchlan, 2000). I also tried to look at the intracellular location of each version of the core protein by co- staining cells with markers for the cytoplasm (phalloidin) or the nucleus (BO-PRO-3), and overlaying these with the core staining. Ad-CE1E2, Ad-Core-195 and probably Ad- Core-124 have expressed the core protein in the cell nucleus and cytoplasm, although higher levels appear to be in the nucleus. This may be due to host cell processing of the full-length core protein (although the exact mechanisms of cleavage of the core protein remain unknown (Reed and Rice, 2000), resulting in an accumulation of core 187 protein within the cells that may have undergone further processing which results in truncated versions of the protein that lack the COOH-domain. These can then be translocated into the cell nucleus. Kawamura et al (Kawamura et al., 1997) have detected nuclear localisation of the core protein in core protein transgenic mice. It was difficult to resolve exactly where in the cytoplasm the core protein was expressed. It is possible it may be different with each version of the core protein; there may be differences between the full-length core protein expressed by Ad-CE1E2 and that expressed by Ad-Core-195. The core protein expressed by Ad-CEI E2 will be in association with the envelope glycoproteins, and this interaction may retain the core protein near the endoplasmic reticulum, as El and E2 have ER retention signals (Reed and Rice, 2000). The core protein expressed by Ad-Core-195 will not be expressed in association with the envelope glycoproteins, therefore it may have a different location in the cell cytoplasm as there will be no ER retention signals present from the El and E2 proteins, as they are absent in this recombinant adenovirus. The preliminary results presented in this chapter, suggest that the truncated version of the core protein expressed by Ad-Core-169 may be restricted to the cell nucleus. However, I cannot be sure as the expression levels with this virus were very low, due to the low MOI of infection. Also, with cells infected with Ad-Core-124, where expression was also low, there were some cells where core expression could only be seen in the nucleus and not in the cytoplasm. From the literature it would be expected that only the truncated version of the core protein (amino acids 1-169) would be expressed in the cell nucleus (Hussy et al., 1996; Kawamura et al., 1997; Liu et al., 1997; Lo et al., 1994). If more time was available I would like to produce purified high titre stocks of the Ad- Core viruses and do higher resolution analysis of the intracellular location of the core protein in cells infected with these three viruses and Ad-CEI E2. The three adenoviruses I constructed could also be used for functional experiments, similar to those carried out with Ad-CEI E2, described in chapters 4 and 5. This could give an 188 insight into the regions of the core protein involved in immunomodulatory effects. But at least I knew that the core protein was expressed in DCs by means of Ad-CE1E2, which enabled further experiments to analyse how the core protein may affect DC activation, functions and susceptibility to apoptosis, as described in chapters 4 and 5. 189 Chapter 4 - Effects of the Hepatitis C Virus Core Protein on Dendritic Cell Activation and Functions 4.1. Introduction Hepatitis C virus is able to evade control by the host immune response in the majority of infected individuals as approximately 80% of people infected with this virus develop a chronic persistent infection. As discussed in chapter 1, multiple mechanisms have been proposed to contribute to HCV’s ability to evade immune control, and a variety of roles for individual viral proteins, including the core protein of HCV have been suggested to be involved. The core protein may mediate immunomodulatory effects by extracellular actions, for example, it has been shown to bind to the complement receptor gC1qR and prevent T cell responses (Kittlesen et al., 2000; Yao et al., 2001a; Yao et al., 2001b). In addition, expression of the core protein within infected cells could contribute to HCV persistence. One way the core protein may do this is by affecting the sensitivity of infected cells to antiviral effector mechanisms, for example, altering their susceptibility to lysis by NK cells and/or CD8* CTLs. This is considered in the next chapter. However, as the cells infected by HCV are thought to include cells of the immune system, the core protein could also contribute to HCV persistence by impairing their functions, thus altering the induction or maintenance of the antiviral immune response. Evidence of HCV infection in PBMCs has been found (Gong et al., 2003; Lerat et al., 1998; Muller et al., 1993) and infection of antigen presenting cells, including DCs, by HCV has also been documented (Goutagny et al., 2003; Navas et al., 2002); therefore expression of viral protein in these cells could alter their functions. There is evidence that HCV infection impairs the function of DCs from chronically infected patients (Bain et al., 2001; Kakumu et al., 2000; Kanto et al., 1999). These studies found the DCs to have a 190 reduced stimulatory capacity and decreased IL-12 production. Bain et al (Bain et al., 2001) detected expression of HCV genome sequences within the DCs; therefore expression of HCV proteins within the cells may be occurring and could be responsible for the defect of the DCs. One possible way the core protein could be mediating these effects is by its ability to bind to the cytoplasmic tail of members of the TNFR superfamily, such as the TNFR-1 (Zhu et al., 1998) and LTpR (Chen et al., 1997; Matsumoto et al., 1997). Core may also be capable of binding to CD40 (Lai and Ware, 2000), although this interaction has not been confirmed as yet. Interacting with these members of the TNFR superfamily that transduce activating signals via signalling components including NFkB for the production of cytokines may disrupt DC activation and functions (Chen et al., 1997; Matsumoto et al., 1997; Zhu et al., 1998). The core protein may also act within the nucleus of infected cells and modulate cellular gene expression by the activation or suppression of promoters (Chang et al., 1998; Ray et al., 1997; Ray et al., 1998b; Shih et al., 1993) which may be related to the development of hepatocellular carcinoma. The goal of the work described in this chapter was to examine the effects of core protein expression within DCs by the use of adenoviral vectors, which as discussed previously, I hoped would not have too many effects on the activation and function of DCs themselves. The work in this chapter focuses particularly on DC activation and functions in response to signalling through members of the TNFR superfamily. Responses to TNFa and anti-CD40 stimulation in comparison to LPS stimulation which is not a member of the TNFR superfamily were examined. TNFa will bind to TNF receptors on the DC surface and activate the cell and anti-CD40 will bind to CD40 and stimulate signalling via the CD40-CD40L pathway, whereas LPS stimulates dendritic cells via toll-like receptors, an innate recognition mechanism. 191 4.2 Maturation of Dendritic Ceiis Dendritic cells are normally present in an immature state. They express low levels of CD83, CD40, CD80, CD86, MHC class I and class II and they have a high ability to take up of antigens by endocytosis. Upon stimulation by a pathogen, cytokines or T cells the DCs undergo maturation and activation. Their ability to take up antigen by endocytosis is reduced and they upregulate expression of surface markers including MHC class I and class II, adhesion molecules and co-stimulatory molecules on their surface to enable them to present antigen to and activate T cells. They also produce IL-12 which enhances T cell activation. Prior to carrying out experiments to address the effects of core protein expression in DCs on their responsiveness to activation via different pathways, initial experiments were carried out to determine the optimal concentrations of different maturation stimuli for use in future experiments. Monocyte-derived DCs were generated as described in chapter 2, by culturing CD14'" cells from normal peripheral blood for 6-7 days in the presence of IL-4 and GM-CSF. The DCs produced in this way were judged to be immature on the basis of the lack of expression of the maturation marker CD83 on the majority of cells and expression of relatively low levels of MHC (class I and class II) and co-stimulatory molecules (CD40, CD80, CD86). Examples of the phenotype of these “immature” DCs are illustrated in figures 4.1-4.3 (untreated control DCs). These examples also illustrate that there was some variation in the phenotype of DCs produced from different donors on different days, for example, whereas CD83 was expressed only on 33% of immature DCs in the experiments shown in figures 4.1 (the range of unstimulated cells expressing CD83 from 6 donors was 4-33%) and 4.2 (the range of unstimulated cells expressing CD83 from 6 donors was 6-33%), it was expressed on 43% of DCs in the experiment shown in figure 4.3 (the range of unstimulated cells expressing CD83 from 3 donors was 6- 192 (b) (a) CD83 CD83 CD83 2 8$ = 0001 0,01 0001 001 LPS Cone (nfl/frt) LPS Cone, (no/ frt) FL-2 Class Class I 0) ™- Class I > 80- Ml g : n n n n 0.001 0.01 0 0001 0.01 0.1 LPS Cone, (ng/ rr«) LPS Cone (ng/rrt) FL-2 Class II Class II Class ■ llll 0 001 0.01 0-001 0.01 0.1 LPS Cone, (ng/ rrt ) LPSConc. (ng/frt) FL-2 CD40 CD40 CD40 Ml n n Hill0 0 001 0.01 0.1 1 0 0.001 001 0 1 LPS Gone (ng/fW) LPSConc. (ng/ rr<) FL-2 CD80 CD80 CD80 Ml :! n n n IXJXI0.001 001 0 0 001 0.01 01 FL-2 LPS Cone (ng/ n i) LPSConc. (ng/rrt) CD86 CD86 CD86 Ml n n n Hill0001 0.01 0 0.001 OOl LPS Cone, (ng/irt) LPSConc. (ng/ r, FL-2 CD95 100 CD95L 80 — Isotype control i - ■I 60 (/) p 40 — Ong/ml LPS Q. 20 — 1 ng/ml LPS 0001 001 0.001 0.01 0 LPS Cone, (ng/ml) Figure 4.1. Maturation of DCs in response to LPS determined by phenotypic surface marker staining. 1x10® uninfected DCs were stimulated with a range of LPS concentrations (0-1 ng/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. Part (a) shows the percentage of cells expressing each marker (red bars) and the mean fluorescence intensity (MFI) of staining (yellow bars). Representative examples of the staining are shown in part (b), with the black line indicating an isotype control, the red line indicating staining of unstimulated cells with the indicated antibody and the purple line indicating staining of DCs stimulated with 1 ng/ml of LPS with the indicated antibody. The results shown are representative of 6 individual experiments. 1 93 (a) (b) CD83 CD83 CD83 □ □ FL-2 Class I Class I Class I M1 T N F a C o n e . ( n g / m l) TNFa Cone (n g /rrt) FL-2 Class II Class II CD40 CD40 CD40 M □ [~l TNFa Cone (ng /frt) TNFa Cofie. (ng/rrt) FL-2 CD80 CD80 CD80 Ml lull□ □ I Cone, (ng /m l) TNFa Cone (ng/rrt) FL-2 CD86 CD86 XIII CD86 TNFa Cone, (ng/rrt) TNFa Cone (ng/rrt) FL-2 CD95 CD95L 0) «0 __ — Isotype control — Ong/ml TNFa g 1 1 1 1 — 200ng/ml TNFa TNFa Cone. Figure 4.2. Maturation of DCs in response to TNFa determined by phenotypic surface marker staining. 1x10® uninfected DCs were stimulated with a range of TNFa concentrations (0- 200ng/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. Part (a) shows the percentage of cells expressing each marker (red bars) and the mean fluorescence intensity (MFI) of staining (yellow bars). Representative examples of the staining are shown in part (b), with the black line indicating an isotype control, the red line indicating staining of unstimulated cells with the indicated antibody and the purple line indicating staning of DCs stimulated with 200ng/ml of TNFa with the indicated antibody. The data shown is representative of 6 individual experiments. 194 (a) (b) CD83 CD83 CD83 Ml S. * ■ III AntUCO40 Cone. (uQ/rrt) »ntl-CO40 Cone, (ug/ rrl) FL-2 Class I Class I Class I A n ti>0040 Cone. {ug/rn) >CD40 Cone, (ug/ rrt) FL-2 Class II Class II Class II I: 3$ 201 FL-2 I-0040 Cone, (ug/ rrt ) I-C040 Cone, (ng/ r t ) CD40 CD40 CD40 I.CD40 Cone (ug/ rrt) FL-2 CD80 CD80 CD80 CL «■ 2) ' IJII 10®k ÎV 10® 10* 10 ‘ Anti-CD40 Cone, (u g /rri) -CD40 Cone, (ug/ frt ) FL-2 CD86 CD86 CD86 Ml J i l l 1.CD40 Cone, (ug/ n4) Anti .0 0 40 Cone, (ug/nri) FL-2 CD95 CD95L — Isotype control — Oug/ml anti-CD40 g § 40- _0 40- Û- _ — 20ug/ml anti- CD40 tl*C040 Cone, (ug/ml) mn#I CD40 Cone, (ug/mi) Figure 4.3. Maturation of DCs in response to anti-CD40 determined by phenotypic surface marker staining. 1x1Q6 uninfected DCs were stimulated with a range of anti-CD40 mAb concentrations (0-20|ig/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. Part (a) shows the percentage of cells expressing each marker (red bars) and the mean fluorescence intensity (MFI) of staining (yellow bars). Representative examples of the staining are shown in part (b), with the black line indicating an isotype control, the red line indicating staining of unstimulated cells with the indicated antibody and the purple line indicating staining of DCs stimulated with 20pg/ml of anti-CD40 with the indicated antibody. The data shown is representative of 3 individual experiments. 195 43%), however, the mean fluorescent intensity (MFI) was similar for all donors (-13). These differences are most likely to be due to donor-donor variation in the extent of maturation triggered under the culture conditions, in response to, for example, low levels of endotoxin present in the culture medium, as the culture conditions were kept constant between experiments. To test the responsiveness of immature DCs to different activation/maturation stimuli, 10® cells were stimulated with a range of concentrations of LPS (0-1 ng/ml), TNFa (0- 200ng/ml) and anti-CD40 (0-20|ig/ml) for 24 hours. Cells were then stained with antibodies against maturation and activation markers to allow determination of their maturation status. Fas (CD95) and Fas-ligand (CD95L) expression were also measured. Antibody staining was analysed by flow cytometry. As shown in figure 4.1, LPS is a very potent inducer of DC maturation. Low levels (0.1 ng/ml) of LPS induced a high proportion of cells to express CD83 (80%). Co stimulatory molecules CD80, CD86 and class I were also upregulated in a dose- dependent manner, as shown by the increase in the MFI. However, there were only very small changes in the percentage of cells expressing MHC class II, CD40 or CD95. Very few cells expressed CD95L. TNFa induced DC maturation in a dose dependent manner as the percentage of CD83^ cells increased as the concentration of TNFa increased (as did the MFI), as shown in figure 4.2. At the doses tested, TNFa did not achieve maturation of as high a proportion as cells did when treated with LPS (60% of cells were CD83 positive at the highest TNFa concentration, compared to 80% with LPS). TNFa also increased the percentage of cells expressing CD80 and CD40. As with LPS the number of cells expressing MHC class I and II did not change dramatically (reflected by the percentage 196 of cells positive and the MFI). The number of cells expressing CD86 also did not change as dramatically as with LPS. Again CD95 was expressed and very few cells expressed CD95L and TNFa maturation did not alter their expression. Anti-CD40 stimulation of DCs was the weakest inducer of DC maturation out of the three stimuli. Figure 4.3 shows the phenotypic marker expression induced by anti- CD40 stimulation. The percentage of CD83^ cells and the MFI of CD83 staining did increase upon anti-CD40 stimulation, but not to the same extent as with LPS (60% positive at the highest anti-CD40 mAb concentration). In this experiment the DCs were in a more mature state than the cells stimulated by LPS and TNFa, as there were more cells expressing CD83 in the unstimulated cells in the LPS and TNFa experiments. However, in duplicate experiments, anti-CD40 was a consistently weaker inducer of DC maturation than LPS and TNFa (data not shown). Class I and class II expression did not alter dramatically when DCs were stimulated with anti-CD40, and only small increases in both the percentage of CD80'" and CD86'" cells and the MFI of staining occurred. CD40 expression could not be detected. This was due to the anti-CD40 antibody used to stimulate the cells blocking the epitope recognised by the anti-CD40 antibody used in flow cytometry. This could have been overcome by co-culturing DCs with cells expressing CD40L to induce activation and maturation rather than ligation of CD40 by an antibody (Omata et al, 2002, Frieta et al, 2003). Again, DCs expressed CD95 and did not express CD95L. In conclusion, immature monocyte-derived DCs could be matured with LPS, TNFa and anti-CD40. There was some donor-donor variation in the extent of maturation levels (CD83 expression) in unstimulated cells, which may be triggered under the culture conditions (for example the difference between the results shown in figures 4.2 and 4.3). Overall, differences in the potency of the three stimuli were seen. LPS was the 197 strongest inducer of DC maturation, followed by TNFa, with anti-CD40 being the weakest inducer of DC maturation, as determined by induction or upregulation of CD83 expression. 4.3. Infection of Dendritic Ceiis with Adenovirus To study effects of HCV core protein expression in immature DCs on their activation state and functional capacity in response to the stimuli tested above, I planned to achieve high levels of core expression in DCs by infecting them with recombinant adenoviral vectors encoding the core (and the envelope glycoproteins, E1 and E2). Before I embarked on these experiments, preliminary experiments were carried out to determine the level of DC transduction achieved by infecting DCs with recombinant adenoviral vectors at different multiplicities of infection (MOI) and to determine effects of the vector on DCs. In an initial experiment, the kinetics of GFP expression in DCs following infection with Ad-GFP were investigated, to determine the optimal time for analysis of the proportion of transduced cells and/or analysis of the effects of HCV core protein within DCs on their activation and functions. DCs were infected at a MOI of 75 with Ad-GFP and the percentage of GFP positive cells and the level of GFP expression in transduced cells were determined after 4, 8,12, 24 and 32 hours by flow cytometry. As shown in figure 4.4, a high proportion (84%) of DCs were GFP positive, however the level of GFP expression in each cell was fairly low (MFI of the GFP-positive cells was 69.00) at 8 hours. By contrast at 24 hours, by which time 98% of cells were GFP positive, the MFI of GFP positive cells had increased to 1074.43. By 32 hours post-infection there was some but not a dramatic further increase in the mean level of GFP expression per cell. Based on these results, a 24 hour infection period was used for all future experiments. 198 0% 27% 84% \ M1 V ^ V ' ■ ■"■T '■■-1 10°10^ 10^10°10* 10°10’ 10^10^10* 10°10^ 10^10^10* FL1 -H FL1 -H FL1 -H Uninfected DC Ad-GFP DC Ad-GFP DC 4 hours 4 hours 8 hours 96% 98% 10°10'10^10°10* 10°10'10^10^10* 10° 1?' To^To° ïb* FL1 -H FL1 -H FL1 -H Ad-GFP DC Ad-GFP DC Ad-GFP DC 12 hours 24 hours 32 hours Figure 4.4. Timecourse of GFP expression following Ad-GFP Infection of Immature DCs. 5x10® immature DCs were infected with Ad-GFP at a MOI of 75. GFP expression within the cells was assessed by flow cytometry at 4,8,12 and 24 hours post infection, as indicated. The percentage of cells expressing GFP in each sample is shown. The data shown is representative of 2 individual experiments. 199 Previous studies have shown that adenoviral vectors transduce monocyte-derived DCs efficiently, although some differences in effects of transduction at different MOIs were reported between studies, ranging from 40%-96% transduction (Jonuleit et al., 2000; Miller et al., 2002; Rea et al., 1999; Rouard et al., 2000; Zhong et al., 1999). To determine the efficiency of infection of DCs by adenoviruses and the optimal multiplicity of infection (MCI) for future experiments, immature and mature (treated with anti-CD40 at Spg/ml, 24 hours) DCs were infected with Ad-GFP at a range of MOIs for 24 hours. Cells were then stained for C D IIc expression (a DC marker) and analysed for GFP expression by flow cytometry. This allowed the percentage of DCs expressing GFP to be determined. Representative results from this type of analysis are shown in figure 4.5. A dose-dependant increase in the percentage of GFP positive cells was seen in both immature and mature DCs as the MCI with Ad-GFP was increased. Similar results were obtained with DCs that had been matured with other stimuli and infected at a range of MOIs (data not shown). Interestingly, not only the percentage of cells transduced, but also the mean level of GFP expression within transduced cells increased with increasing MOIs, for example, the 21.2% of immature DCs that expressed GFP after infection at a MOI of 10 had an MFI of 25.9; whereas the 87.3% of cells expressing GFP after infection at a MOI of 1000 had a MFI of 333.2 (data not shown). It is unclear why there is this difference. It may be at lower MOIs infection was more asynchronous with some cells infected more recently than others, hence less GFP per cell however this is unlikely. It is probably due to cells infected at higher MOIs being infected with multiple adenovirus particles, resulting in more gene delivery and higher expression of GFP. It was also observed that mature DCs were infected slightly better than immature DCs, for example, following infection at a MOI of 100, 71% of immature DCs were GFP positive at 24 hours post infection whilst 79% of mature DCs were GFP positive at this time point. 200 9 8 .3% 0 .8% 9 6 .5% 31% 91.0% 8.3% 78.3% 2 1 .2% & Immature DC 10“ 10 ’ 10“ 10“ 10‘ 10“ 10’ 10^ 10^ 10‘ 10“ 10’ 10^ 10“ 10* 10“ 1 0 ’ 10“ 10“ 1 0 ‘ FL1-H 0.0% 1.0% FL1-H 0.0% 0.4% FL1-H 0 0% 0 .6% FL1-H 0.0% 98.5% 0 .8% 94.( 5 .3% 8 6 .0% 13.4% 2 3 .7 % Mature DC & 1 0 ‘ 0.7% FL1-H 0 .0% FL1-H0.7% 0 .0% 0.5% FL1-H 0 .0% 0.8% FL1-H 0.0% MOI 0 MOI 1 MOI 3 MOI 10 o Q O 42.9% 71.7% 12.0% 86.3% 9.9% 87.3% Immature DC 10“ 1 0 ’ 10“ 10“ 1 0 ' FL1-H 0.1% 0.4% FL1-H 0 3% 10“ 1 0 ’ 10“ 1 0 “ 1 0 ' 1 0 ’ 10 “ 10 “ 1 0 ' 6 FL1-H 1.2% FL1-H 2.3% 19.9% 79.1% 48 2% 51 0% % 84.7% 8 8 .0% & Mature DC 10“ 1q1 10“ 10“ 10' 10“ 1 0 ’ 10“ 10“ 1 0 ' FL1-H 0.2% 10“ 1 0 ’ 10“ 10“ 1 0 ' 0.7% FL1-H 0.4% 10“ 10 ’ 10“ 10 “ 1 0 * 0.5% FL1-H 1.4% FL1-H 4.2% MOI 30 MOI 100 MOI 300 MOI 1000 GFP Figure 4.5. Infection of Immature and mature DCs with Ad-GFP at a range of MOIS. 1x10^ immature or mature (5^g/ml of anti-CD40 for 24 hours) DCs were infected with Ad-GFP at the indicated MOIs. 24 hours later cells were stained with a PE-labelled antibody to CDIIc, to identify DCs, and analysed for GFP (FL-1) expression by flow cytometry. The percentage of CD11c^ and GFP* cells is shown in the upper right quadrant. 201 There are differences between the percentage of GFP positive cells at 24 hours between experiments shown in figures 4.4 (98% GFP* at a MOI of 75) and 4.5 (71% GFP* at a MOI of 100). This is most likely to be due to donor-donor variation. Typically at a MOI of 200, approximately 80% DCs were transduced and GFP positive, therefore this MOI was used for future experiments at this time point. As the MOI increased above 300, the infection of DCs started to have a detrimental effect on DC viability as assessed by changes in the forward and side scatter profiles which represent the size and granularity of cells (data not shown). Adenoviruses are generally reported to achieve high levels of DC transduction with minimal effects on DC activation and functions, but there are some studies suggesting that adenovirus infection of monocyte-derived DCs induces some level of DC activation (Hirschowitz et al., 2000; Korst et al., 2002; Miller et al., 2002; Morrelli et al., 2000; Rea et al., 1999; Rouard et al., 2000). I examined effects of adenovirus infection on the expression of CD83, MHC (class I and class II) and co-stimulatory molecules (CD40, CD80 and CD86) on DCs and also on DC maturation in response to LPS, TNFa and anti-CD40. The data for CD83 expression is shown in figure 4.6; changes in other activation markers paralleled this data, although they were less dramatic. This figure represents results from 6 different donors out of 20 donors analysed in total. The figure illustrates the donor-to-donor variation in the responses made to maturation stimuli: DCs from donor 1 responded better to LPS stimulation than those from donor 2. Similarly DCs from donor 3 responded better than those from donor 4 to TNFa stimulation. Generally, there was little effect of adenovirus on the activation/maturation state of immature DCs, although occasionally a slight increase in the percentage of cells expressing CD83 (and also MHC class I and class II and co-stimulatory molecules) occurred, as seen in donor 4 in figure 4.6 when cells were unstimulated. In total, 5/20 donors underwent an increase of <5% and DCs from 2/20 donors underwent 202 LPS stimulation Donor 1 Donor 2 0) 100 n 80 - Uhinfected R 60 n □ Uninfected Ad-G FP £ o A d-G FP on r>n n-. I~ln FTl 0 0.001 0.01 0.1 1 0 0.001 0.01 0.1 1 LPS Cone, (ng/ml) LPS Cone, (ng/ml) TNFa stimulation Donor 3 Donor 4 100 100 - 80 - 80 i 60 , Uninfected 60 J □ Uhinfected 40 - Ad-GFP 40 1 ■ A d-G FP I 20 : 20 H 0 I- 0 5 50 200 0 5 50 200 TNFa Cone, (ng/ml) TNFa Cone, (ng/ml) Anti-CD40 stimulation Donor 5 Donor 6 4, 100 .> 80 ■s 60 □ Uninfected Uhinfected ■ A d-G FP Ad-G FP JZL 0 5 10 20 0 0.5 1 5 anti CD40 Cone, (ug/ml) anti-CD40 Cone, (ug/ml) Figure 4.6. Effect of Adenovirus on DC maturation in response to LPS, TNFa and anti- CD40 stimulation. Uninfected monocyte-derived DCs and DCs that had been infected with Ad-GFP at a MOI of 200 for 24 hours were stimulated with LPS, TNFa and anti-CD40 mAh at the concentrations indicated for 24 hours. Cells were then stained with an antibody against CD83 (DC maturation marker) and analysed by flow cytometry. The percentage of cells expressing CD83 is shown. Data is shown for 6 individual donors out of 20 donors analysed. 203 an increase of >10% in the percentage of CD83^ cells following infection Ad-GFP. Adenovirus infection frequently had little effect on the response to maturation stimuli, although it did sometimes reduce responses, as seen in donors 1 and 3 in figure 4.6. Altogether, Ad-GFP infection caused a reduction >10% in the percentage of cells stimulated to express CD83 in response to treatment with the highest concentration of maturation stimuli in 9/20 donors. This slight donor-to-donor and experiment-to- experiment variation made it important to repeat experiments with a minimum of three different donor DCs, to be certain of the reproducibility of any effects observed. In conclusion, DCs were efficiently transduced with GFP by adenovirus vectors (70- 80%) and this could be achieved with only minimal effects on DC activation and functions. A MOI of 200 was chosen for future experiments as this gave high levels of infection with minimal effects on DC viability. Donor to donor variation and experiment to experiment variation in the efficiency of adenovirus infection and responses to maturation stimuli was observed, so experiments were repeated a minimum of three times to be certain observed effects were reproducible. 4.4 Effects of the HCV core protein on the ability of DCs to uptake antigen One of the functions of immature DCs is to take up antigen, which can then be presented on the cell surface in the context of MHC class I or class II. One possible means for a virus to impair induction of the antiviral immune response would be to reduce the uptake of antigen by antigen presenting cells and thus reduce the presentation of viral antigens to T cells and reduce T cell activation. Therefore, it was of interest to determine whether the core protein from HCV could affect the ability of DCs to take up antigen. 204 DCs were infected with Ad-GFP and Ad-CE1E2 and their ability to take up antigen was compared to that of uninfected DCs. 10® DCs were incubated with 20pg/ml of dextran alexa-fluor-647 at 4°C and 37°C for 0,5,10 and 20 minutes. The percentage of cells containing dextran was determined by flow cytometry. As can be seen in figure 4.7, DCs do not take up dextran at 4°C, whilst at 37°C the percentage of dextran positive cells increases over time. Adenovirus (Ad-GFP) and the HCV structural proteins (Ad- CE1E2) have no effect on the antigen uptake capacity of immature DCs. This experiment was also repeated with mature DCs (which take up antigen less efficiently, but still do to some extent) and no effect of adenovirus or the HCV structural proteins was observed (data not shown). In conclusion, the core protein has no effect on the ability of DCs to uptake antigen. 4.5 Effects of the HCV core protein on DC maturation In response to different stimuli Reports that the HCV core protein can interact with the cytoplasmic tail of members of the TNFR superfamily, including the TNFR-1 and LT(3R, and may modulate signal transduction via them, led to investigation into the effects of the core protein expression in DCs on their maturation and activation in response to these and other stimuli. Initially, experiments were performed to assess whether core protein expression in immature DCs had any effect on their maturation and activation. It was unlikely that the core protein would induce maturation, as this would have resulted in a decrease in the capacity of immature DCs to take up antigens, which was not seen in the previous experiments (figure 4.7). Phenotypic analysis of the DCs infected with Ad-CE1E2 confirmed this, when compared to uninfected DCs that were untreated, as shown in figure 4.8. Adenovirus infection by Ad-GFP had a tendancy to decrease surface 205 (a) 100 - I 80 ! 60 □ Uninfected c (0 ■ Ad-GFP V 40 H ■ Ad-CE1E2 20 5 10 20 Time (mins) (b) 100 o 80 > '55 ■ Uninfected s . □ Ad-GFP s % 40 ■ Ad-CE1E2 o O m 5 10 20 Time (mins) Figure 4.7. Antigen uptake capacity of immature DCs infected with Ad-GFP & Ad- CE1E2. 1x10® DCs were incubated with 20p.g/ml of dextran Alexa-Fluor-647 for 0,5,10 and 20 minutes at (a) 4°C and (b) 37°C. Cells were washed and the percentage positive for dextran was determined by flow cytometry. The data shown is representative of 3 individual experiments. 206 CD83 100 80 I □ Uninfected "5) 60 o □ Ad-GFP Q. 40 ■ Ad-CE1E2 20 0 I 0.001 0.01 0.1 LPS Cone, (ng/ml) J* 20 lllll S' 20 0 0.001 0.01 0 1 1 hull0 0.001 0.01 0 1 1 LPS Cone, (ng/ml) LPS Cone, (ng/ml) D Uninfected I Uninfected ■ Ad-GFP I Ad-GFP UMU S' 20 |Ad-CE1E2 0 0.001 0.01 01 1 lllll0 0.001 001 01 1 0 0.001 0.01 0.1 LPS Gone, (ng/ml) LPS Cone, (ng/ml) LPS Cone, (ng/ml) 100 . a 80 - I Uninfected I :! I Ad-GFP |Ad-CE1E2 s' 20 4 mi0 0.001 0.01 0.1 0 0.001 0.01 0.1 1 LPS Cone, (ng/ml) LPS Cone (ng/ml) Figure 4.8. Effect of HCV core protein expression in DCs on their phenotypic maturation in response to LPS stimulation. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) DCs were stimulated with a range of LPS concentrations (0-1 ng/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. The results shown are the percentage of cells expressing each marker. The data shown is representative of 6 individual experiments. Statistical analysis of variance performed with a two-way ANOVA test determined no significant differences between marker expression by uninfected or adenovirus infected (Ad-GFP or Ad-CE1E2) DCs at each LPS concentration. 207 marker expression more frequently than Ad-CE1E2. This effect was possibly due to effects of residual caesium chloride in the purified Ad-GFP having detrimental effects on DC viability. Experiments were then carried out to look at whether the core protein would affect maturation of DCs in response to a stimulus not delivered via a TNFR superfamily ligand. Effects of the core protein on maturation responses to LPS were looked at. Immature uninfected, Ad-GFP or Ad-CE1E2 infected DCs were stimulated with a range of LPS concentrations (0-1 ng/ml) for 24 hours. Cells were stained for CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L expression which was analysed by flow cytometry. LPS induced maturation of the DCs as determined by the increase in CD83"^ cells, illustrated in figure 4.8. LPS maturation also increased the percentage of cells expressing class I, class II and CD40 (the MFI of staining for each marker also increased with LPS stimulation, data not shown). There was little change in the number of cells expressing CD80, CD86 or CD95 and no cells expressed CD95L (no changes were seen in the MFI of staining, data not shown). Adenovirus infection and the expression of the HCV structural proteins did not alter the phenotypic maturation of DCs in response to LPS, as statistical analysis of variance performed with a two-way ANOVA test revealed no significant differences. IL-12 p40 production by uninfected, Ad-GFP and Ad-CE1E2 infected DCs in response to LPS was also measured by ELISA. LPS induced IL-12 production in a dose-dependent manner in all cells. Interestingly, adenovirus infection enhanced IL-12 production in response to LPS, as shown in figure 4.9. However, the HCV structural proteins had had no effect on LPS- stimulated IL-12 production by DCs. These results were expected as LPS is a very strong maturation stimulus, so even if the core protein had effects on downstream signalling pathways, LPS may be able to override them. 208 18000 - 16000 ^ 14000 ^ 1 12000 - ? □ Uninfected 5 10000 - o ■ Ad-GFP Q. 8000 - ■ Ad-CE1E2 V 6000 - 4000 - 2000 0 - 0 0.001 0.01 0.1 LPS Cone, (ng/ml) Figure 4.9. Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad-CE1E2 infected DCs in response to LPS stimulation. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected DCs were stimulated with a range of LPS concentrations for 24 hours. Supernatants were harvested and IL-12 production was measured by an IL-12 p40 ELISA. The data shown is representative of 4 individual experiments. 209 Effects of the core protein on the DC maturation response to TNFa were looked at next. Immature uninfected, Ad-GFP or Ad-CE1E2 infected DCs were stimulated with a range of TNFa concentrations (0-200ng/ml) for 24 hours. Cells were stained for CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L expression which was analysed by flow cytometry. TNFa also induced DC maturation shown by the increase in the percentage of CD83"^ cells, illustrated in figure 4.10 (the MFI of staining also increased, data not shown). Class I, class II, CD80, CD80 and CD40 expression also increased (shown by both the percentage of cells expressing each marker and the MFI of staining). CD95 and CD95L expression were unchanged. Adenovirus infection significantly altered the expression of CD83 and MHC class II of unstimulated DCs and DCs stimulated with 5ng/ml of TNFa, as indicated by a * on figure 4.10 (as determined by a two-way ANOVA test). However there were no effects on the expression of the other surface markers and the expression of the HCV structural proteins did not alter the phenotypic maturation of DCs in response to TNFa. IL-12 p40 production by uninfected, Ad-GFP and Ad-CE1E2 infected DCs was also measured in response to TNFa maturation by ELISA. TNFa induced IL-12 production in a dose-dependent manner. Adenovirus infection reduced the levels of IL-12 production, as seen in figure 4.11. This contrasts with the effect of adenovirus infection on the response to LPS, where IL-12 production was enhanced (see figure 4.10). Again, however, the HCV structural proteins had no effect on IL-12 production in response to TNFa. Finally, effects of the core protein on the DC maturation response to anti-CD40 were examined. Immature, uninfected, Ad-GFP and Ad-CE1E2 infected DCs were stimulated with a range of anti-CD40 concentrations (0-20pg/ml) for 24 hours. Cells were stained for CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L expression which was analysed by flow cytometry. Anti-CD40 also induced maturation of the DCs, but to a lesser extent than LPS or TNFa, as determined by the proportion 210 CD83 100 1 Q) 80 - > □ Uninfected 60 - (A □ Ad-GFP £ 40 ^ 55 20 - ■ Ad-Œ1E2 0 - i l 0 5 50 200 TNFa Conc. (ng/ml) Class I Class II I Uninfected I Ad-GFP |Ad-CE1E2 illli0 5 50 200 0 5 50 200 TNFa Conc. (ng/ml) TNFa Conc. (ng/ml) CD66 CD40 I I Uninfected Uninfected Uninfected I Ad-GFP I Ad-GFP |Ad-CE1E2 |Ad-CE1E2 0 5 50 200 5 50 200 0 5 50 200 TNFa Conc. (ng/ml) TNFa Conc. (ng/ml) TNFa Conc. (ng/ml) I Uninfected 100 80 I Uninfected I Ad-GFP 60 I Ad-GFP |Ad-CE1E2 40 20 |Ad-CE1E2 0 5 50 200 0 5 50 : TNFa Conc. (ng/ml) TNFa Conc. (ng/ml) Figure 4.10. Effect of HCV core protein expression in DCs on their phenotypic maturation in response to TNFa stimulation. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) DCs were stimulated with a range of TNFa concentrations (0-200ng/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. The results shown are the percentage of cells expressing each marker. The data shown is representative of 6 individual experiments. Statistical analysis of variance performed with a two-way ANOVA test determined a significant difference (p<0.05) between marker expression by uninfected or adenovirus infected (Ad-GFP or Ad-CE1E2) DCs at the TNFa concentrations indicated by * . 211 7000 - 6000 - 5000 - 2 4000 ■ Uninfected □ Ad-GFP I “ ■ 3000 CM ■ Ad-CEI E2 2000 1000 0 CEE i W l 5 50 200 TNFa Conc. (ng/ml) Figure 4.11. Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad-CEIE2 Infected DCs In response to TNFa stimulation. 1x10® uninfected, Ad-GFP or Ad-CEI E2 infected DCs were stimulated with a range of TNFa concentrations for 24 hours. Supernantants were harvested and IL-12 production was measured by an IL-12 p40 ELISA. The data shown is representative of 3 individual experiments. 212 of cells stimulated to express CD83. Figure 4.12 shows the phenotypic marker expression on DCs matured with anti-CD40. There was no change in the level of expression of any marker except CD83 (no changes occurred in the MFI of staining, data not shown). There was no significant effect of adenovirus infection on DC surface marker expression when cells were matured with anti-CD40 (as determined by a two- way ANOVA test). CD40 expression was not detected; again this is due to the antibody used to mature the cells blocking the epitope recognised by the antibody used for flow cytometric analysis. Anti-CD40 maturation also induced IL-12 p40 production, as shown in figure 4.13. Adenovirus infection enhanced IL-12 production in response to anti-CD40, as had been seen with LPS. However, the HCV structural proteins had no effect on the stimulation of IL-12 production from DCs by anti-CD40. It has not been confirmed that the core protein interacts with the cytoplasmic domain of CD40; therefore the lack of effects may have been due to the core protein not interacting with this member of the TNFR superfamily. In conclusion, neither adenovirus infection of DCs nor the expression of HCV structural proteins within these cells had any effect on the phenotypic maturation of DCs in response to LPS, TNFa or anti-CD40. Adenovirus infection enhanced LPS and anti- CD40 stimulated IL-12 production by DCs, but decreased IL-12 production in response to TNFa. However, the HCV structural proteins had no effect on IL-12 production by DCs in response to LPS, TNFa or anti-CD40 stimulation. 4.6 Effects of the core protein on IL-12 production by Macrophages As described above, expression of the HCV core protein within DCs was not found to have any effect on their production of IL-12 in response to several different stimuli. However, a study by Lee at al (Lee et al., 2001 ) had suggested that expression of the 213 CD83 100 - 0) 80 " > □ Uninfected 60 - S □ Ad-GFP Q. 40 - □ Ad-CE1E2 20 - 0 - 0 5 10 20 anti-CD40 Conc. (ug/ml) I Uninfected I Uninfected I Ad-GFP I Ad-GFP |Ad-CE1Ë2 |Ad-CE1E2 0 5 10 20 0 5 10 20 antl-CD40 Conc. (ug/ml) anti-CD40 Conc. (ug/ml) 100 80 ■ Uninfected 60 ■ Ad-GFP 40 s' 20 ■ Ad-CE1E2 20 l i u 0 H 0 5 10 20 Mil0 5 10 20 0 5 10 20 anti CD40 Conc. (ug/ml) anti-CD40 Conc. (ug/ml) anti-CD40 Conc. (ug/ml) ■ Uninfected I Uninfected I Ad-GFP I Ad-GFP |Ad-CE1E2 |Ad-CE1E2 antl CD40 Conc. (ug/ml) anti-CD40 Conc. (ug/ml) Figure 4.12. Effect of HCV core protein expression in DCs on their phenotypic maturation in response to anti-CD40 stimulation. 1x10® uninfected, Ad-GFP or Ad-CEI E2 infected (MOI 200) DCs were stimulated with a range of anti-CD40 concentrations (0-20ug/ml) for 24 hours. Cells were stained with antibodies against CD83, class I, class II, CD80, CD86, CD40, CD95 and CD95L and analysed by flow cytometry. The results shown are the percentage of cells expressing each marker. The data shown is representative of 4 individual experiments. Statistical analysis of variance performed with a two-way ANOVA test determined no significant differences between marker expression by uninfected or adenovirus infected (Ad-GFP or Ad- CEI E2) DCs at each anti-CD40 concentration. 214 1600 - 1400 - I * 1200 - Q.g> 1000 Uninfected I 800 Ad-GFP CM 600 I Ad-CEI E2 400 200 0 f - r m 0 5 10 20 anti-CD40 Conc. (ug/ml) Figure 4.13. Induction of IL-12 p40 production by uninfected, Ad-GFP & Ad-CEIE2 infected DCs in response to anti-CD40 stimulation. 1x10® uninfected, Ad-GFP or Ad-CEIE2 infected DCs were stimulated with a range of anti-CD40 mAb concentrations for 24 hours. Supernatants were harvested and IL-12 production was measured by an IL-12 p40 ELISA. The data shown is representative of 3 individual experiments. 215 core protein in macrophage cell lines inhibited their ability to produce IL-12 in response to activation with LPS for 30 hours following pre-treatment with IFNy. It was therefore decided to investigate whether expression of the HCV structural proteins within macrophages may affect their ability to produce IL-12. 5x10® monocyte-derived macrophages were infected at a MOI of 1000 (a higher MOI was required to achieve the same level of infection of macrophages as DCs, data not shown) with Ad-GFP or Ad-CEI E2 or left uninfected for 24 hours prior to stimulation with a range of LPS concentrations for a further 24 hours. The culture supernatants were assayed for IL-12 p40 production by ELISA. As can be seen in figure 4.14, LPS induced IL-12 production by uninfected, Ad-GFP and Ad-CEI E2 infected macrophages. There was no difference in the levels of IL-12 produced by macrophages expressing the HCV structural proteins (Ad-CEI E2 infected). In summary, the HCV structural proteins had no effect on IL-12 production by monocyte-derived macrophages in response to LPS stimulation. 4.7 Effects of the core protein on the ability of DCs to stimulate allogenic & antigen-specific T ceil responses One of the functions of DCs is to present antigen to and activate T cells in order to induce and maintain an immune response. It has been reported that DCs from chronically infected HCV patients have a reduced stimulatory capacity (Auffermann- Gretzinger et al., 2001; Bain et al., 2001; Kakumu et al., 2000; Kanto et al., 1999) and it has been suggested that expression of the HCV core protein may have a role in this (Dolganiuc et al., 2003). Therefore it was of interest to determine whether DCs expressing the HCV structural proteins had a reduced T cell stimulatory capacity. 216 1200 1000 800 I □ Uninfected 600 1 ■ Ad-GFP CM ■ Ad-CEI E2 400 200 0 50 100 150 200 250 300 LPS conc. (ng/mi) Figure 4.14. IL-12 p40 production by uninfected, Ad-GFP and Ad-CEIE2 infected macrophages in response to LPS stimulation. 5x10^ monocyte-derived macrophages were infected at an MOI 1000 with Ad-GFP or Ad-CEI E2 for 24 hours prior to simulation with a range of LPS concentrations (0-300ng/ml) for a further 24 hours. Supernatants were harvested and assayed for IL-12 p40 production by ELISA. The data shown is representative of 3 individual experiments. 2 1 7 Initially, effects of the HCV protein expression within DCs on their ability to stimulate allogenic T cell responses were addressed. Uninfected, Ad-GFP and Ad-CE1E2 infected DCs were cultured with allogenic T cells, and T cell activation was assessed in three ways: by measuring proliferation, IL-2 production and activation marker expression. Figure 4.15 shows the allogenic T cell proliferative responses stimulated by uninfected, Ad-GFP and Ad-CEI E2 infected DCs after 3, 4 and 5 days of co-culture, measured in a standard ^H-thymidine incorporation assay. There was a significant difference (determined by a one-way ANOVA test and Tukey’s pairwise comparison) in the ability of uninfected and adenovirus infected DCs to stimulate T cell proliferation as indicated by a * on figure 4.15. The adenovirally infected DCs had a reduced stimulatory capacity when compared to uninfected DCs. The expression of the HCV structural proteins in DCs altered the T cell stimulatory capacity of DCs with the highest number of stimulating DCs (1x10^) on day 4, where they were more stimulatory than uninfected or Ad-GFP infected DCs and on day 5 when 2x10^ stimulating DCs were present, where there was a decrease in the abilty of Ad-CEI E2 infected DCs to stimulate T cell proliferation compared to the uninfected and Ad-GFP infected DCs. However, as these effects of the core protein are not consistently observed at any time point, it is unlikely that the core protein is affecting the ability of the DCs to stimulate T cell proliferation. To determine whether there may have been subtle effects on initial T cell activation that were not revealed by analysis of T cell proliferation after several days of culture, early T cell activation was also assessed by analysing T cell production of IL-2 on days 1 and 2 following stimulation with allogenic DCs. The kinetics of upregulation of CD69 and CD25 on T cells in response to allogenic stimulation was also analysed. The production of IL-2 by T cells was measured from culture supernatants by ELISA. As shown in figure 4.16, co-culture of T cells with uninfected allogenic DCs stimulated IL-2 218 (a) 160000 ^ 140000 -i I 120000 - U 100000 - • Uninfected ro 80000 - .Ad-GFP I 60000 - . Ad-CEI E2 40000 - 20000 - 0 . . . ^ ^ \+ «o'- N't- <5^ <5'- n+ DC Number (b) 160000 1 _ 140000 - £ 120000 - (-) 100000 - • Uninfected § 80000 - • Ad-GFP 2 60000 - . Ad-CEI E2 40000 - 20000 - 0 ^ ^ ^ ^ ^ ^ N+ if «ÿ- if \f if (C) 160000 1 _ 140000 t 120000 - • Uninfected 100000 - 1 80000 • Ad-GFP 2 60000 • Ad-CEI E2 40000 20000 0 \+ n::f- <5^ N+ <5^ \+ T+ «dF DC Number Figure 4.15. Determination of the allostimuiatory capacity of uninfected, Ad-GFP & Ad-CE1E2 infected DCs by measurement of CDS* T cell proliferation. Uninfected, Ad-GFP & Ad-CE1E2 infected (MOI 200) DCs were incubated at a range of concentrations with 1x10® CDS* T cells. Cells were pulsed with ®H-thymidine on days (a) 3, (b) 4 and (c) 5, harvested and proliferation measured in counts per minute (cpm). The data shown is representative of 4 individual experiments. Statistical analysis of variance was performed with a one-way ANOVA and Tu key's pairwise comparison. A significant difference (p<0.05) between uninfected DCs and adenovirus infected (Ad-GFP or Ad-CEIE2) DCs is indicatedby*. A significant difference between Ad-GFP and Ad-CEI E2 infected DCs is indicated by A. 219 Day 1 150 I 100 I Uninfected I I Ad-G FP M 50 I A d-C E I E2 0 I 1x10e3 1x10e4 1x10e5 DC Num ber Day 2 140 . I 90 I □ Uninfected I □ Ad-G FP 21 40 - ■ A d-CEI E2 -10 1x10e3 1x10e4 1x10e5 DC Num ber Figure 4.16. Determination of the allostimuiatory capacity of uninfected, Ad-GFP & Ad-CEIE2 infected DCs by measurement of IL-2 production. 1x10® CDS'" T cells were stimulated by culture with uninfected, Ad-GFP or Ad-CEI E2 infected allogenic DCs at the indicated numbers per well. Supernatants were harvested on day 1 and day 2 and assayed for IL-2 content by ELISA. The data shown is representative of 3 individual experiments. 220 production in a dose-dependent fashion. Supernatant IL-2 levels did not increase between days 1 and 2, likely because the T cells were using as well as producing IL-2. Adenovirus infection of DCs did not alter their stimulation of IL-2 production by allogenic T cells; and expression of the HCV structural proteins in DCs also had no major effect on their ability to stimulate IL-2 production by T cells. Although there appears to be a lower level of IL-2 in the supernatant of T cell-Ad-CE1E2 DC co cultures than that of T cell-uninfected DC co-cultures on day 1 in the experiment depicted in figure 4.16, this effect was not reproduced in other experiments. Expression of the T cell activation markers CD69 and CD25 was measured on T cells co-cultured with uninfected, Ad-GFP and Ad-CE1E2 infected DCs by flow cytometry after 1, 2 and 3 days of incubation. The percentage of CD4+ and CD8+ cells expressing the early T cell activation marker CD69 (figure 4.17) and the level of expression of CD25 (IL-2 receptor) on CD4* and CD8+ T cells (figure 4.18) increased as the T cells were co-cultured with increasing numbers of allogenic DCs, and with increasing length of culture. Adenovirus infection of DCs and expression of the HCV structural proteins in DCs did not alter their ability to induce T cell activation marker expression. In conclusion, adenovirus infection and the expression of the HCV core protein did not alter the ability of DCs to stimulate allogenic immune responses as measured by T cell proliferation, IL-2 production and T cell activation marker expression. A further series of experiments were carried out to address the effects of expression of the HCV core protein in DCs on their ability to stimulate antigen-specific CD4* and CD8* T cell responses. EJIVR blood donors were initially screened for reactivity various antigens (by IFNy ELISPOT assay). DCs were then generated from donors who had strong responses to specific antigens, and T cells isolated from the same donor the following week to allow the ability of these DCs (uninfected or infected with 221 CD4+ CD8* 50 . 4 0 - t 20 4 Is., B U ninfected I U ninfected Day 1 8 ■ A d -G F P lA d -G F P I 20 4 B A d -C E 1 E 2 |A d -C E 1 E 2 i - m r r n 1x10e3 1x10e4 1x10e3 1x10e4 D C N u m b e r D C N u m b e r 50 - 4 0 1 I U ninfected 5 30 □ Uninfected Day 2 lA d -G F P □ A d -G F P Lo |A d -C E 1 E 2 ■ Ad-CE1E2 LŒD Jld ItB 1. Ix10e3 1x10e4 1x10e3 1x1064 D C N u m b e r D C N u m b e r 5 0 ] 4 0 4 1 ? 1 30 i □ Uninfected □ Uninfected Day 3 □ A d -G F P □ A d -G F P 20 i I □ Ad-C E1E2 □ Ad-CE1E2 oi m D 1x10e3 1x10 64 0 IxlOeS 1x1064 D C N u m b e r D C N u m b e r Figure 4.17. Determination of the aliostimulatory capacity of uninfected, Ad-GFP & Ad- CEI E2 infected DCs by measurement of T cell activation marker expression. 1x10® CDS* T cells were stimulated by culture with uninfected, Ad-GFP or Ad-CEI E2 infected allogenic DCs at the indicated numbers per well. Cells were stained with antibodies against CDS & CD4 or CDS & CDS plus CD69 on days 1, 2 and S and analysed by flow cytometry. The percentage of CD69* CD4* and CDS* I cells are shown. The data shown is representative of S individual experiments. 222 CD4+ CD8^ 6 0 1 50 1 ■ Uninfected I Uninfected I 4 0 H ■ A d -G F P Day 1 I A d -G F P 3 0 . ■ Ad-CE1E2 |A d -C E 1 E 2 20 j 10 J nniin 1x10e3 1x10e4 1x10 63 1x10@4 D C N u m b e r D C N u m b e r 40 35 3 0 □ U ninfected 2 5 i □ Uninfected Day 2 □ A d -G F P 20 ■ A d -G F P ■ Ad-C E1E2 15 ■ Ad-CE1E2 10 5 0 1x10e3 1x10e4 1x10e3 1x10e4 D C N u m b e r D C N u m b e r 4 0 . 35 30 I 2 5 ^ I U ninfected Day 3 20 i lA d -G F P 15 ^ |A d -C E 1 E 2 10 5 0 1x10e3 1x10e4 1x10 63 1x10e4 D C N u m b e r D C N u m b e r Figure 4.18. Determination of the allostimuiatory capacity of uninfected, Ad-GFP & Ad-CEI E2 infected DCs by measurement of T cell activation marker expression. 1x10® CD3^ T cells were stimulated by culture with uninfected, Ad-GFP or Ad-CEIE2 infected allogenic DCs at the indicated numbers per well. Cells were stained with antibodies against CDS & CD4 or CDS & CDS plus CD25 on days 1, 2 and S and analysed by flow cytometry. The mean fluorescence intensity (MFI) of CD25^ CD4* and CDS* I cells is shown. The data shown is representative of S individual experiments. 223 Ad-GFP or Ad-CE1E2) to stimulate syngenic antigen-specific T cell responses to be measured. The ability of uninfected, Ad-GFP and Ad-CE1E2 infected DCs to stimulate CD4* T cell responses to PPD (purified protein derivative from M. tuberculosis', the majority of BCG vaccinated individuals will respond to this antigen) was initially addressed using IFNy ELISPOT assays to read out CD4* T cell responses. As shown in figure 4.19 uninfected DCs stimulated CD4* T cells to produce IFNy in a PPD-specific fashion; but adenovirus-infected DCs stimulated a large number of CD4^ T cells to produce IFNy when PPD was absent from the cultures, with only a small PPD-specific response being apparent above this “background” response. The “background” response may have been due to CD4* T cell production of IFNy in response to recognition of adenoviral antigens, and/or to adenovirus infection activating DCs to stimulate CD4* T cells to produce IFNy in an antigen-independent fashion. As non-specific T cell production of cytokines may be more readily triggered than T cell proliferation, proliferation assays were used as an alternative read out of CD4* T cell responses in an attempt to overcome the problem of high “background” in the IFNy ELISPOT assay. As shown in figure 4.20, uninfected DCs stimulated some proliferation of CD4* T cells in the absence of PPD, but a PPD-specific CD4* T cell response was apparent above this. Neither adenovirus infection of DCs nor the expression of the HCV structural proteins within them had an effect on the ability of DCs to stimulate antigen-specific CD4'" T cell responses. The ability of uninfected, Ad-GFP and Ad-CEI E2 infected DCs to stimulate CD8* T cells to respond to a HLA-A2-restricted CMV epitope was assessed by IFNy ELISPOT assay. Purified CD8* T cells from a HLA-A2 positive donor known to recognise this CMV epitope were stimulated by culture with syngenic DCs in the presence or absence 224 700 _ 600 I 500 S Uninfected Without PPD I □ Uninfected With PPD a 400 ISAd-GFP Without PPD BAd-GFP With PPD Z 300 c SAd-CE1E2 Without PPD (0 ® 200 ■ Ad-CE1E2 With PPD 100 5x10' 2x10' 1x10' DC Number Figure 4.19. Comparison of the ability of uninfected, Ad-GFP & Ad-CE1E2 infected DCs to stimulate CD4"^ T cell responses to PPD, as measured by an IFNy ELISPOT assay. 1x10^ purified CD4* T cells were stimulated by culture with the indicated number of uninfected, Ad-GFP or Ad-CE1E2 infected DCs in the presence (solid bars) or absence (striped bars) of 5pg/ml of PPD for 24 hours. The number of cells stimulated to produce IFNy spots was determined by ELISPOT assay. The results shown are the mean (of duplicate wells) number of cells/well stimulated to produce IFNy (the number of IFNy spots), i.e. the number of responding cells per 10^ CD4* T cells. The results shown are representative of 3 individual experiments. 225 90000 1 80000 - 70000 - S 60000 - 0 50000 - - - Uninfected No PPD 1 40000 ♦ Uninfected With PPD - -àr - Ad-GFP No PPD I 30000 - — &— Ad-GFP With PPD 20000 - - - Ad-CE1 E2 No PPD — Ad-CE1 E2 With PPD 10000 - 0 1 r 1 1 1 1 1 1 1x10= 2x10^ 5x10' 1x10" 2x10" 5x10" 1x10* 2x10* DC Number Figure 4.20. Comparison of the ability of uninfected, Ad-GFP & Ad-CE1E2 infected DCs to stimulate CD4* T cell responses to PPD, as measured by a proliferation assay. 1.6x10^ purified CD4* T cells were stimulated by culture with the indicated number of uninfected, Ad-GFP or Ad-CE1E2 infected DCs in the presence (solid lines) or absence (dashed lines) of Spg/ml of PPD. Cells were pulsed with ^H-thymidine on day 5 and harvested 24 hours later for analysis of "H-thymidine incorporation. The results shown are the mean (of triplicate wells) counts per minute (cpm). The data shown is representative of 3 individual experiments. 226 of 10'^M CMV peptide for 24 hours and the number of cells producing IFNy spots was determined. As shown in figure 4.21, there was little or no production of IFNy by CD8" T cells cultured in the presence or absence of DCs in the absence of the CMV peptide. Addition of the CMV peptide to T cells alone led to the production of IFNy by a small number of cells, which may have been due to the T cells presenting the peptide to one and another. The number of responding cells increased when DCs were added. Adenovirus infection and the expression of the HCV structural proteins within the DCs did not alter their ability to stimulate peptide-specific CD8* T cell production of IFNy. In conclusion, these results suggested that the ability of DCs to stimulate both CD4* and CD8* antigen-specific T cell responses was not altered by adenovirus infection or the expression of the HCV core protein within these cells. 4.8. Discussion This chapter presents results from experiments addressing the effects of expression of the HCV core protein (together with El and E2) in DCs on their activation in response to different stimuli and their ability to stimulate T cell responses. Adenoviral vectors were chosen as a means of achieving core protein expression within DCs. In line with expectations based on reports in the literature, it was found that a high proportion of DCs could be transduced using adenoviral vectors, with minimal effects on DC activation and functions. Expression of the HCV structural proteins in DCs was not found to have any effect on their activation and functions in any of the assay systems used here. This was surprising considering the prior observations in the literature and recent reports suggesting that the core protein does have immunomodulatory effects on DCs. Possible explanations for this are discussed below. 227 250 I 200 - ■ B B 0 Uninfected No Peptide •g B B B ■ Uninfected With Peptide I I I 0 Ad-GFP No Peptide 0 I I I □ Ad-GFP With Peptide c ■ ■ ■ I I I SAd-CE1E2 No Peptide 1 50 ■ ■ ■ ■ III III ■Ad-CE1E2 With Peptide 0 lUiJiJLl 0 1x10^ 1x10^ 1x10' DC Number Figure 4.21. Comparison of the ability of uninfected, Ad-GFP & Ad-CE1E2 infected DCs to stimulate CDS"^ T cell responses to a CMV epitope, as measured by an IFNy ELISPOT assay. 5x10* purified CD8'' T cells were stimulated by uninfected, Ad-GFP infected or Ad-CE1E2 infected DCs in the presence (solid bars) or absence (open bars) of 10'^ M CMV peptide (NLVPMVATV) for 24 hours. The number of cells stimulated to produce IFNy spots was determined by ELISPOT assay. The results shown are the mean (of duplicate wells) number of cells/well stimulated to produce IFNy (the number of IFNy spots), i.e. the number of responding cells per 5x10* CD8* T cells. The results shown are representative of 3 individual experiments. 228 I found that high levels of gene expression could be achieved in a high proportion of monocyte-derived DCs using these vectors (70-80% of transduced cells being GFP positive at a MOI of 200 after 24 hours of infection with Ad-GFP). There was some donor-donor variation seen in the efficiency of transduction of DCs. This may be due to the levels of expression of the receptors adenoviruses use to infect cells. Initial attachment of adenovirus to host cells is via high affinity binding of the adenovirus capsid protein to the coxsackie virus receptor (Bergelson et al., 1997; Hidaka et al., 1999). Internalisation of adenoviral particles occurs by receptor mediated endocytosis, involving cellular aV(33 or aVpS integrins (Wickham et al., 1993). Other molecules may also act as receptors for adenovirus entry into host cells, including MHC molecules (Hong et al., 1997) and integrins on the surface of cells of a haematopoietic origin (Huang et al., 1996). Therefore any differences in the levels of expression of these receptors between donors will influence the ability of adenoviruses to infect their cells. Most studies in the literature agree with my findings that DCs are efficiently transduced by adenoviral vectors (Dietz and Vuk-Pavlovic, 1998; Jonuleit et al., 2000; Rouard et al., 2000; Zhong et al., 1999), but not all (Rea et al., 1999). I chose to use a MOI of 200 for infection of DCs, as this gave a high level of transduced cells without detrimental effects on the viability of the cells. I found that infection at higher MOIs resulted in a decrease in viability (assessed by changes in forward and side scatter (representing cell size and granularity) by flow cytometry). This was probably not due to the adenovirus itself, but is more likely to be due to residual caesium chloride in the purified virus preparations. Steps were taken to reduce the levels of caesium chloride following gradient purification, by increasing the period of dialysis with more frequent changes of dialysis buffer in order to remove as much caesium chloride as possible without reducing the titre of the virus. I found that a MOI of 200 routinely gave greater than 80% of DCs expressing GFP; in retrospect it may have been better to use a higher MOI to give higher levels of protein 229 expression. This would have also made me more confident that I was transducing DCs with Ad-CE1E2. The drawback of this study was that this adenovirus did not co express GFP, so I could not check the transduction efficiency in each experiment. This problem would be overcome by the use of the adenoviruses I generated, as they co express the core protein and GFP. I found that adenovirus infection resulted in some activation of DCs (shown by the slight increase in CD83, CD80 and CD86 expression). This also varied with donors, whose baseline activation state and/or responsiveness to maturation stimuli varied. Therefore to be certain of effects, all assays were repeated a minimum of three times with different donor DCs. This mild activation of DCs agreed with other studies which have observed either no effects (Jonuleit et al., 2000; Zhong et al., 1999) or mild activation as I did (Rea et al., 1999; Rouard et al., 2000). Studies which did observe effects of the adenoviral vector on DC activation (Hirschowitz et al., 2000; Korst et al., 2002; Miller et al., 2002; Morrelli et al., 2000) were carried out in murine DCs. The difference between these studies could be due to variation between species. The only major effect of adenovirus infection on DCs that I saw was a dramatic decrease in IL- 12 production in response to TNFa. Adenovirus infection did not block IL-12 production by DCs (I actually saw an increase in IL-12 production in response to LPS and anti-CD40 stimulation, as did Rea et al (Rea et al., 1999), although they also saw an increase in CD83 expression); the impairment was only in response to TNFa. This may be due to the adenoviral vectors not having the entire E3 region deleted from the genome. The adenoviral E3 region contains immunomodulatory genes which target the TNFa pathway for activation and apoptosis (Burgert et al., 2002; Wold et al., 1999). These genes encode three proteins, which are alternatively spliced during transcription. They are the E3-10.4K, E3-14.5K and E3-14.7K proteins, which protect adenovirally infected cells from TNF-mediated cytolysis (Gooding et al., 1988; Gooding et al., 1991; 230 Horton et al., 1991). Ad-CE1E2 and Ad-GFP both have deletions in the E3 region of the adenoviral genome which disrupts the coding sequences for these three genes (Cladaras and Wold, 1985), therefore it was surprising that I saw an effect of the adenovirus on the response to TNFa, potentially the deletion in the E3 region may not have been complete in these vectors resulting in one of the proteins still being produced and exerting effects on the cells infected with these vectors. Overall, the effects of adenovirus on DCs were minimal and should not have masked any effects of the HCV proteins expressed within DCs. My findings do not support the hypothesis that the HCV core protein has immunomodulatory effects on DCs. I did not see any effects of expression of the HCV structural proteins in DCs on their ability to take up antigen, their phenotypic maturation and cytokine production in response to different stimuli, or their ability to stimulate T cell responses, either allogenic or antigen-specific. This is in contrast to findings made in recent studies that have also addressed immunomodulatory effects of the HCV core protein. A number of studies have been carried out to address this hypothesis in both murine (Hiasa et al., 1998; Kim et al., 2002a; Large et al., 1999; Liu et al., 2002) and human (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Dolganiuc et al., 2003; Kanto et al., 1999; Sarobe et al., 2002) systems. A variety of cells including monocyte-derived DCs (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Kanto et al., 1999; Sarobe et al., 2002), bone marrow-derived DCs (Hiasa et al., 1998; Kim et al., 2002a) and cell lines (Lee et al., 2001) have been used, as have different systems for expressing the core protein (either alone or in combination with the envelope proteins) within cells (Dolganiuc et al., 2003; Hiasa et al., 1998; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002). There has been some variation in the results, but overall a remarkably consistent message comes across; there is no effect on the expression of MHC and 231 co-stimulatory molecules (Dolganiuc et al., 2003; Hiasa et al., 1998; Kim et al., 2002a; Sarobe et al., 2002). No effects have been seen on the phenotypic maturation in response to LPS (Dolganiuc et al., 2003; Sarobe et al., 2002) or to TNFa (Sarobe et al., 2002), although only one study has looked at responses to TNFa and the results are not shown. No studies have looked at the maturation of DCs in response to other stimuli, such as CD40, and it is still unclear whether the core protein can affect this pathway. However, despite these studies with no effects on the phenotype of DCs, a consistent decrease in IL-12 production has been observed in response to a range of stimuli including; LPS, TNFa, Staphylococcus aureas and IFNy (Auffermann- Gretzinger et al., 2001; Bain et al., 2001; Hiasa et al., 1998; Kanto et al., 1999; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002) and a decrease in the allostimulatory capacity of DCs has been observed (Hiasa et al., 1998; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002), which in the study by Dolanguic et a! (Dolganiuc et al., 2003) was associated with a decrease in IL-12 and an increase in IL-10 production by T cells. Overall, these results suggest that the core protein may impair the ability of DCs to induce and maintain T cell responses. Further evidence for this comes from the study by Large et a! (Large et al., 1999), who found the core protein when expressed in a vaccinia virus construct, inhibited the induction of vaccinia virus specific CTL in mice, resulting in death of the mice. Liu et a! (Liu et al., 2002) in contrast did not see any effect on virus-specific CTL responses and liver damage. The differences between these studies may be due to the genotype of the core protein used. In the studies with no effect, genotype 1b core protein was used and other studies used a genotype la core protein. Studies have also shown that DCs from chronically infected HCV patients have an impaired ability to stimulate T cell responses and have a reduced capacity to produce IL-12 (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Kakumu et al., 2000; 232 Kanto et al., 1999). it is possible that the DCs are infected with HCV, so there is a potential role of the core protein in impairing DC functions. The apparent defect in DC function may be contributing to the lack of maintenance of strong HCV-specific I cell responses in the chronic phase of infection. There is a consensus in the literature that the expression of the core protein within DCs impairs IL-12 production and their T cell stimulatory capacity, which may be of in vivo importance. The mechanisms involved in these effects are unclear. Potentially, signalling through molecules such as CD40 may be impaired, although this has not been directly addressed by the studies in the literature and it is unclear whether the core protein can bind to CD40. Alternatively, core may be mediating effects on downstream signalling components involved in DC activation, for example, NFkB. The core protein may also be affecting DC functions by having effects within the cell nucleus, such as activating or suppressing promoters. However, I did not see any effects of the core protein on DC activation or functions. The lack of effects of the core protein in my studies may be due to the fact that I used an adenovirus expressing the envelope glycoproteins as well as the core protein (Ad- CE1E2). The presence of the envelope glycoprotein may alter the way the core protein interacts with host cell components and could also affect the form the core protein is expressed in, as the core protein may act differently when it is independent of the envelope glycoproteins. However, it could be argued that the association of the core protein with the envelope glycoproteins is more physiologically relevant than expressing each protein individually, as HCV virions will contain the core protein and envelope glycoproteins in association with each other, as has been determined by the production of virus-like particles (Baumert et al., 1999). However, I feel that this is unlikely to be the reason as other studies in the literature also had the core protein expressed in the presence of either El alone or El and E2 (Hiasa et al., 1998; Kim et 233 al., 2002a; Sarobe et al., 2002). It could also be due to the genotype of the core protein, as difference have been observed between genotypes, however the core protein I used is of genotype 1a, which is the same genotype used in studies where effects have been observed (Auffermann-Gretzinger et al., 2001; Lee et al., 2001). It may be possible that I was not efficiently transducing cells with Ad-CE1E2, so not all cells may have been expressing the core protein. I could not check directly the levels of transduction with Ad-CE1E2 as this virus did not co-express the marker GFP, however by comparison with Ad-GFP it was expected to have approximately 80% of cells transduced. The confocal experiments described in chapter 3 confirmed that the core protein was expressed within DCs infected with Ad-CE1E2 at fairly high levels, but conceivably the levels of expression could be lower than those achieved with Ad-GFP. If less than 50% of cells were transduced with Ad-CE1E2, then in assays such as T cell stimulation and cytokine production, minor effects of the core protein may not be apparent as the untransduced cells could mask any effects on the cells expressing the HCV proteins. This problem could be overcome by the use of the adenoviral vectors I constructed, as they co-express GFP and the core protein, transduced cells could be selected by sorting so only cells expressing the core protein would be present in assays. Potentially the levels of the core protein expression by Ad-CE1E2 transduced cells may not have been high enough to exert effects on the cells. The experiments looking at expression of GFP by Ad-CE1E2 infected DCs suggest that as a higher MOI is used, the levels of GFP expression are higher as the MFI of GFP increased. So if the levels of transduction were low with Ad-CE1E2 this could potentially be a problem. The confocal experiments described in chapter 3 did show the core protein was expressed; however the majority of the protein appeared to be in the cell nucleus, with lower amounts in the cytoplasm. Therefore the location of the core protein within the cells could also affect its actions. In future experiments, a higher MOI could be used to achieve higher levels of core expression within transduced cells. Also, the use of the 234 adenovirus I generated which expresses the mutated version of the core protein (Ad- Core-124) may give higher levels of core protein expression, as this version of the core protein is not cleaved at a maturation site, which results in over expression of the protein. Future experiments could address the recent reports in the literature that the core protein has immunomodulatory effects. It would be interesting to look at effects on the T cell response further and to try and identify the mechanisms in which core can impair the ability of DCs to stimulate T cell responses. It would be interesting to explore further the effects the core protein may have on DC stimulation of T cells through the CD40 pathway and activation pathways such as NFkB, as this area has not been studied in much detail in HCV infection. The use of the recombinant adenoviruses I generated would allow the regions of the core protein involved in immunomodulatory effects on DCs to be determined. This would allow a greater insight into the role the core protein may be playing in the establishment of persistent infection in the majority of people infected with HCV. 235 Chapter 5 - Effects of the Hepatitis 0 Virus Core Protein on the Susceptibiiity of DCs to Induction of Apoptosis via Different Pathways 5.1 introduction As described previously (chapter 1 and chapter 4), there is evidence to suggest that HCV infects cells within lymphoid tissues, principally antigen presenting cells (DCs, macrophages and B cells), raising the possibility that expression of viral proteins within these cells may affect their activation, function or lifespan in ways that lead to impaired control of HCV replication by the host immune response and promote viral persistence. In the previous chapter, the hypothesis that the expression of the HCV core protein within DCs may affect their activation and functions (and hence the induction and maintenance of antiviral immune responses) was investigated. This chapter addresses whether expression of the HCV core protein within DCs (and also macrophages) may affect their sensitivity to apoptosis-inducing stimuli, and how this may impact on in vivo control of viral replication. There are several pathways which all lead to the induction of apoptosis within cells; these are shown in figure 5.1. There is an intrinsic pathway of apoptosis (suicide pathway) which is involved in the development of many organ systems (especially the nervous system) as well as eliminating cells which have developed abnormally or that have genetic errors. This pathway is triggered by a lack of trophic receptor stimulation; for example, the loss of growth factors, DMA damage or inappropriate loss of contact with neighbouring cells. This results in the loss of mitochondrial function which then releases cytochrome c, which activates the “apoptosome” (a complex of Apaf-1 and caspase-9) resulting in further activation of the caspase cascade that ultimately results in DMA fragmentation and apoptosis. 236 NK CTL LTa1p2 INTRISIC PATHWAY TRAIL Loss of growth factors Fas-L perforin & granzymes TNFa LTa2p1 DMA damage Fas DR5 Loss of cell-cell TNF R1 LTa3 contact, LTpR DD DD DD FADD FADD FADD Grb Cytochrome C Granzyme B Apoptosome Procaspases Caspase Cascade APOPTOSIS Figure 5.1. A schematic outline of apoptosis pathways. Apoptosis of cells can be induced via several pathways. The intrinsic pathway is induced by the lack of growth factors or cell-cell contact, or by DNA damage, which results in a loss of mitochondrial function causing the release of cytochrome c. This activates the apoptosome, resulting in activation of the caspase cascade that leads to apoptosis. Fas-Ligand binds to Fas, TRAIL binds to the receptor DR5, and TNFa, LTa1(32 or LTa2p1 bind to the TNFR-1. These all recruit an adapter protein FADD, which binds to death domains (DD) on these receptors. This leads to the activation of procaspases that initiate a caspase cascade which results in apoptosis of the target cell. LTa3 binds to the LTpR which recruits adapter molecules belonging to the TRAF family which initiate apoptosis within the cell. NK cells and CTL release perforin which forms a pore in the target cell membrane for the entry of granzymes. These activate an adapter protein called Grb which activates granzyme B, leading to DNA fragmentation and apoptosis of the target cell. 238 CTL lysis can also occur in a calcium-dependent, contact-dependent manner, by perforin and granzymes (Russel and Ley, 2002). Once the CTL has identified the target cell by cell-cell contact, signals are induced which lead to the release of granules containing perforin and granzymes. Perforin is polymerised in a calcium-dependant manner and enters the target cell membrane, creating a passage for granzymes to enter the target cell. The granzymes, particularly granzyme B, cleave proteins within the target cell resulting in the activation of caspases involved in the apoptotic pathways, ultimately resulting in lysis of the target cell. NK cells also use the perforin/granzyme pathway to mediate lysis of virally infected cells. The Fas-Fas Ligand pathway of apoptosis is a calcium-independent, contact- dependent process (Rouvier et al., 1993). Fas, expressed by the target cell has death domain (DD) regions intracellularly. Upon ligation with Fas-ligand expressed by CTL, reformation of a death-inducing signalling complex (DISC) occurs. This complex contains adapter proteins such as FADD (Fas-associated death domain protein), which activate caspases (in particular, caspase-8) in a cascade that results in the induction of apoptosis within the target cell (details of the pathway are in Wajant, 2002). Apoptosis may also be induced within target cells by other pathways involving members of the TNF receptor superfamily, such as TRAIL (TNF related apoptosis inducing ligand), lymphotoxin (LTaS, LTa1p2 or LTa2p1) and TNFa. These all act in a similar way to induce apoptosis within target cells. Binding of the ligand by an appropriate receptor (for example, TRAIL binds to DR5, LTaS binds to the LTpR, LTaip2, LTa2pi and TNFa all bind to the TNF-R1) results in the recruitment of adapter proteins which recognise death domain regions of the receptors. This results in the activation of caspase cascades which induce apoptosis within the target cell. A 239 schematic overview of the pathways is shown in figure 5.1 (further details can be found in Abe et a!., 2000; Schulze-Osthoff et al., 1998). Previous studies have shown that the core protein can affect the susceptibility of cells to apoptosis. However, these studies have provided a confusing picture as they have used different forms and genotypes of the core protein in transformed cell lines to look at apoptosis in response to different stimuli. Some studies report an increase, whilst others report a decrease in sensitivity to apoptosis. It has been shown that the core protein can suppress TNFa mediated apoptosis in certain cell lines (Marusawa et al., 1999; Ray et al., 1998a; Shrivastava et al., 1998), whilst other groups have shown that the core protein can sensitise cells to apoptosis (Dumoulin et al., 1999; Ray et al., 1996; Zhu et al., 1998; Zhu et al., 2001). Core may also sensitise cells to Fas-mediated apoptosis (Hahn et al., 2000; Moorman et al., 2003; Ruggieri et al., 1997). Potential mechanisms that may be involved could be effects of the core protein on signalling through the TNFR family members (Zhu et al., 1998; Zhu et al., 2001), effects on downstream signalling events (Hahn et al., 2000; Machida et al., 2001; Otsuka et al., 2002) or effects in the cell nucleus (Alisi et al., 2003; Isoyama et al., 2002; Yamanaka et al., 2002). If the core protein either up- or down-regulated apoptosis, how could this contribute to viral persistence? Many viruses have strategies for blocking the “suicide” pathway of apoptosis and/or killing by various immune effector mechanisms in order to prolong their replication (see chapter 1). If the core protein is capable of mediating similar inhibitory effects on apoptosis, this would be most important in hepatocytes, which are the major site of HCV replication. Core may likewise also prevent or delay apoptosis in antigen presenting cells, although they are “naturally” relatively resistant to lysis (Ashany et al., 1999; Funk et al., 2000; Medema et al., 2001). Conversely, the core protein might increase the susceptibility of antigen presenting cells to lysis. If these 240 cells were rendered more susceptible to apoptosis via immune effector mechanisms, they might be destroyed and induction and maintenance of the immune response impaired, which would promote viral persistence. The aim of the work in this chapter was to express the core protein in primary cell types (DCs and macrophages), which may be more physiologically relevant than the transformed cell lines used in previous studies, and to investigate the effects this had on their susceptibility to lysis, especially lysis triggered by immune effector pathways. 5.2 Effects of the HCV core protein on apoptosis induced by the TNFR famiiy moiecuies Reports that the core protein can modulate the sensitivity of transformed cell lines to apoptosis induced by TNFR family molecules, through binding of the core protein to the cytoplasmic tail of these molecules and altering signalling pathways (Dumoulin et al., 1999; Hahn et al., 2000; Marusawa et al., 1999; Moorman et al., 2003; Ray et al., 1996; Ray et al., 1998a; Ruggieri et al., 1997; Shrivastava et al., 1998), prompted investigation of its effects on susceptibility of DCs and macrophages to apoptosis induced via the Fas-FasL pathway (triggered by an agonistic anti-Fas antibody), the TNF pathway (triggered by TNFa, recombinant lymphotoxin alpha 1 beta 2 and lymphotoxin alpha 2 betal) including the TRAIL pathway (triggered by recombinant TRAIL, TRAIL receptors 1 and 2). As DCs and macrophages may be relatively resistant to killing via these pathways, initial experiments were carried out with Jurkat cells (a human lymphoblastoid T cell line), which were known to be susceptible to apoptosis induced via an agonistic anti- Fas antibody (Peter et al., 1995). Jurkat cells were efficiently infected by adenovirus. 241 with 83% of cells expressing GFP 24 hours after infection at MOI 200 with Ad-GFP (data not shown). Jurkat cells were infected with Ad-GFP or Ad-CE1E2 for 24 hours before stimulation with a range of concentrations of an agonistic anti-Fas antibody, recombinant TRAIL and recombinant lymphotoxin (LTaip2 and LTa2p1) for a further 24 hours. Cells then were stained with annexin V to determine the percentage of apoptotic cells by flow cytometry. Annexin V binds to phosphatidyl-serine which becomes exposed as the cell membranes are reversed during death of the cells. As can be seen in figure 5.2, there was a dose-dependant induction of apoptosis in response to stimulation with the agonistic anti-Fas antibody, as the percentage of annexin V positive cells increases with the concentration of the agonistic anti-Fas antibody. Adenovirus infection did not alter the susceptibility of the cells to apoptosis induced via this pathway. The expression of the HCV core protein by Ad-CE1E2 has also not altered the susceptibility of Jurkat cells to apoptosis. Jurkat cells were also susceptible to apoptosis induced by TRAIL, as can be seen in figure 5.3. There is a dose-dependant induction of apoptosis, and adenovirus infection or the expression of the core protein has not altered the susceptibility of the cells to death. However, the Jurkat cells were resistant to apoptosis induced via lymphotoxin (both LTa1p2 and LTa2p1), as can be seen in figure 5.4. Again, adenovirus infection or the expression of the core protein by Ad-CE1E2 infection did not alter their susceptibility to apoptosis. Similar experiments were then carried out using two hepatoma cell lines; Hep16 and Hep3B (liver hepatocellular carcinoma cell lines). Previous studies using similar hepatoma cell lines, such as HepG2, as well as Hep3B cells had found contrasting effects of the core protein on susceptibility of these cell lines to apoptosis. Some found core sensitised cells to apoptosis (Kalkeri et al., 2001; Ruggieri et al., 1997; Yoshida et al., 2001), whilst others found it inhibited apoptosis (Otsuka et al., 2002; Yang et al., 2002). 242 (a) 100 80 > 60 □ Uninfected (/> o □ Ad-GFP Q. 40 ■ Ad-CE1E2 20 0 m 0 0.1 1 10 100 1000 Agonistic anti-Fas Ab Cone (ng/ml) (b) At Ong/m of an agonistic anti-Fas Ab FSC-H FL-2 lOOOng/m of an agonistic anti-Fas Ab FSC-H FL-2 Figure 5.2. Induction of apoptosis in Jurkat cells by an agonistic anti-Fas antibody, measured by annexin V staining. 1x1 OG uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) Jurkat cells were stimulated for 24 hours with a range of agonistic anti-Fas Ab concentrations (0- lOOOng/ml). Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. Part (a) shows the percentage of cells judged to be apoptotic by this method. Part (b) shows the forward/side scatter (FSC-H/SSC- H) and annexin V fluorescence (measured in the FL-2 channel) of uninfected Jurkat cells stimulated with Ong/ml or lOOOng/ml of an agonistic anti-Fas antibody, to illustrate examples of the results obtained. The data shown is representative of results obtained in 3 independent experiments. 243 100 80 □ Uninfected I4-* 60 ■ Ad-GFP I 40 ■ Ad-CE1E2 20 0 0 1000 10 100 500 Fas rTRAIL Cone, (ng/ml) Figure 5.3. Induction of apoptosis in Jurkat cells by TRAIL, measured by annexin V staining. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) Jurkat cells were stimulated for 24 hours with a range of recombinant TRAIL concentrations (0-500ng/ml), or lOOOng/ml of an agonistic anti-Fas antibody (Fas) as a positive control. Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 244 (a) 1 0 0 , 80 i I 60 - Uninfected CL Ad-GFP 40 - I Ad-CE1E2 20 - _ _ _ M- 0 1000 Fas 10 100 500 Lymphotoxin alpa2 betal Cone, (ng/ml) Ong/ml LTa2pi 500ng/ml LTa2pi 10® 1 0 ' 10- 1 0 * 1 0 * 10® 1 0 ’ 1 0 “ 1 0 * 10 FL2-H FL2-H (b) 100 - I Uninfected o I Ad-GFP Q. I Ad-CE1E2 rn ril 0 1000 Fas 10100 500 Lymphotoxin alphal beta2 Cone, (ng/ml) Ong/ml LTa2pi 500ng/ml LTa1p2 // : 10 1 0 “ 1 0 ' 1 0 “ 1 0 ’ 1 0 * FL2-H Figure 5.4. Induction of apoptosis in Jurkat cells by LTa2p1 & LTaip2, measured by annexin V staining. 1x1Q6 uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) Jurkat cells were stimulated with a range of concentrations of (a) lymphotoxin a2pi and (b) lymphotoxin aip2 or with 1000ng/ml of an agonistic anti-Fas antibody (Fas) as a positive control, for 24 hours. Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. Examples of the forwards/side scatter (FSC- H/SSC-H) and annexin V staining (in the FL-2 channel), of cells stimulated with Ong/ml or 500ng/ml of LTa2pi (part a) or LTa1p2 (part b) are also shown. The data shown is representative of results obtained in 3 independent experiments. 245 Herzer et al (Herzer et al., 2003) also found that the core protein altered the sensitivity of hepatoma cell lines to NK lysis. Both HepSB and He16 cells were efficiently infected by adenovirus with 80% of Hep16 and 89% of HepSB being GFP positive 24 hours following infection with Ad-GFP at a MOI of 100 (data not shown). I found that there was a higher spontaneous background apoptosis of HepSB and Hep16 cells than with the Jurkat cells; however neither cell line was susceptible to apoptosis induced by any of the ligands tested, as shown in figures 5.5 and 5.6. Infection with Ad-GFP appeared to have led to a reduction in the percentage of annexin V positive cells as compared to uninfected cells, but this is probably not the case. During acquisition of the samples by flow cytometry, problems were encountered with compensating between the FITC (GFP) and PE (annexin V- CyS) fluorescence. The GFP fluorescence was very bright and quenched the fluorescence in the PE channel, making detection of annexin V staining difficult, resulting in the lower levels of annexin V staining being detected. Expression of the HCV structural proteins in both Hep16 and HepSB cells did not alter their susceptibility to apoptosis induced via the Fas-Fas ligand, TRAIL or lymphotoxin pathways. What was of greatest interest to determine was whether the sensitivity of primary cells to apoptosis induced via different pathways was altered by the core protein. An initial series of experiments were carried out to look at the susceptibility of DCs to apoptosis induced via a variety of pathways. Uninfected DCs were stimulated for 24 hours with a range of concentrations of each apoptosis-inducing ligand and the percentage of apoptotic cells was determined by annexin V staining, assessed by flow cytometry. As can be seen in figure 5.7, DCs were relatively resistant to apoptosis induced by the agonistic anti-Fas antibody when compared to Jurkat cells. However, there is a slight increase in the percentage of apoptotic cells at the highest anti-Fas concentrations. Figure 5.8 shows that DCs are also resistant to apoptosis induced via the TRAIL 246 (a) (b) 100 1 80 - Uninfected > uninfected 5 60 - Ad-GFP (fl Ad-GFP g 40 - Ad-CE1E2 I Ad-CEI E2 20 i 0 1 J 0 10 100 500 0 10 100 500 Fas Cone, (ng/ml) TRAIL Cone, (ng/ml) (C) (d) 100 1 100 80 ' 80 - Uninfected e uninfected 1 60 60 i A d-GFP % □ Ad-GFP I 40 I §. Ad-CE1E2 40 55 Ad-CE1E2 20 , 20 - 0 ! UUUll 0 - 0 10 100 500 0 10 100 500 LTa2b1 Cone, (ng/ml) LTa1b2 Cone, (ng/ml) Figure 5.5. Induction of apoptosis In uninfected, Ad-GFP & Ad-CE1E2 Infected Hep16 cells measured by annexin V staining. 5x10^ uninfected, Ad-GFP or Ad-CE1E2 infected Hep16 cells were stimulated with a range of concentrations of (a) agonistic anti-Fas antibody, (b) TRAIL, (c) lymphotoxin a2p1 and (d) lymphotoxin aip2 for 24 hours. Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 247 (a) (b) 100 80 ^ Uninfected 60 -1 a Uhinfected A d-G FP 40 H n Ad-GFP I Ad-CE1E2 20 . ■ Ad-CE1E2 0 ; 10 100 500 0 10 100 500 Fas Cone, (ng/ml) TRAIL cone, (ng/ml) (C) (d) 100 80 100 Uninfected 60 80 Ad-G FP Uninfected 40 60 Ad-CE1E2 Ad-G FP 20 40 Ad-CE1E2 0 20 0 10 100 500 0 T 0 10 100 500 LTa2b1 Cone, (ng/ml) LTa1b2 Cone, (ng.ml) Figure 5.6. Induction of apoptosis in uninfected, Ad-GFP & Ad-CE1E2 infected HepSB ceiis measured by annexin V staining. 5x10^ uninfected, Ad-GFP or Ad-CE1E2 infected HepSB cells were stimulated with a range of concentrations of (a) agonistic anti-Fas antibody, (b) TRAIL, (c) lymphotoxin a2(31 and (d) lymphotoxin a1(32 for 24 hours. Apoptotic cells were detected by staining with annexin V-CyS, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 248 100 -1 80 - 60 - '(/) DC o CL Jurkat ^ 404 20 4 0 0 10 100 1000 Agonistic anti-Fas Ab Cone, (ng/ml) Figure 5.7. Induction of apoptosis in uninfected DCs by an agonistic anti-Fas antibody measured by annexin V staining. 1x10® DCs or Jurkat cells were stimulated for 24 hours with a range of concentrations of an agonistic anti-Fas antibody (0-1000ng/ml). Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 2 4 9 (a) 100 80 0) le o o CL 40 20 0 0.05 0.1 0.2 1 TRAIL R1 Cone, (ug/ml) (b) 100 80 0) :-E(/) 60 o 20 0 0 0.05 0.1 0.2 1 2 10 TRAIL R2 Gone, (ug/ml) (C) 100 -1 80 - 60 - DC 40 - • Jurkat 20 - 0 10 100 500 rTRAIL Cone, (ng/ml) Figure 5.8. Induction of apoptosis in uninfected DCs via the TRAIL pathway, measured by annexin V staining. 1x10® DCs were stimulated for 24 hours with a range of concentrations of (a) TRAIL R1 antibody (0-2ug/ml), (b) TRAIL R2 antibody (0-10ug/ml) or (c) recombinant TRAIL (0-500ng/ml). Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 250 pathway, as stimulation with recombinant TRAIL and with TRAIL receptor antibodies (TRAIL R1 and TRAIL R2) did not increase the percentage of annexin V positive cells. DCs were also found to be resistant to apoptosis induced by lymphotoxin and TNFa at the concentrations tested, as shown in figure 5.9. Experiments were then carried out to look at effects of adenovirus infection and expression of the core protein within DCs on their susceptibility to apoptosis induced via these pathways. As can be seen in figures 5.10 and 5.11 adenovirus infection of DCs and expression of the HCV core protein in them via Ad-CE1E2 did not increase their susceptibility to apoptosis induced via the Fas, TRAIL, TNFa or lymphotoxin pathways. Similar experiments were also carried out with monocyte-derived macrophages. As shown in figure 5.12, there was a slight induction of apoptosis in these cells in response to all four stimuli tested, but like DCs they were relatively resistant to apoptosis induced via these pathways, as shown in figure 5.12. Adenovirus infection appears to have increased slightly the levels of spontaneous apoptosis, but there was no effect of the core protein expressed by Ad-CE1E2 on the susceptibility of macrophages to apoptosis induced via any of the pathways tested. In conclusion, Jurkat cells were susceptible to apoptosis induced via the Fas and TRAIL pathways and resistant to apoptosis induced via the lymphotoxin pathway. Hepatoma cell lines were resistant to apoptosis induced by all the pathways tested. Finally, DCs and macrophages were relatively resistant to apoptosis induced via the Fas, TRAIL, TNFa and lymphotoxin pathways. Expression of the core protein did not alter the susceptibility of any of the cells tested to apoptosis induced via these pathways. 251 (a) 100 n 80 - Q) > ■(75 60 - O CL 4 0 - 20 - 0 - 50 200 5 00 (b) TNFa Conc. (ng/ml) 100 80 (D H 60 (/) DC O • Jurkat 20 0 0 10 100 500 Lymphotoxin alphal beta2 Conc. (ng/ml) (b) 100 n 80 - (U > 60 - ■(Ô DC o 0_ 40 - • Jurkat 20 - 0 10 100 500 Lymphotoxin alpha2 beta1 Conc. (ng/ml) Figure 5.9. Induction of apoptosis in uninfected DCs by TNFa, LTa1(32 & LTa2(31, measured by annexin V staining. 1x10® DCs or Jurkat cells were stimulated for 24 hours with a range of concentrations of (a) TNFa(0-500ng/ml), (b) Lymphotoxin a1(32 (0-500ng/ml) or (c) Lymphotoxin a2(31 (0-500ng/ml). Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 252 (a) 100 i 80 - ■ Uninfected 60 - □ Ad-GFP 40 : ■ Ad-CE1E2 20 : 0 0 10 100 1000 anti-Fas Ab Conc. (ng/ml) (b) 100 ^ 80 - 0) ■> 60 ■ Uninfected □ Ad-GFP 0- 40 ■ Ad-CE1E2 20 j UMU 10 100 1000 anti-Fas Ab Conc. (ng/ml) Figure 5.10. Effect of the HCV core protein on induction of apoptosis in immature & mature DCs by an agonistic anti-Fas antibody, measured by annexin V staining. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) (a) immature or (b) TNFa matured DCs were stimulated for 24 hours with a range of agonistic anti-Fas Ab concentrations (0-1000ng/ml), Apoptotic cells were detected by staining with annexin V- Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 4 independent experiments. 253 (a) 60 ■ Uninfected □ Ad-GFP □ A d-G FP ■ Ad-CE1E2 ■ Ad-CE1E2 AM 0 1000 5 50 200 500 10 100 500 Fas TNFa Conc. (ng/ml) rTRAIL Conc. (ng/ml) (c) (D 20 □ Uninfected □ Uninfected Ü3 15 ■ Ad-GFP □ A d-GFP ■ Ad-CE1E2 ■ Ad-CE1E2 10 100 500 Lymphotoxin alpha2 betal Conc. (ng/ml) Lymphotoxin alphal beta2 Conc. (ng/ml) Figure 5.11. Effect of the HCV core protein on induction of apoptosis of immature DCs by TNFa, TRAIL, LTo2pi & LTa1p2, measured by annexin V staining. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected (MOI 200) DCs were stimulated with a range of concentrations of (a) TNFa, (b) recombinant TRAIL, (c) lymphotoxin a2pi and (d) lymphotoxin a1p2 , or lOOOng/ml of an agonistic anti-Fas antibody (Fas) as a positive control for 24 hours. Apoptotic cells were detected by staining with annexin V-Cy3, which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 2 5 4 (a) (b) 100 ~ 100 - 0) 80 - ■ Uninfected (D 80 4 ;■! 60 - ■ Uninfected (fl □ Ad-GFP è 60 1 O 4 0 ^ CO □ A d-G FP ■ Ad-CE1E2 g 40^ ^ 20^ ■ Ad-CE1E2 0 ! rfirfiiïin 0 10 100 500 0 ^ 10 100 500 Fas Conc. (ng/ml) TRAIL Conc. (ng/ml) (c) (d) 100 100 9 80 0) 80 □ Uninfected □ Uninfected :■! 60 ■ Ad-GFP = 40 ■ A d-G FP ■ Ad-CE1E2 20 dl ■ Ad-CE1E2 10 100 500 0 10 100 500 LTa2b1 Conc. (ng/ml) LTa1b2 Conc. (ng/ml) Figure 5.12. Effect of the HCV core protein on induction of apoptosis in uninfected, Ad-GFP & Ad-CE1E2 infected monocyte-derived macrophages, measured by annexin V staining. 1x10® uninfected, Ad-GFP or Ad-CE1E2 infected macrophages were stimulated with a range of concentrations of (a) agonistic anti-Fas antibody, (b) TRAIL, (c) lymphotoxin a2p1 and (d) lymphotoxin a1p2 for 24 hours. Apoptotic cells were detected by staining with annexin V-Cy3 which was quantitated by flow cytometric analysis. The results shown are the percentage of cells judged to be apoptotic by this method. The data shown is representative of results obtained in 3 independent experiments. 255 5.3 Effect of the HCV core protein on apoptosis induced via the perforin & granzyme pathway Further experiments were carried out to iook at the susceptibility of DCs and macrophages to lysis by NK cells and CD8* cytotoxic T cells (CTL). These experiments aimed to address the sensitivity of DCs and macrophages to apoptosis induced by the perforin and granzyme pathways; as experiments described above had shown that these cells were relatively resistant to killing via the majority of the other pathways by which NK cells and CTL are known to mediate target cell lysis, any lysis seen in these experiments would therefore be likely to be mediated via perforin and granzymes. Whether the HCV core protein altered the susceptibility of DCs and macrophages to lysis by NK cells and CTL was also addressed. Initial NK cell lysis experiments were carried out using K562 cells, a cell line known to be a good target for NK cells (West et al., 1977). The effect of infection of these target cells with Ad-GFP or Ad-CE1E2 on their susceptibility to lysis by NK cells was assessed using a standard chromium release assay. K562 cells were efficiently infected by adenovirus, as 93% of cells were GFP positive after 24 hours of infection with Ad-GFP at MOI 200 (data not shown). An increasing percentage of specific chromium release (this represents cell lysis) was observed when K562 cells were incubated with increasing numbers of NK cells, as shown in figure 5.13. However, adenovirus infection (Ad-GFP) and expression of the core protein did not alter the susceptibility of K562 cells to NK lysis. Further experiments used hepatoma cell lines as NK cell targets. There have been reports in the literature suggesting that the hepatoma cell line Hep3B is more susceptible to NK cell lysis than the Hep16, as it expresses lower levels of class I 256 100 - 90 - o (/) 80 - (0 0) 70 - 2 60 - Uninfected ^ 5 0 - 'Ad-GFP '% 40 - Ad-CE1E2 0) 30 - 20 - 10 - 10:1 25:1 50:1 100:1 E:T Ratio Figure 5.13. NK cell lysis of uninfected, Ad-GFP & Ad-CE1E2 infected K562 target cells. 1x10^ labelled uninfected, Ad-GFP or Ad-CE1E2 infected K562 target cells were incubated at a range of effector:target (E:T) ratios with PBMCs taken from a donor with a high frequency of NK cells and lysis was assessed in a standard 5 hour chromium release assay. NK cell lysis of the target cells is expressed as the percentage specific cytotoxicity, calculated as described in the materials and methods. The results shown are representative of 3 independent experiments. 257 (Herzer et al., 2003). Here, experiments were carried out to address whether the core protein altered the susceptibility of each of these cell lines to lysis by NK cells. Uninfected, Ad-GFP or Ad-CE1E2 infected Hep16 and HepSB cells were incubated with NK cells at a range of effector:target ratios in a standard chromium release assay. As the difference in susceptibility of the two hepatoma cell lines to NK cell lysis is thought to relate to their relative levels of MHC class I expression, class I levels on these cells were also monitored by staining and analysis by flow cytometry. In agreement with the published observations I found low levels of NK cell lysis of uninfected HepSB cells at high E:T ratios, whilst the uninfected Hep16 cells were resistant to NK lysis, as shown in figure 5.14. Both cell lines were much more resistant to NK lysis than K562 cells. As shown in figure 5.15, the Hep16 cells which were less susceptible to NK lysis, expressed higher levels of MHC class I than the HepSB cells. Adenovirus infection enhanced the susceptibility of both cell lines to NK lysis, which might be expected as the role of NK cells is to kill virally-infected cells. This enhanced susceptibility was not due to a reduction in surface MHC class I expression levels (figure 5.15). However, the expression of the core protein in the hepatoma cell lines by Ad-CE1E2 has did not alter their susceptibility to NK lysis. As can be seen in figure 5.15, the levels of class I expression by these cells also did not alter following infection with Ad-CE1E2. The susceptibility of DCs and macrophages to NK lysis was then examined. Uninfected, Ad-GFP or Ad-CE1E2 infected DCs or macrophages were incubated with NK cells at a range of effector:target ratios in a standard chromium release assay. As can be seen in figures 5.16 and 5.17, both the DCs and the macrophages were resistant to lysis by NK cells (the peak of lysis of uninfected monocyte-derived macrophages at an E:T of 25:1 in figure 5.17 was an anomaly and was not reproduced in independent experiments). Adenovirus infection of DCs and macrophages and 258 (a) 100 -1 (D 10:1 25:1 50:1 80:1 E:T ratio (b) 100 -1 80 - s ■ Uninfected Ô ■ AJ-GFP ■ Ad-CE1E2 ^ 40 - ■K562 ^ 20 - 10:1 25:1 50:1 80:1 E;T ratio Figure 5.14. NK cell lysis of uninfected, Ad-GFP & Ad-CE1E2 Infected hepatoma cell lines. 2x10^ labelled uninfected, Ad-GFP or Ad-CE1E2 infected (a) HeplB or (b) HepSB cells, or uninfected K562 cells were incubated at a range of effector:target (E:T) ratios with PBMC taken from a donor with a high frequency of NK cells in a standard 5 hour chromium release assay. NK cell lysis of the target cells is expressed as the percentage specific cytotoxicity, calculated as described in the materials and methods. The results shown are representative of 3 independent experiments. 259 Isotype Control Class GFP (a) 7.42% 95.59% 95.08% 98.02% 42.33% M1 ■E : 1 ; , à I ; , JL ^1, ? UJ lluJ. , 10° 10'10“ 10^ 10* lo n ^ ^ ^ H 'o * 10 ° 1 0 ' 10 “ 10 " 1 0 * 10 ° 1 0 ' 10 “ 10 " 10 * FL2-H 10 ° 1 0 ' 10 “ 1 0 " 1 0 * FL2-H FL2-H FL2-H FL1-H K» Oo\ Uninfected Ad-GFP Ad-CE1E2 ml 89.38% 74.99% 45.78% 64.37% I> UJ D '5' 10° 10' 10“ 10" 10* ° ' “ " * 10° 1 0 ' 10“ 10 " 10 * 10 1 0 10 10 1 0 10 ° 1 0 ' 10 “ 10 " 1 0 * 10° 1 0 ' 10“ 10 " 10 ' FL2-H FL2-H FL2-H FL2-H FL1-H Figure 5.15. Expression of MHC class I by uninfected, Ad-GFP & Ad-CE1E2 infected Hepatoma cell lines. Uninfected, Ad-GFP or Ad-CE1E2 infected (a) Hep16 or (b) HepSB cells were stained for class I expression and analysed by flow cytometry. The percentage of class I positive cells is shown within Ml, set against the isotype control staining in FL-2. The level of infection was determined by GFP expression in FL-1, with the percentage of GFP positive cells (Ml) being gauged by comparison with the FL-1 fluorescence of uninfected cells. The data shown is representative of 2 independent experiments. Q) (A (00) £ Uninfected DC ü Ad-GFP DC Ad-CE1E2DC I O K562 & co 0 - 1 5:1 10:1 25:1 50:1 100:1 E:T Ratio Figure 5.16. NK cell lysis of uninfected, Ad-GFP & Ad-CE1E2 infected DCs & K562 target cells. 1x10^ ^^Cr labelled uninfected, Ad-GFP or Ad-CE1E2 infected DCs or uninfected K562 cells were incubated at a range of effectontarget (E:T) ratios with PBMCs taken from a donor with a high frequency of NK cells in a standard 5 hour chromium release assay. NK cell lysis of the target cells is expressed as the percentage specific cytotoxicity, calculated as described in the materials and methods. The results shown are representative of 3 independent experiments. 261 100 - (/)(D 80 i CD0) 2 60 - Ô Uninfected m Ad-GFP ü Ad-CE1E2 0) c/îCL 20 K562 10:1 25:1 50:1 80:1 E:T Ratio Figure 5.17. NK cell lysis of uninfected, Ad-GFP & Ad-CE1E2 infected monocyte-derived macrophages. 2x10^ labelled uninfected, Ad-GFP or Ad-CE1E2 infected macrophages or uninfected K562 cells were incubated at a range of effectontarget (E:T) ratios with PBMCs taken from a donor with a high frequency of NK cells in a standard 5 hour chromium release assay. NK cell lysis of the target cells is expressed as the percentage specific cytotoxicity, calculated as described in the materials and methods. The results shown are representative of 3 independent experiments. 262 expression of the core protein in these cells using Ad-CE1E2 did not alter their susceptibility to NK lysis. Finally, experiments were carried out to determine whether expression of the core protein in target cells altered their ability to be lysed by CTL. Initial experiments used EBV-BLCL as target cells. These were infected with adenovirus, with -50% being GFP positive 24 hours post infection with Ad-GFP at a MOI of 200 (data not shown). HLA- A2^ and HLA-A2 EBV-BLCL target cells were infected with Ad-GFP or Ad-CE1E2 and their susceptibility to lysis by CD8* T cells with a specificity for a HLA-A2 restricted CMV peptide (NLVPMVATV) was compared by assessing lysis in the presence of a range of peptide concentrations at an effectontarget ratio of 10:1, using a standard chromium release assay. Uninfected HLA-A2^ target cells were efficiently killed by the CTL (90% specific chromium release at the highest peptide concentration of 10‘®M) and the levels of killing titrated out with the peptide concentration, as can be seen in figure 5.18. Lysis of the HLA-A2 target cells was 0.3%, demonstrating that this lysis was MHC-restricted as well as peptide-dependent. Adenovirus infection and expression of the HCV core protein by Ad-CE1E2 did not alter the susceptibility of the B cell target cells to CTL lysis. Further experiments addressed whether DCs (which were resistant to apoptosis via the Fas, TRAIL, TNFa and lymphotoxin pathways and to lysis by NK cells) were susceptible to CTL lysis, and how expression of the HCV core protein within these cells could affect this. Uninfected, Ad-GFP and Ad-CE1E2 infected HLA-A2"^ and HLA-A2' DCs were incubated with a range of concentrations of a HLA-A2 restricted CMV peptide in the presence of CMV-specific CD8^ T cells, and lysis was assessed in a standard chromium release assay. As shown in figure 5.19, HLA-A2'’ DCs were lysed in a peptide-specific fashion and this lysis was also MHC-restricted (data not shown). DCs were relatively resistant to CTL lysis compared to the EBV-BLCL target cells, for 263 1 0 0 1 80 - 60 - Ô Uninfected ü Ad-GFP «= Ad-CE1E2 % 40 - coQ. 20 - 0 Peptide Conc. (M) Figure 5.18. CDS* cytotoxic T cell lysis of uninfected, Ad-GFP & Ad-CE1E2 infected B cell targets. 2x10^ labelled uninfected, Ad-GFP or Ad-CE1E2 infected B cells (HLA-A2*) were incubated in the presence of decreasing CMV peptide concentrations with CMV-specific CD8* T cells in a standard 5 hour chromium release assay. CTL lysis is expressed as percentage cytotoxicity, calculated as described in the materials and methods. The percentage specific ®^Cr release from HLA-A2' EBV-BLCLs incubated with lO^M peptide in this experiment was 0.3%. The results shown are representative of 2 independent experiments. 2 6 4 100 80 % 03 Q) Uninfected 2 60 Ô Ad-GFP m Ad-CE1E2 0 Uninfected B-LCL 0) 1 20 Peptide Conc. (M) Figure 5.19. CD8* cytotoxic T cell lysis of uninfected, Ad-GFP & Ad-CE1E2 infected DC targets. 2x10^ labelled uninfected, Ad-GFP or Ad-CE1E2 infected DCs (HLA-A2^) were incubated in the presence of decreasing CMV peptide concentrations with CMV-specific CD8^ T cells in a standard 5 hour chromium release assay. CTL lysis is expressed as percentage cytotoxicity, calculated as described in the materials and methods. The percentage specific ^^Cr release from HLA-A2' DCs incubated with 10'^M peptide in this experiment was 22%. The results shown are representative of 1 experiment. 265 example, there was 80% of specific chromium release from EBV-BLCL incubated with peptide at 10'^M peptide concentration, but only 50% specific chromium release from DCs at the same peptide concentration. Adenovirus infection increased the amount of lysis of DCs in the absence of peptide and at the lower peptide concentrations (at no peptide, 20% of Ad-GFP infected and 57% of Ad-CE1E2 infected DCs are killed), compared to uninfected cells where there is no background killing. Expression of the core protein within DCs has not altered their susceptibility to lysis by CTL. In conclusion, DCs and macrophages were resistant to lysis by NK cells, but DCs were killed by CTL, although to a lesser extent than EBV-BLCL target cells. This suggests that the DCs may not have triggered NK cell recognition; and/or that the effector pathways employed by CTL and NK cell lysis differ and DCs are more resistant to the pathways used by the latter. The HCV core protein did not decrease the susceptibility of cells to lysis which would allow more time for viral replication, nor did it increase the susceptibility to lysis which would impair immune functions. 5.4 Discussion The aim of the experiments carried out in this chapter was to determine whether expression of the HCV core protein within primary cells such as dendritic cells altered their susceptibility to apoptosis induced by different pathways. Previous studies had suggested that the core protein could modulate apoptosis, however these studies used transformed cell lines and had given somewhat confusing and conflicting results. Here the susceptibility of DCs (and macrophages) to induction of apoptosis via the Fas, TRAIL, TNFa and lymphotoxin pathways and to lysis by NK cells and CTL was assessed. These cells were found to be resistant/relatively resistant to killing via all 266 these pathways, which may make them “protected” sites for HCV replication. The hypothesis that core protein expression within these cells may either make then more resistant to apoptosis (giving them a longer time-frame for viral replication), or may make them more sensitive to apoptosis (leading to their destruction and hence impairment of the antiviral immune response) were investigated. However, no effects of HCV core protein (and the envelope glycoproteins, E1 and E2) expression within DCs on their resistance or sensitivity to apoptosis was observed. Possible explanations for this are discussed. I found that DCs were resistant to induction of apoptosis via the Fas, TRAIL, TNFa and lymphotoxin pathways, and were also resistant to lysis by NK cells. They could be lysed by MHC-restricted peptide-specific CTL, although the levels of CTL lysis were lower than that of EBV-BLCL target cells. Given the resistance of DCs to Fas and TRAIL-mediated apoptosis, it is likely that most of the lysis by CTLs was mediated via the perforin and granzyme pathway. The lack of killing of DCs by NK cells (which can also kill cells using the perforin and granzyme pathway) likely reflected the fact that the DCs did not trigger NK cell recognition. These findings are in agreement with results from other studies (some carried out with human monocyte-derived DCs, and others in murine systems), which have also suggested that DCs are relatively resistant to apoptosis induced via the Fas-FasL pathway (Ashany et al., 1999; McLellan et al., 2000; Willems et al., 2000) and to apoptosis induced via TNFRs (Funk et al., 2000), but have shown that immature DCs can be killed by CTL (Hermans et al., 2000; Knight et al., 1997; Loyer et al., 1999). Mature DCs have been shown to be protected from CTL lysis by expression of a serine protease inhibitor (Medema et al., 2001), this prevents the DC from being lysed by the CTL it has just activated, allowing the DC to activate other cells. 267 It is perhaps not surprising that antigen presenting cells are fairly resistant to apoptosis, as they are important initiators of appropriate immune responses required for clearance of infections. If they were destroyed before they had activated immune responses, the pathogen would have a replicative advantage. However, there needs to be a balance between resistance and susceptibility to apoptosis of cells, as once the pathogen has been cleared the immune response needs to be terminated, in order to prevent inappropriate responses that could cause further inflammation and lead to immunopathology once the pathogen has been cleared. It has been observed that antigen presenting cells are rapidly destroyed once they have activated T cell responses (Hermans et al., 2000; Loyer et al., 1999), however, Wong and Pâmer (Wong and Ramer, 2003) have seen that enhanced survival of antigen presenting cells such as DCs can occur in the absence of perforin, which may result in an increase in the recruitment of antigen-specific T cells. Also, if DCs are an important site of viral replication, then they may need to be killed in order to control infection, although this can lead to a generalised immunosuppression. Altering the susceptibility of host cells to apoptosis is an immune evasion mechanism that is used by many viruses in order to avoid elimination and to gain more time for replication and spread of infection. In many cases, viruses decrease the susceptibility of infected cells to apoptosis to give a longer time frame for production of new virus particles, for example, HSV-1 and EBV encode homologues of the anti-apoptotic molecule Bcl-2 to prevent cells from dying (Henderson et al., 1993; Kroemer, 1997). However, if viruses infect cells of the immune system, it may be advantageous for them to increase susceptibility to apoptosis, to impair immune control of the infection. In some cases, viruses may directly trigger apoptosis, for example, HIV triggers apoptosis of CD4* T cells (Grant et al., 1993; Shankar et al., 1999). Or perhaps viruses could enhance the susceptibility to apoptosis by immune effector pathways by increasing the 268 expression of Fas ligand or TRAIL receptors, or by enhancing signalling. Viruses could also increase the susceptibility to NK cell and CTL recognition. A number of previous studies have reported effects of the HCV core protein on the sensitivity of cells to apoptosis. It has been suggested to affect apoptosis sensitivity and resistance by a variety of different mechanisms. Core has been shown to bind to both the lymphotoxin p receptor (Chen et al., 1997; Matsumoto et al., 1997) and the TNFR-1 (Zhu et al., 1998) at the sites of the death domains, which is where downstream signalling molecules bind to these receptors. Therefore binding of the core protein to these domains could prevent or reduce binding of signalling molecules and prevent or enhance apoptosis. Several studies have found the core protein to enhance susceptibility to TNFa-mediated apoptosis (Zhu et al., 1998) and to Fas- mediated apoptosis (Hahn et al., 2000; Moorman et al., 2003; Ruggieri et al., 1997). Core has also been shown to bind to downstream signalling molecules, and this has also been implicated in modulating apoptosis (Park et al., 2001; Yang et al., 2002; Zhu et al., 2001). In addition, core has been reported to alter the sensitivity of hepatoma cell lines to lysis by NK cells by affecting the level of class I expression via effects on p53 expression. The upregulation of class I relies upon the function of TAP which is dependent on p53, and Herzer et al (Herzer et al., 2003) found the core protein increased TAP upregulation, thus enhancing class I expression. This resulted in a decrease in lysis of hepatoma cell lines (HepG2) by NK cells but not by CTL. They suggest that the core protein and p53 act in synergy to evade the non-antigenic specific effector mechanisms such as NK lysis against HCV-infected hepatocytes. The results published in the literature are somewhat confusing and contrasting, regarding effects of the core protein on the susceptibility of cells to apoptosis. There are several possible explanations for these differences. Different inducers of apoptosis 269 have also been used in these studies, and the physiological ligand has not always been used. Compounds such as cisplatin and actinomycin-D have been used to induce apoptosis in cells, either in the presence or absence of other cytokines such as IFNy (Fujita et al., 1996; Ray et al., 1996). Serum starvation of cells has also been used to induce apoptosis (Honda et al., 2000; Takamatsu et al., 2001), which is more likely to induce the intrinsic pathway of apoptosis via the release of cytochrome c from the mitochondria, than induce apoptosis via the TNFR family members such as the Fas pathway. As these inducers of apoptosis act via different pathways, it is likely that different effects will be observed. All of the previous studies have used transformed cell lines, particularly hepatoma cell lines, which may be considered relevant as the primary site of HCV replication is the liver. These studies also found contrasting results; some found the core protein sensitised cells to apoptosis (Kalkeri et al., 2001; Ruggieri et al., 1997; Yoshida et al., 2001), whilst others found it inhibited apoptosis (Otsuka et al., 2002; Yang et al., 2002). Studies where a suppression of apoptosis was observed (Marusawa et al., 1999; Ray et al., 1996; Ray et al., 1998a) used different cell lines to those where enhancement of apoptosis was observed. One of the cell lines used by Ray et al (Ray et al., 1998a) where no effects were observed was a human breast carcinoma line (MCF7), which has been shown to be deficient in caspase-3 (Kagawa et al., 2001), which is an important effector caspase in apoptotic pathways. Therefore the impairment of apoptosis is more likely due to this missing caspase rather than being an effect of the core protein. Many of the studies have used transformed cell lines, both human and murine, and studies carried out in mice have used different strains of mice and transgenic mice expressing different HCV proteins. Therefore, differences between cell types and mouse strains could also account for the differences reported in the published studies. 270 Different genotypes of the HCV core protein have been used in the published studies, which may account for some of the differences observed. Studies that found no effects of the core protein on apoptosis (Giannini et a!., 2002; Liu et a!., 2002; Sun et a!., 2001) used core protein from genotype 1b, whereas studies where effects were observed used core protein from genotype la. The intracellular location of the core protein may also influence results obtained. Moorman et al (Moorman et al., 2001) found that the full-length version of the core protein was located in the cytoplasm of cells in order to induce apoptosis in Jurkat cells. Cells expressing a truncated version of the core protein lacking the C-terminal domain were not as susceptible to apoptosis. This may be due to the truncated core protein being translocated into the nucleus (Lo et al., 1995; Yasui et al., 1998), however, after prolonged core expression (over 48 hours opposed to 24 hours) cells did undergo low levels of apoptosis. They found residual core expression in the nucleus but also an accumulation of core protein in the cytoplasm over time, which may then exceed the threshold for activation of apoptosis by the Fas pathway. Therefore the location of the core protein could alter the sensitivity of cells to apoptosis. My studies focused on primary cells (DCs and macrophages) and I looked at induction of apoptosis by exogenous signals, however I did not observe any effects of the core protein on the susceptibility of these cells to apoptosis induced by the Fas, TRAIL, TNFa and lymphotoxin pathways. The question of why I did not see any effects of the core protein on the susceptibility of cells to apoptosis remains, considering previous studies had implicated a role of the core protein in modulating apoptosis. The reasons for seeing no effects are similar to those discussed in chapter 4, where I did not see any effects of the core protein on DC activation and function. Differences could be due to the genotype of the core protein, as already mentioned studies which saw no effects of the core protein on apoptosis used a genotype 1b core protein. However, the core 271 protein I used was type 1a, which is the genotype used in studies where effects are reported. The differences may be due to the amount of the core protein expressed in the different systems. Most of the previous studies transiently transfected cells with plasmids expressing the core protein, whereas I used a recombinant adenovirus to express the core protein, so there may be differences in the levels of core protein expression. Again, the different expression systems could result in differences in the glycosylation of the core protein, which may alter how it interacts with cellular proteins and receptors and could account for differences in effects observed between studies. In my studies the envelope glycoproteins were expressed in conjunction with the core protein, as all my studies were carried out with Ad-CE1E2. This could also affect the form the core protein is expressed in, as the core protein may act differently when it is independent of the envelope glycoproteins. However, it could be argued that the association of the core protein with the envelope glycoproteins is more physiologically relevant than expressing each protein individually, as HCV virions will contain the core protein and envelope glycoproteins in association with each other, as has been determine by the production of virus-like particles (Baumert et al., 1999). Other HCV proteins have also been implicated in altering the susceptibility of cells to apoptosis, for example, Ciccaglione et al (Ciccaglione et al., 2003) have found that the C-terminal domain of the El protein is essential for apoptosis, when cells were infected with a baculovirus expressing El. Therefore, if both core and El can induce apoptosis in cells, then it would have been expected to see effects in my studies, as both core, El and E2 were present in infected cells. The intracellular location of the core protein expressed by Ad-CE1E2 may also account for the lack of effects. Moorman et a! (Moorman et al., 2001) showed that a cytoplasmic location of the core protein was required for it to have effects on apoptosis. As shown in chapter 3, I found the core protein to be expressed predominantly in the nucleus of DCs infected with Ad-CE1E2, although some expression in the cytoplasm was also detected. This could account for the lack of effects on the susceptibility of cells to apoptosis when they were infected 272 with Ad-CE1E2, if the majority of the core protein was expressed in the cell nucleus, then it would not be in an appropriate location to interfere with apoptotic signalling pathways. Overall, it is difficult to draw definitive conclusions from the negative results I obtained in the experiments carried out in this chapter, with DCs and macrophages. However, based on the reports in the literature it would be expected that the core protein can alter the susceptibility of cells to apoptosis. In the future, these experiments could be repeated using a higher MOI to infect target cells, to give higher levels of protein expression within the cells. It would also be interesting to use the adenoviruses I generated in chapter 3; the adenovirus expressing the mutated version of the core protein may result in higher levels of protein expression within target cells, making it easier to determine effects of the core protein. It would also be interesting to compare effects of the full-length core protein to those induced by the truncated version of the core protein, as the study by Moorman et al (Moorman et al., 2001) has suggested there may be differences in the ability of the different versions of the core protein to induce apoptosis. 273 Chapter 6 - General Discussion Approximately 80% of individuals infected with HCV fail to clear the virus and develop chronic infection which suggests that HCV has strategies for modulating or evading control by the host immune response. As described in the introduction, HCV persistence is likely facilitated by the concerted action of multiple mechanisms impairing or avoiding immune defences. One of the factors that may contribute to HCV persistence may be its ability to infect DCs and potentially impair their functions. The work carried out in this thesis focused on examining the effects of expression of the HCV core protein within DCs, to address whether/how this may affect the host immune response. Given the central role played by DCs in orchestrating both innate and adaptive immune responses, it is perhaps not surprising that multiple viruses have developed strategies for impairing or modulating DC functions to prolong their replication in the host cells. These target many aspects of DC biology and functions. Some viruses reduce the number of functional DC precursors, for example, HTLV-1 is known to infect monocytes and impair their differentiation into DCs (Makino et al., 2000). Alternatively, viruses may interfere with the recruitment of DCs to sites of infection, for example, pox viruses are large DMA viruses that have genes encoding viral chemokine and cytokine homologues which can “mop up” chemokines and cytokines that would recruit DCs to the site of infection. It is unlikely that HCV uses this approach, as it has a much smaller genome. Viruses may also destroy DCs at the site of infection to reduce activation of T cell responses, for example, pox viruses such as canarypox induce rapid apoptosis of DCs; although this strategy is only effective if a high proportion of DCs are infected, as it does not prevent cross-priming. Again, it is 274 unlikely that HCV is using this approach, as DCs are still found in chronically infected patients; they are just not as functional as DCs from uninfected individuals. Many viruses are known to impair DC antigen uptake and processing in order to prevent the induction of T cell responses by presentation of viral antigens in MHC class I. For example, HSV-1 encodes a protein called ICP47, which inhibits MHC class I presentation by blocking TAP activity, causing MHC retention and accumulation in the endoplasmic reticulum (Fruh et al., 1995; Hill et al., 1995; York et al., 1994). As yet, no HCV proteins have been identified to have the ability to impair antigen processing or presentation, although it remains possible this occurs. Viruses may also impair DC maturation and migration to lymphoid organs, again to prevent the initiation of immune responses; examples of viruses capable of this are HSV-1 and vaccinia virus. HSV-1 not only impairs DC maturation, it also impairs the upregulation of CCR7 within DCs which prevents DC migration (Salio et al., 1999). Finally, viruses may interfere with the activation of T cells by DCs. In measles virus infection, DCs undergo maturation but are not capable of inducing T cell activation. There is a defect in IL-12 production by DCs and upregulation of TRAIL expression in DCs is induced by the virus. This leads to DCs inducing apoptosis in T cells rather than presenting viral antigens and activating T cell responses, thus reducing the number of virus-specific T cells. Alternatively, viruses may alter the type of T cell response activated by DCs. If DCs activate regulatory T cells which produce IL-10 and are capable of downregulating the virus-specific immune response, this would promote viral persistence. There is evidence that HCV does affect the interaction of DCs with T cells. Several studies have found DCs from chronically infected patients to be impaired in their ability to stimulate T cell responses and to have a reduced production of IL-12, compared to DCs 275 from uninfected individuals (Auffermann-Gretzinger et a!., 2001; Bain et a!., 2001; Kakumu et a!., 2000; Kanto et a!., 1999). Inadequate T cell responses are also found in HCV patients with chronic infections, which may in turn be due to impaired functions of DCs. However the exact mechanisms HCV is using to impair host responses are still unclear. This thesis addresses effects that expression of the HCV core protein within DCs may have on their activation, functions and survival. Given reports that the core protein is able to, one, bind to the cytoplasmic tail of members of the TNFR family, potentially modulating signalling through these receptors (Chen et al., 1997; Matsumoto et al., 1997; Ruggieri et al., 1997; Zhu et al., 1998): two, to affect the regulation of gene expression (Alisi et al., 2003; Isoyama et al., 2002; Yamanaka et al., 2002); and three, to alter the sensitivity of cells to apoptosis (Dumoulin et al., 1999; Marusawa et al., 1999; Ray et al., 1996; Ray et al., 1998a; Shrivastava et al., 1998; Zhu et al., 1998; Zhu et al., 2001), two possible types of effects of the core protein on DCs were explored. First (addressed in chapter 4), the ability of the core protein to modulate DC activation in response to LPS, TNFa and anti- CD40, and to stimulate I cell responses was explored, focusing on the hypothesis that core protein expression within DCs may be responsible for impairing the ability of DCs to stimulate strong type 1 I cell responses. Second (addressed in chapter 5), whether the core protein might affect the resistance of DCs to apoptosis was addressed, concentrating particularly on the hypothesis that the core protein may render these cells more susceptible to destruction by immune-mediated lytic pathways, hence again leading to impairment in the induction or maintenance of the immune response to HCV. The approach taken was to construct replication-deficient adenoviral vectors expressing the full-length core protein and fragments thereof and to use core-expressing adenoviral vectors to transducer DCs in order to examine potential immunomodulatory effects of core 276 protein expression in these cells. Core protein expression within DCs was not found to affect their activation, antigen uptake, cytokine production or ability to stimulate T cell responses. Likewise, the core protein did not alter the susceptibility of DCs (or other cells) to apoptosis via pathways including Fas, TRAIL, TNFa, lymphotoxin and the perforin/granzyme lytic pathways of CTL and NK cells. These results do not suggest that HCV core protein expression within DCS has any immunomodulatory effects. However, several other studies that have also recently addressed this have reached different conclusions. A consistent decrease in IL-12 production by DCs in response to a range of stimuli has been reported (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Hiasa et al., 1998; Kanto et al., 1999; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002) and a decrease in their stimulatory capacity (Hiasa et al., 1998; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002), suggesting that the core protein is capable of impairing the ability of DCs to induce and maintain T cell responses. If the HCV core protein does have effects on DCs (and other cell types) there are several possibilities as to why I did not observe this. The level of core protein expression achieved within cells is likely to have been a particularly important factor. I did show that the core protein was expressed in DCs infected with Ad-CE1E2, however the level of core protein expression achieved at the MOIs I was using may have been too low for it to have a noticeable effect. In addition, although experiments performed with Ad-GFP suggested that a high proportion of DCs were transduced at the MOIs I used for infection, I could not readily check the efficiency of DC transduction by Ad-CEI E2. If not all cells were expressing the core protein, subtle effects of the core protein on DCs could have been missed. Higher levels of protein expression within target cells would be achieved by infecting cells at a higher MCI, however a balance between the level of infection and the viability of cells has to be achieved, as if too high a MCI is used the viability of DCs decreases so they are unlikely to function normally. Another factor that may have affected the results I obtained may have 277 been the processing and intracellular location of the core protein when expressed together with the E1 and E2 from an adenoviral vector. Most of the core protein expressed by Ad- CEI E2 in DCs was in the nucleus, with small amounts in the cytoplasm of some cells. There was thus probably little protein available to interact with, for example, the cytoplasmic tail of members of the TNFR family or downstream components of the apoptotic signalling pathways. To overcome some of these problems, future experiments could be carried out with the recombinant adenoviruses I generated that co-express GPP and different forms of the core protein, including a version that may accumulate at higher levels within the cytoplasm. Adenoviral recombinants expressing different versions of the core protein could potentially also be used to identify regions of the core protein responsible for immunomodulatory effects. Whether the HCV core protein has any effect on the resistance of DCs to induction of apoptosis via immune effector pathways has not been addressed in any other studies. However, the core protein has been shown to alter the susceptibility of transformed cell lines to apoptosis in response to a variety of stimuli, in some systems rendering cells more susceptible to lysis (Dumoulin et al., 1999; Hahn et al., 2000; Moorman et al., 2003; Ray et al., 1996; Ruggieri et al., 1997; Zhu et al., 1998; Zhu et al., 2001), and in others rendering cells resistant to lysis (Marusawa et al., 1999; Ray et al., 1998a; Shrivastava et al., 1998). The latter is unlikely to be of importance to DCs, but if HCV is able to increase the resistance of other cell types, such as hepatocytes, to apoptosis, this might be of importance in vivo, providing more time for viral replication. A limited number of experiments carried out with hepatocyte cell lines to address this again did not reveal any effects of the core protein on resistance to lysis, although a recent study did suggest that the core protein could increase the resistance of hepatoma cell lines to NK lysis by increasing the levels of class I expression on these cells (Herzer et al., 2003). 278 Although my results did not reveal any immunomodulatory effects of the core protein expression within DCs (or other cell types), other studies together suggest that the core protein may make an important contribution to HCV persistence, acting in more than one way to do this. First, when expressed within DCs it may impair their activation and interaction with T cells and possibly other cell types (Herzer et al., 2003; Kim et al., 2002a; Lee et al., 2001; Sarobe et al., 2002). Second, there may be external interactions of the core protein on T cells via interactions with the complement receptor gClqR (Kittlesen et al., 2000; Yao et al., 2001a; Yao et al., 2001b). Given the central role played by DCs in both innate and adaptive responses, effects of the core protein on DC functions could impact on the immune response at multiple levels, potentially affecting the HCV-specific immune response induced in primary infection (Chen et al., 1999; Lechner et al., 2000a) and being responsible for the alterations in NK cell function (Corado et al., 1997; Herzer et al., 2003) and impairment in HCV-specific T cell responses seen in many chronic HCV patients (Auffermann-Gretzinger et al., 2001; Bain et al., 2001; Kakumu et al., 2000; Kanto et al., 1999). In addition, the core protein may confer resistance on infected cells (such as hepatocytes) to NK/CTL lysis by impairing NK cell recognition of target cells by its ability to upregulate class I expression (Herzer et al., 2003) and by rendering target cells resistant to apoptosis induced by lytic effector mechanisms used by CTL and NK cells (Bonavita et al., 1993; Castiglione et al., 2001; Gabrielli et al., 1995; Par et al., 2002). Coupled with the other immune evasion strategies employed by HCV (including impairment of type 1 interferon production (Pfiugheber et al., 2002; Polyak et al., 2001) and resistance to type 1 interferon control (Basu et al., 2001; Gale et al., 1997); and antigenic variation to avoid recognition by virus-specific antibody and T cell responses), this virus has an armoury of strategies for achieving persistence in vivo. 279 Understanding the mechanisms HCV uses to establish chronic persistent infections is crucial to the design of effective vaccines and therapies to combat HCV infection. A prophylactic vaccine for HCV would be a great benefit to public health, even though transmission has been reduced since the introduction of blood screening tests. Ideally, a prophylactic vaccine should induce sterilising immunity but this may be difficult to achieve, and may not in fact be necessary. A vaccine which allowed a “transient infection” (sub- clinical or of limited acuity), whilst preventing the development of a chronic infection could be as beneficial. The core protein could be used as a vaccine component, and several investigators are already exploring this option (Alvarez-Obregon et al., 2001; Duenas- Carrera et al., 2001; Hunzulker et al., 2002; Kamei et al., 2000; Ou-Yang et al., 2002; Polakos et al., 2001). However, if the core protein is modulating host responses, particularly by suppressing DC functions this could have implications for its use as an immunogen. Particularly if the core protein is given in some form of vector that targets expression to DCs; it could impair the function of cells expressing it. This might not matter, as not all DCs would be affected, so a good response could be induced by cross presentation. However, any immunomodulatory effects of the core protein could be overcome by using a mutated version of the core protein that lacked regions responsible for immunomodulatory effects as an immunogen. As mentioned above, these could be determined by using replication-deficient adenoviruses expressing different versions of the core protein. If a prophylactic vaccine could induce a good neutralising antibody response and strong memory CD4"^ and CD8"^ T cell responses, these could hopefully block or rapidly eliminate infection before there was too much impairment of DC function. If the core protein renders cells resistant to CTL lysis, hopefully by pre-priming other effector mechanisms such as antibodies and cytokine production, elimination of the infection could still be achieved. 280 As the major burden of HCV infection on healthcare is due to the complications of chronic infection, such as cirrhosis and hepatocellular carcinoma, and there are many infected individuals worldwide, therapeutic vaccines (and other forms of therapy) are perhaps more urgently required than a prophylactic vaccine. Therapeutic vaccines could aim to eliminate the infection or reduce the severity of infection-associated disease. They could potentially be used in combination with other antiviral therapies. If the core protein is modulating DC functions, this is more likely to have implications for the development of therapeutic strategies which would need to induce an antiviral immune response in the context of an impaired immune system. One possible way of overcoming this problem would be to use a therapeutic approach to reduce the viral load before attempting vaccination. Another approach that could be considered would be that of immunotherapy using DCs. Optimism has arisen from recent findings by Bhardwaj et al (Bhardwaj and Walker, 2003), who have found evidence that this approach may work in a model of HIV infection, where viral replication was controlled by therapeutic immunisation. They immunised monkeys with a combination of DCs and chemically inactivated SIV (simian immunodeficiency virus) and found that a dramatic reduction in the viral loads in the blood and an increase in the number of CD4"^ T cells, which are essential in the activation and maintenance of CDS"” T cell responses involved in viral clearance, was achieved. The use of DCs generated in vitro and delivered in vivo has also been effective in inducing protective responses against tumours (Banchereau et al., 2000) in animal models. A third option may be to enhance in vivo patient DC functions to enhance the activation of T cells. This could possibly be achieved by incorporating cytokines such as GM-CSF into vaccines to promote DC maturation and activation in the face of viral impairment (Dranoff et al., 1993; Pardoll, 2002). Alternatively, cytokines such as IL-12, which infected DCs may be deficient in producing, could be incorporated into therapeutic vaccines. 281 If the core protein is affecting apoptotic pathways and impairing lysis of cells, a possible way of getting around this may be to target lytic pathways that are not affected by the core protein. This may not be possible if the core protein is targeting downstream signalling molecules that are involved in multiple apoptotic pathways, such as components of the caspase cascade. Compounds which block the core protein from binding to signalling components could potentially be designed for therapeutic use, to prevent core affecting apoptotic pathways. Alternatively, if lysis of cells is affected by the core protein this could be overcome by the use of “curative” cytokines, such as interferons and TNFa that will cure cells of viral infection and enhance antiviral immune responses. Although there is potential for these and other approaches to be employed to combat HCV infection, the lack of suitable model systems for studying HCV hampers the development of vaccination strategies. There is thus a great need for better model systems of HCV infection, disease progression and pathogenesis. In summary, there are still many challenges in developing vaccines and therapeutics for HCV infection. In order to combat this virus a greater understanding of the virus, the types of immune responses essential for viral clearance and the strategies the virus is taking to evade elimination in order to establish chronic persistent infections is required. 282 References Abe, K., Kurakin, A., Mohseni-Maybodi, M., Kay, B., and Khosravi-Far, R. (2000). The complexity of INF-related Apoptosis Inducing Ligand. Ann NY Acad Sci 926, 52-63. Abe, K., Kurata, T., Teramoto, Y., Shiga, J., and Shikata, T. (1993). Lack of susceptibility of various primates and woodchucks to hepatitis C virus. J Med Primatol 22, 433-434. Afford, 8. C., Randhawa, S., Eliopoulos, A. G., Hubscher, 8. G., Young, L. 8., and Adams, D. H. (1999). CD40 activation induced apoptosis in cultured human hepatocytes via induction of Fas ligand expression and amplifies Fas-mediated hepatocyte death during allograft rejection. J Exp Med 189, 441-446. Agnello, V. (1997). The etiology and pathophysiology of mixed crypglobulinemia secondary to hepatitis 0 virus infection. Spring Semin Immunopathol, 111-129. Agrati, 0., D'Offizi, G., Narciso, P., Abrignani, S., Ippolito, G., Collizi, V., and Poccia, F. (2001a). Vdeltal T lymphocytes expressing a Thi phenotype are the major gammadelta T cell subset infiltrating the liver of HCV infected persons. Mol Med 7, 11-19. Agrati, 0., D'Offizi, G., Narciso, P., Selva, 0., Pucillo, L. P., Ippolito, G., and Poccia, F. (2001b). Gammadelta T cell activation by chronic HIV infection may contribute to intrahepatic vdeltal compartmentalization and hepatitis 0 virus disease progression independently of highly active antiretroviral therapy. AIDS Res Hum Retroviruses 17, 1357-1363. Ahn, K., Gruhler, A., Galocha, B., Jones, T. R., Wiertz, E. J., Ploegh, H., Peterson, P. A., Yang, Y., and Fruh, K. (1997). The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP. Immunity 6, 613-621. Alberti, A., Cavalletto, D., Pontisso, P., Chemello, L., Tagariello, G., and Belussi, F. (1988). Antibody response to pre-S2 and hepatitis B virus induced liver damage. Lancet 1, 1421- 1424. Alberti, A., Morsica, G., Chemello, L., Cavalletto, D., Noventa, F., Pontisso, P., and Ruol, A. (1992). Hepatitis C virus viraemia and liver disease in symptom free individuals with anti-hepatitis C virus. Lancet 340. Alcami, A., and Smith, G. L. (1996). Receptors for gamma-interferon encoded by poxviruses: implications for the unknown origin of vaccinia virus. Trends Microbiol 4, 321- 326. All, N., Tard iff, K. D., and Siddiqui, A. (2002). Cell-free replication of the hepatitis C virus subgenomic replicon. J Virol 76, 12001-12007. Alisi, A., Giambartolomei, S., Cupelli, F., Merlo, P., Fontemaggi, G., Spaziani, A., and Balsano, C. (2003). Physical and functional interaction between HCV core protein and the different p73 isoforms. Oncogene 22, 2573-2580. 283 Almasio, P., Provenzano, G., Sciemeni, M., Cascio, G., Craxi, A., and Pagliaro, L. (1992). Hepatitis C virus and Sjogren's syndrome. Lancet 339, 989-990. Almeida, J. D., and Waterson, A. P. (1969). The morphology of virus-antibody interactions. Adv Virus Res 15, 307-338. Alter, H. J., Purcell, R. H., Holland, P. V., and Popper, H. (1978). Transmissible agent in non-A non-B hepatitis. Lancet 1, 459-463. Alvarez-Obregon, J. C., Duena-Carrera, S., Valenzuela, C., and Grille, J. M. (2001). A truncated HCV core protein elicits a potent immune response with a strong participation of cellular immunity components in mice. Vaccine 19, 3940-3946. Andersson, M., Paabo, S., Nilsson, T., and Peterson, P. A. (1985). Impaired intracellular transport of class I MHC antigens as a possible means for adenovirus to evade immune surveillance. Cell 43, 215-222. Ando, K., Hiroishi, K., and Kaneko, T. (1997). Perforin, fas/fas ligand, and TNF alpha pathways as specific and bystander killing mechanisms of the hepatitis C virus-specific human CTL. J Immunol 158, 528-591. Ashany, D., Savir, A., Bhardwaj, N., and Elkon, K. B. (1999). Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway. J Immunol 163, 5303-5311. Auffermann-Gretzinger, S., Keeffe, E. B., and Levy, S. (2001). Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection. Blood 97, 3171-3176. Back, S. H., Kim, J. E., Rho, J., Hahm, B., Lee, T. G., Kim, E. E., Cho, J. M., and Jang, S. K. (2000). Expression and purification of an active, full-length hepatitis C viral NS4A. Protein Expr Purif 20, 196-206. Bain, C., Fatmi, A., Zoulim, F., Zarski, J. P., Trepo, C., and Inchauspe, G. (2001). Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gasteroenterology 120, 512-524. Bain, C., Fatmi, A., Zoulin, F., Zarski, J. P., C., T., and Inchauspe, G. (1999). Paper presented at: 6th International Symposium of hepatitis C virus and related viruses. (Washington, USA). Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J., Pulendran, B., and Palucka, K. (2000). Immunobiology of dendritic cells. Ann Rev Immunol 18, 767-811. Barba, G., Harper, F., Harada, T., Kohara, M., Goulinet, S., Matsuura, Y., Eder, G., Schaff, Z., Chapma, M. J., Miyamura, T., and Brechot, C. (1997). Hepatitis C virus core protein shows a cytoplasmic localisation and associates to cellular lipid storage droplets. Proc Natl Acad Sci USA 94, 1200-1205. Bartenschlager, R. (2002). In Vitro Models for Hepatitis 0. Virus Res 82, 25-32. Bartenschlager, R., and Lohmann, V. (2000). Replication of hepatitis C virus. J Gen Virol 81, 1631-1648. 284 Barth, H., Schafer, C., Adah, M. I., Zhang, F., Linhardt, R. J., Toyoda, H., Kinoshita- Toyoda, A., Toida, T., Van Kuppevelt, T. H., Depla, E., et al. (2003). Cellular binding of hepatitis 0 virus envelope glycoprotein E2 requires cell surface heparan sulfate. J Biol Chem. Bartocsh, B., Vitelli, A., Granier, 0., Goujon, 0., Dubuisson, J., Pascale, S., Scarselli, E., Cortese, R., Nicosia, A., and Cosset, F. L. (2003a). Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem. Bartosch, B., Dubuission, J., and Cosset, F. L. (2003b). Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197, 633-642. Bassett, 8. E., Brasky, K. M., and Lanford, R. (1998). Analysis of hepatitis C virus- innoculated chimpanzees reveals unexpected clinical profiles. J Virol 72, 2589-2599. Basu, A., Meyer, K., Ray, R. B., and Ray, R. (2001). Hepatitis C virus core protein modulates the interferon-induced transacting factors of Jak/Stat signalling pathway but does not affect the activation of downstream IRF-1 or 561 gene. Virology 288, 379-390. Battegay, M., Fikes, J., Di Bisceglie, A. M., Wentworth, P. A., Sette, A., Cells, E., Ching, W.-M., Grakoui, A., Rice, C. M., Kurokochi, K., et ai. (1995). Patients with chronic hepatitis C have circulating cytotoxic T cells which recognise hepatitis C virus-encodes peptides binding to HLA-A2.1 molecules. J Virol 49, 2462-2470. Battegay, M., Moskophidis, D., and Waldner, H. (1993). Impairment and delay of neutralising antiviral antibody responses by virus-specific cytotoxic T cells. J Immunol 151, 5408-5415. Baumert, T. F., Ito, S., Wong, D. T., and Liang, T. J. (1998). Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J Virol 72, 3827-3836. Baumert, T. F., Vergalla, J., Satoi, J., Thomson, M., Lechmann, M., Herion, D., Greenberg, H. B., Ito, S., and Liang, I. J. (1999). HCV-virus like particles synthesised in insect cells as a potential vaccine candidate. Gasteroenterology 117, 1397-1407. Beard, M. R., Abell, G., Honda, M., Carroll, A., Gartland, M., Clarke, B., Suzuki, K., Lanford, R., Sangar, D. V., and Lemon, S. M. (1999). An infectious molecular clone of a Japanese genotype 1b hepatitis C virus. Hepatology 30, 316-324. Bennett, S. R., Carbone, F. R., Karamalis, F., Flavell, R. A., Miller, J. F., and Heath, W. (1998). Help for cytotoxic T-cell responses is mediated by CD40 signalling. Nature 393, 478-480. Bergelson, J. M., Cunningham, J. A., Droguett, G., Kurt-Jones, E. A., Krithivas, A., Hong, J. S., Horwitz, M. S., Crowell, R. L., and Finberg, R. W. (1997). Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275,1320-1323. Bertoletti, A., D'Elios, M. M., Boni, C., De Carli, M., Zignego, A. L., Durazzo, M., Missale, G., Penna, A., Fiaccadori, F., Del Prete, G., and Ferrari, C. (1997). Different cytokine 285 profiles of intrahepatic T cells in chronic hepatitis B and C virus infections. Gasteroenterol 112, 193-199. Bett, A. J., Haddara, W., Prevec, L, and Graham, F. L. (1994). An efficient and flexible system for construction of adenovirus vectors with insertions or delections in early regions 1 and 3. Proc Natl Acad Sci USA 91, 8802-8806. Bett, A. J., Prevec, L, and Graham, F. L. (1993). Packaging capacity and stability of human adenovirus type 5 vectors. J Virol 67, 5911-5921. Bevegnu, L, Pontisso, P., Cavalletto, D., Noventa, F., Chemello, L, and Albert, A. (1997). Lack of correlation between hepatitis C virus genotypes and clinical course of hepatitis C virus-related cirrhosis. Hepatology 25, 211-215. Bhardwaj, N., and Walker, B. D. (2003). Immunotherapy for AIDS virus infections: Cautious optimism for cell-based vaccine. Nat Med 9, 13-14. Biron, C. A. (1998). Role of early cytokines, including alpha and beta interferons (IFN- alpha/beta), in innate and adaptive immune responses to viral infections. Semin Immunol 10, 383-390. Biron, C. A. (1999). Initial and innate responses to viral infections - pattern setting immunity or disease. Curr Opin Microbiol 2, 374-381. Biron, C. A. (2001). Interferons alpha and beta as immune regulators - a new look. Immunity 14, 661-664. Blight, K. J., McKeating, J. A., Marcotrigiano, J., and Rice, C. M. (2003). Efficient replication of hepatitis C virus genotype la RNAs in cell culture. J Virol 77, 3181-3190. Bode, J. G„ Ludwig, S., Eherhardt, A., Albrecht, U., Schaper, F., Heinrich, P. C., and Haussinger, D. (2003). IFN-alpha antagonistic activity of HCV core protein involves induction of suppressor of cytokine signalling-3. FASEB J 17, 488-490. Bodenheimer, H. C. J., Lindsay, K. L., Davis, G. L., Lewis, J. H., Thung, S. N., and Seef, L. B. (1997). Tolerance and efficacy of oral ribavirin treatment of chronic hepatitis C: a multicentre trial. Hepatology 26, 473-477. Bonavita, M. S., Franco, A., Paroli, M., Santilio, I., Benvenuto, R., De Petrillo, G., Levrero, M., Perrone, A., Balsano, C., and Barnaba, V. (1993). Normalisation of depressed natural killer activity after interferon-a therapy is associated with a low frequency of relapse in patients with chronic hepatitis C. Int J Tiss React XV, 11-16. Borrow, P., and Oldstone, M. B. A. (1994). Viruses. In Samter's Immunological Diseases, M. M. Frank, K. F. Austen, H. N. Claman, and E. R. Unanue, eds. (Boston, Little, Brown & Co.), pp. 1379-1392. Borrow, P., and Shaw, G. M. (1998). Cytotoxic T-lymphocyte escape viral variants: how important are they in viral evasion of immune clearance in vivo. Immunological Reviews 164, 37-51. 286 Bosivert, J., Kunkel, E. J., Campbell, J. J., Keefe, E. B., Butcher, E. 0., and Greenberg, H. B. (2003). Liver-infiltrating lymphocytes in end-stage hepatitis 0 virus: Subsets, activation status and chemokine receptor phenotypes. J Hepatol 38, 67-75. Botarelli, P., Brunetto, M. R., Minutello, M. A., Calvo, P., Unutmaz, D., Weiner, A. J., Choo, Q. L., Shuster, J. R., Kuo, G., Bonino, F., et al. (1993). T-lymphocyte response to hepatitis 0 virus in different clinical courses of infection. Gasteroenterology 104, 580-587. Bourgeois, 0., Rocha, B., and Tanchot, 0. (2002). A role for CD40 expression on CD8+ T cells in the generation of CD8+ T cell memory. Science 297, 2060-2063. Bradley, D. W., Maynard, J. E., Popper, H., Cook, E. H., Ebert, J. W., McCaustland, K. A., Schable, C. A., and Fields, H. A. (1983). Posttransfusion NANB hepatitis: physiochemical properties of two distinct agents. J Infect Dis 148, 254-265. Bradley, D. W., Maynard, J. E., Popper, H., Ebert, J. W., Cook, E. H., Fields, H. A., and Kemler, B. J. (1981). Persistent NANB hepatitis in experimentally infected chimpanzees. J Infect Dis 143, 210-218. Brinster, C., Chen, M., Boucreux, D., Paranhos-Baccala, G., Liljestrom, P., Lemmonier, F., and Inchauspe, G. (2002). Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunisation with DNA or recombinant Semliki Forest Virus particles. J Gen Virol 83, 369-381. Brinster, C., Muguet, S., Lone, Y. C., Boucreux, D., Renard, N., Fournillier, A., Lemonnier, F., and Inchauspe, G. (2001). Different hepatitis C virus nonstructural protein 3 (Ns3)-DNA expressing vaccines induce in HLA-A2.1 transgenic mice stable cytotoxic I lymphocytes that target one major epitope. Hepatology 34,1206-1217. Bruna-Romero, O., Lasarte, J. J., Wilkinson, G., Grace, K., Clarke, B., Burras-Cuesta, F., and Prieto, J. (1997). Induction of cytotoxic T cell response against HCV structural antigens using a defective recombinant adenovirus. Hepatology 25, 479-. Bukh, J., Miller, R. H., and Purcell, R. H. (1995). Genetic heterogenity of HCV: Quasispecies and genotypes. Semin Liver Dis 15, 41-63. Bukh, J., Pietschmann, T., Lohmann, V., Krieger, N., Faulk, K., Engle, R. E., Govindarajan, S., Shapiro, M., St Claire, M., and Bartenschlager, R. (2002). Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevents productive replication in chompanzees. Proc Natl Acad Sci USA 99, 14416-14421. Bukh, J., Purcell, R. H., and Miller, R. H. (1993). At Least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative El gene of isolates collected workdwide. Proc Natl Acad Sci USA 90, 8234-8238. Bukowski, J. F., Warner, J. F., Dennert, G., and Welsh, R. M. (1985). Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo. J Exp Med 161, 40- 52. Burgert, H. G., and Kvist, S. (1985). An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell 41, 987-997. 287 Burgert, H. G., Ruzsies, Z., Obermeier, S., Hilgendorf, A., Windheim, M., and EIsing, A. (2002). Subversion of host defence mechanisms by adenoviruses. Curr Top Microbiol Immunol 269, 273-318. Byrne, J. A., and Oldstone, M. B. A. (1984). Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo. J Virol 51, 682- 686 . Cacciarelli, I. V., Martinez, O. M., Gish, R. G., Villanuesa, J. 0., and Krams, S. M. (1996). Immunoregulatory cytokines in chronic hepatitis 0 virus infection: Pre- and post- treatment with inteferon alpha. Hepatology 24, 6-9. Carding, S. R., Allan, W., McMickle, A., and Doherty, P. C. (1993). Activation of cytokine genes in I cells during primary and secondary murin influenza pneumonia. J Exp Med 177, 475-482. Carrere-Kremer, S., Montpellier-Pala, C., Cocquerel, L., Wychowski, C., Penin, F., and Dubuisson, J. (2002). Subcellular localization and topology of the p7 polypeptide of hepatitis C virus. J Virol 76, 3720-3730. Carroll, S. S., Tomassini, J. E., Bosserman, M., Getty, K., Stahlhut, M. W., Eldrup, A. B., Bhat, B., Hall, D., Simcoe, A. L, LaFemina, R., et al. (2003). Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs. J Biol Chem 276, 11979-11984. Castiglione, G., Guglietta, A., and Lirussi, F. (2001). Natural killer (NK) cell cytotoxicity in chronic HCV infection. Relationship with clinical and virological features and response to interferon (IFN). J Hepatol 34, 131. Cerny, A., McHutchinson, J. G., Pasquinelli, C., Brown, M. E., Brothers, M. A., Grabscheid, B., Fowler, P., Houghton, M., and Chisari, F. V. (1995). Cytotoxic I lymphocyte response to hepatitis C virus - derived peptides containing the HLA-A2.1 binding motif. J Clin Invest 95, 521-530. Chang, J., Yang, S.-H., Cho, Y.-G., Hwang, S. B., Hahn, Y. S., and Sung, Y. C. (1998). Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J Virol 72, 3060-3065. Chang, K. M., Rehermann, B., McHutchinson, J. G., Pasquinelli, C., Southwood, S., Sette, A., and Chisari, F. V. (1997). Immunological significance of cytotoxic T lymphocyte epitope-variants in patients chronically infected by the hepatitis C virus. J Clin Invest 100, 2376-2385. Chang, K. M., Thimme, R., Melpolder, J. J., Oldach, D., Pemberton, J., and Moorhead- Loudis, J. (2001). Differential CD4+ and CD8+ T-cell responsiveness in hepatitis C virus infection. Hepatology 33, 267-276. Chang, M., Williams, O., Mittler, J., Quintanilla, A., Carithers, R. L. J., Perkins, J., Corey, L., and Gretch, D. R. (2003). Dynamics of hepatitis C virus replication in human liver. Am J Pathol 163, 433-444. 288 Charlton, M., Seaberg, E., Wiesner, R., Everhart, J., Zetterman, R., Lake, J., Detre, K., and Hoofnagle, J. H. (1998). Predictors of patient and graft survival following liver transplantation for hepatitis 0. Hepatology 28, 823-830. Chartier, 0., Degryse, E., Gantzer, M., Dieterle, A., Pavirani, A., and Mehtali, M. (2996). Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coii. J Virol 70, 4805-4810. Chen, C.-M., You, L. R., Hwang, C.-H., and Lee, Y. H. (1997). Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta-receptor modulates the signal pathway of the lymphotoxin beta pathway. J Virol 71, 9417-9426. Chen, M., Sallberg, M., Sonnerborg, A., Weiland, O., Mattson, L., Jin, L., Birkett, A., Peterson, □., and Milich, D. (1999). Limited humoral immunity in hepatits C virus infection. Gasteroenterology 116,135-143. Chien, □., Choo, Q. L., and Tabrizi, A. (1992). Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture antibodies: Re- evaluation of the role of HCV in liver disease. Proc Natl Acad Sci USA 89,10011-10015. Cho, J. H., Lee, S. W., and Sung, Y. C. (1999). Enhanced cellular immunity to HCV non structural proteins by co-delivery of GM-CSF gene in intramuscular DNA immunisation. Vaccine 17, 1136-1144. Choi, J., Xu, Z., and Ou, J. H. (2003). Triple decoding of hepatitis C virus RNA by programmed translational frameshifting. Mol Cell Biol 23,1489-1497. Choo, Q. L., Kuo, G., Ralston, R., Weiner, A., Chein, D., Van Nest, G., Han, J., Berger, K., Thudium, K., and Kuo, C. (1994). Vaccination of chimpanzees against infection by HCV. Proc Natl Acad Sci USA 91,1294-1298. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne Non-A, Non-B viral hepatitis genome. Science, 359-362. Christensen, J. P., Bartholdy, C., Wordarz, D., and Thomsen, A. R. (2001). Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus. J Immunol 166, 3384-3391. Christensen, J. P., Marker, O., and A.R., T. (1994). The role of CD4+ T cells in cell- mediated immunity to LCMV: studies in MHC class I and class II deficient mice. Scand J Immunol 40, 373-382. Cianciolo, G. J., Bogerd, H., and Snyderman, R. (1988). Human retrovirus-related synthetic peptides inhibit T lymphocyte proliferation. Immunol Lett 19, 7-13. Ciccaglione, A. R., Costantino, A., Marcantonio, C., Equestre, M., Geraci, and Rapicetta, M. (2001). Mutagenesis of hepatitis C virus El protein affects its membrane- permeabilizing activity. J Gen Virol 82, 2243-2250. 289 Ciccaglione, A. R., Marcantonio, G., Costantino, A., Equestre, M., Geraci, A., and Rapicetta, M. (2000). Expression and membrane association of hepatitis C virus envelope 1 protein. Virus Genes 21, 223-226. Ciccaglione, A. R., Marcantonio, C., Costantino, A., Equestre, M., and Rapocetti, M. (2003). Expression of HCV E1 protein in baculovirus-infected cells: effects on cell viability and apoptosis induction. Intervirology 46,121-126. Cladaras, C., and Wold, W. S. (1985). DNA sequence of the early E3 transcription unit of adenovirus 5. Virology 140, 28-43. Cocquerel, L., Meunier, J. C., Op de Beek, A., Bonte, D., Wychowski, C., and Dubuisson, J. (2001). Coexpression of hepatitis C virus envelope proteins El and E2 in cis improves the stability of membrane insertion of E2. J Gen Virol 82,1629-1635. Cocquerel, L, Meunier, J. C., Pillez, A., Wychowski, C., and Dubuission, J. (1998). A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72, 2183-2191. Cocquerel, L., Op de Beeck, A., Lambot, M., Roussel, J., Delgrange, D., Pillez, A., Wychowski, C., Penin, P., and Dubuission, J. (2002). Toplogical chnages in the transmembrane domains of the hepatitis C virus envelope glycoproteins,. EMBO 21, 2893- 2902. Cocquerel, L, Quinn, E. R., Flint, M., Hadlock, K. G., Foung, S. K., and Levy, S. (2003). Recognition of native hepatitis C virus E1E2 heterodimers by human monoclonal antibody. J Virol 77, 1604-1609. Cohen, J. (1999). The Scientific Challenge of Hepatitis C. Science 285, 26-30. Colombo, M., Kuo, G., Choo, Q. L., Donato, M. F., Del Ninno, E., Tommasini, M. A., Dioguardi, N., and Houghton, M. (1989). Prevalance of antibodies to hepatitis C virus in Italian patients with hepatocellular carcinoma. Lancet 2,1006-1008. Colombo, M., Rumi, M. G., Donato, M. F., Tommasini, M. A., Del Ninno, E., Ronchi, G., Kuo, G., and Houghton, M. (1991). Hepatitis C antibody in patients with chronic liver disease and hepatocellular carcinoma. Dig Dis Sci 36, 1130-1133. Conry-Cantelena, C., Van Raden, M., Gibble, J., Melpolder, J., Shakil, A. O., Viladomia, L., Cheung, L., DiBisceglie, A., Hoofnagle, J., Shih, J. W., et al. (1996). Routes of infection, viraemia and liver disease in blood donors found to have HCV infection. N Eng J Med 334, 1691-1696. Cook, D., Beck, M., Coffman, T., Kirby, S., Sheridan, J., Pragnell, I., and Smithies, O. (1995). Requirement of Mlp-1 alpha for an inflammatory response to viral infection. Science 269, 1583-1586. Cooper, S., Erikson, A. L., Adams, E. J., Kansopon, J., Weiner, A. J., Chien, D. Y., Houghton, M., Parham, P., and Walker, C. M. (1999). Analysis of a successful immune response against hepatitis C virus. Immunity 10, 439-449. 290 Corado, J., Toro, F., Rivera, H., Bianco, N. E., Deibis, L., and De Sanctis, J. B. (1997). Impairment of natural killer (NK) cytotoxic activity in hepatitis 0 virus (HCV) infection. Clin Exp Immunol 109, 451-457. Couzigou, P., Richard, L, Dumas, P., Schouler, L, and Fleury, H. (1993). Detection of HCV-RNA in saliva of patients with chronic hepatitis C. Gut 34, 859-60. Crabb, C. (2001). Hard-Won Advances Spark Excitement about Hepatitis C. Science 294, 506-507. Cramp, M. E., Rossol, S., Chokshi, S., Carucci, P., Williams, R., and Naoumov, N. V. (2000). Hepatitis C virus-specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C. Gasteroenterol 118, 346-355. Crotta, S., Stilla, A., Wack, A., D'Andrea, A., Nuti, S., D'Oro, U., Mosca, M., Filliponi, P., Brunetto, R. M., Bonino, P., et al. (2002). Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med 195, 35- 41. Crouzet, J., Naudin, L., Orsini, C., Vigne, E., Perrero, L., Le Roux, A., Benoit, P., Latta, M., Torrent, C., Branellec, D., et si. (1997). Recombinantional construction in Escherichia coii of infectious adenoviral genomes. Proc Natl Acad Sci USA 94,1414-1419. D'Amico, G., and Pornasien, A. (1995). Cryoglobulinémie glomerulnephritis: a membranoproliferative glomerulonephritis induced by hepatitis C virus. Am J Kidney Dis 25, 361-369. Davis, G. L. (2000). Current therapy for chronic hepatitis C. Gasteroenterology 118, S104- S114. Davis, G. L., Balart, L. A., Schiff, E. R., Lindsay, K., Bodenheimer Jr, H. C., Perrillo, R. P., Carey, W., Jacobson, I. M., Payne, J., Dienstag, J. L., et al. (1989). Treatment of chronic hepatitis C with recombinant interferon alfa. A multicenter randomized, controlled trial. N Eng J Med 321, 1501-1506. Davis, G. L., Esteban-Mur, R., Rustgi, V., Hoefs, J., Gordon, S. C., Trepo, C., Shiftman, M. L., Zeuzem, S., Craxi, A., Ling, M. H., and Albrecht, J. (1998). Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. International Hepatitis Intervention Group. N Eng J Med 339,1493-1499. De Vita, S., Sansonno, D., Dolcetti, R., Perraccioli, G., Carbone, A., Cornacchiulo, V., Santini, G., Crovatto, M., Gloghini, A., and Dammacco, P. (1995). Hepatitis C virus within a malignant lymphoma lesion in the course of type II mixed cryoglobulinaemia. Blood 86, 1887-1892. Deleersnyder, V., Pillez, A., Wychowski, C., Blight, K., Xu, J., Hahn, Y. S., Rice, C. M., and Dubuisson, J. (1997). Formation of native hepatitis C virus glycoprotein complexes. J Virol 71, 697-704. Di Bisceglie, A., Martin, P., Kassianides, C., Lisker-Melman, M., Murray, L., Waggoner, J., Goodman, Z., Banks, S. M., and Hoofnagle, J. H. (1989). Recombinant interferon alfa 291 therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med 321, 1506-1510. DiBisceglie, A. M., and Shindo, M. (1992). A pilot study of ribavirin therapy for chronic hepatitis C. Hepatology 16, 649-654. Diepolder, H., Scholz, S., and Pape, G. (1999). Influence of HI_A alleles on outcome of hepatitis 0 virus infection. Lancet 354, 2094-2095. Diepolder, H. M., Zachoval, R., Hoffmann, R. M., Wierenga, E. A., Santantonio, T., Jung, M. C., Eichenlaub, D., and Pape, G. R. (1995). Possible mechanism involving T- lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. The Lancet 346, 1006-1007. Dietz, A. B., and Vuk-Pavlovic, S. (1998). High efficiency adenovirus-mediated gene transfer to human dendritic cells. Blood 91, 392-398. Dittmann, S., Roggendorf, M., Durkop, J., Wiese, M., Lorbeer, B., and Deinhardt, F. (1991). Long-term persistence of hepatitis C virus antibodies in a single source outbeak. J Hepatol 13, 323-327. Dolganiuc, A., Kodys, K., Kopasz, A., Marshall, C., Do, T., Romics, L., Mandrekar, P., Zapp, M., and Szabo, G. (2003). Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation. J Immunol 170, 5615-5624. Domingo, E., and Holland, J. J. (1994). Muatation rates and rapid evolution of RNA viruses. In Evolutionary Biology of Viruses, M. S.S., ed. (New York, Raven Press), pp. 161-184. Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, D., Pardoll, D. M., and Mulligan, R. C. (1993). Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 90. Dubuisson, J., Hsu, H. H., Cheung, R. 0., Greenberg, H. B., Russell, D. G., and Rice, 0. M. (1994). Formation and intracellular localisation of hepatitis 0 virus envelope glycoprotein complexes expressed by recombinant vaccinia and Sindbis viruses. J Virol 68, 6147-6160. Dubuisson, J., and Rice, 0. M. (1996). Hepatitis 0 virus glycoprotein folding: Disulphide bond formation and association with calnexin. J Virol 70, 718. Duenas-Carrera, S., Alvarez-Lajonchere, L., Alvarez, J. 0., Ramos, T., Pichardo, D., and Morales, J. (2001). Repeated administration of hepatitis C virus core-encoding plasmid to mice does not necessarily increase the immune repense generated against this antigen. Biotechnol AppI Biochem 33, 47-51. 292 Dumas, E., Staedel, C., Colombat, M., Reigadas, S., Chabas, S., Astier-Gin, T., Cahour, A., Litvak, S., and Ventura, M. (2003). A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR. Nucleic Acids Res 31,1275-1281. Dumoulin, F. L., von dem Bussche, A., Sohne, J., Sauerbruch, T., and Spengler, U. (1999). Hepatitis C virus core protein does not inhibit apoptosis in human hepatoma cells. E J Clin Invest 29, 940-946. Dusheiko, G., Main, J., Thomas, H., Reichard, O., Lee, 0., Dhillon, A., Rassam, S., Fryden, A., Reesink, H., Bassendine, M., etal. (1996). Ribavirin treatment for patients with chronic hepatitis c: results of a placebo-controlled study. J Hepatol 25, 591-598. Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H., Moradapour, D., and Bienz, K. (2002). Expression of hepatitis 0 virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 5974-5984. Enomoto, N., Sakuma, I., Asahina, Y., Kurosaki, M., Murakami, T., Yamamoto, 0., Izumi, N., Marumo, F., and Sato, 0. (1995). Comparison of full-length sequences of interferon- sensitive and resistant hepatitis C virus 1b. J Clin Invest 96, 224-230. Enomoto, N., Sakuma, I., Asahina, Y., Kurosaki, M., Murakami, T., Yamamoto, C., Ogura, Y., Izumi, N., Marumo, F., and Sato, C. (1996). Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 334, 77-81. Erdtmann, L., Franck, N., Lerat, H., Le Seyec, J., Gilot, D., Cannie, I., Gripon, P., Hibner, U., and Guguen-Guillouzo, C. (2003). The hepatitis C virus NS2 protein: an inhibitor of CIDE-B- induced apoptosis. J Bio Chem 278,18256-18264. Erikson, A. L., Houghton, M., Choo, Q. L., Weiner, A. J., Ralston, R., Muchmore, E., and Walker, C. M. (1993). Hepatitis C virus-specific CTL response in the liver of chimpanzees with acute and chronic hepatitis C. J Immunol 151, 4189-4199. Esteban, J. I., Gomez, J., Martell, M., Cabot, B., Quer, J., Camps, J., Gonzalez, A., Otero, T., Moya, A., and Esteban, R. (1996). Transmission of hepatitis C virus by a cardiac surgeon. N Eng J Med 334, 555-560. Esumi, M., Rikihisa, T., Nishimura, S., Goto, J., Mizuno, K., Zhou, Y. H., and Shikata, T. (1999). Experimental vaccine activities of recombinant El and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol 144, 973- 980. Farci, P., Alter, H., Govindarajan, S., Wong, D. C., Engle, R., and Lesniewski, R. R. (1992). Lack of protective immunity against reinfection with hepatitis C virus. Science 258, 135-140. Farci, P., Alter, H. J., Wong, D., Miller, R. H., Shih, J. W., Jett, B., and Purcell, R. H. (1991). A long term study of hepatitis C virus replication in non-A non-B hepatitis. N Eng J Med 325, 98-104. 293 Farci, P., Alter, H. J., Wong, D. C., Miller, R. H., Govindarajan, S., Engle, R., Shapiro, M., and Purcell, R. H. (1994). Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vivo neutralisation. Proc Natl Acad Sci USA 91, 7792-7796. Farci, P., and Purcell, R. H. (2000). Clinical Significance of Hepatitis 0 Virus Genotypes and Quasispecies. Semin Liver Dis 20,103-124. Farci, P., Shimoda, A., Coiana, A., Diaz, G., Peddis, G., Melpolder, J. 0., Strazzera, A., Chein, D. Y., Munoz, S. J., Balestrieri, A., et al. (2000). The Outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 288, 339-344. Farci, P., Shimoda, A., Wong, D. C., Cabezon, T., De Gioannis, D., Strazzera, A., Shimizu, Y., Shapiro, M., Alter, H. J., and Purcell, R. H. (1996). Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 glycoprotein. Proc Natl Acad Sci USA 93, 15394-15399. Feinstone, S. M., and Alter, H. J. (1981). NANB hepatitis in chimpanzees and marmosets. J Infect Dis 144, 588-598. Feinstone, S. M., and Mihalik, K. B. (1983). Inactivation of HCV and NANB hepatitis by chloroform. Infect Immun 41, 816-821. Ferlazzo, G., Tsang, M. L., Moretta, L., Melioli, G., Steinman, R. M., and Munz, C. (2000). Human dendritic cells activate resting natural killer (NK) cells and re recognised via the NKp30 receptor by activated NK cells. J Exp Med 195, 343-351. Flint, M., and McKeating, J. A. (2000). The role of hepatitis C virus glycoproteins in infection. Rev Med Virol 10, 101-117. Fong, T. L., Di Bisceglie, A. M., Waggoner, J. C., Banks, S. M., and Hoofnagle, J. H. (1991). The significance of antibody to hepatitis C virus in patients with chronic hepatitis B. Hepatology 14, 64-67. Forns, X., Emerson, S. U., Tobin, G. J., Mushahwar, I. K., Purcell, R. H., and Bukh, J. (1999). DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface. Vaccine 17, 1992- 2002. Forns, X., Payette, P. J., Ma, X., Satterfield, W., Eder, G., Mushahwar, I. K., Govindarajan, S., Davis, H. L., Emerson, S. U., Purcell, R. H., and Bukh, J. (2000). Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV. Hepatology 32, 618-625. Foy, E., Li, K., Wang, C., Sumpter, R. J., Ikeda, M., Lemon, S. M., and Gale, M. J. J. (2003). Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300,1145-1151. Frank, C., Mohamed, M. K., Strickland, G. T., Lavanchy, D., Arthur, R. R., Magder, L. S., El Khoby, T., Abdel-Wahab, Y., Aly Ohn, E. S., Anwar, W., and Sallam, I. (2000). The role 294 of parental anti-schistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 355, 887-891. Friebe, P., and Bartenschlager, R. (2002). Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 76, 5326-5338. Frieta, D., Lin, T.J., Quezada, S.A., Wade, T.K., Barth, R.J., Noelle, R.J., Wade, W.F., (2003), Distinctive maturation of in vitro versus in vivo anti-CD40 mAh matured dendritic cells in mice, J. Immunother., 26, 72-84. Fruh, K., Ahn, K., Djaballah, H., Sempe, P., Van Endert, P. M., Tanmpe, R., Peterson, P. A., and Yang, Y. (1995). A viral inhibitor of peptide transporters for antigen presentation. Nature 375, 415-418. Fujita, T., Ishido, S., Muramatsu, S., Itoh, M., and Hotta, H. (1996). Suppression of actinomycin D-induced apoptosis by the NS3 protein of hepatitis C virus. Biochem Biophys Res Comm 229, 825-831. Fukuda, K., Tsushihara, K., Hijikata, M., Nishiguchi, S., Kuroki, T., and Shimotohno, K. (2001). Hepatitis 0 virus core protein enhances the activation of the transcription factor, Elk-1, in response to mitogenic stimuli. Hepatology 33,159-165. Funk, J. O., Walczak, H., Voigtlander, 0., Berchtold, S., Baumeister, T., Rauch, P., Rossner, S., Steinkasserer, A., Schuler, G., and Lutz, M. B. (2000). Cutting Edge: Resistance to apoptosis and continuous proliferation of dendritic cells deficient for TNF receptor-1. J Immunol 165, 4792-4796. Gabrielli, A., Mazin, A., Candela, M., Caniglia, M. L., Paolucci, S., Daniel, G., and Clementi, M. (1994). Active hepatitis C virus infection in bone marrow and peripheral blood mononuclear cells from patients with mixed cryoglobulinaemia. Clin Exp Immunol 97, 87- 93. Gabrielli, A., Sambo, P., Zhang, Z.-X., Candela, M., Savoldi, S., Manzin, A., Clementi, M., Amoroso, A., Sallberg, M., and Danieli, G. (1995). Humoral immune response and natural killer activity in patients with mixed cryoglobulinemia. Clin Exp Rheumatol 13, S95-99. Gale, M. J. J., Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R., and Katze, M. G. (1997). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217-227. Gamadia, L. E., Remmerswaal, E. B., Weel, J. F., Bemelman, F., Van Lier, R. A., and Ten Berge, I. J. (2003). Primary immune responses to CMV: a critical role for IFN-gamma- producing CD4+ T cells in protection against CMV disease. Blood 101, 2686-2692. Garson, J. A., Whitby, K., Watkins, P., and Morgan, A. J. (1997). Lack of susceptibility of the cotton top tamarin to hepatitis C virus infection. J Med Virol 52, 286-288. Geijtenbeek, T. B., and van Kooyk, Y. (2003). DC-SIGN: a novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol 276, 31-54. 295 Geissler, M., Geisen, A., Tokushige, K., and Wands, J. R. (1997). Enhancement of cellular and humoral immune responses to hepatitis C virus core protein using DNA based vaccines augmented with cytokine expressing plasmids. J Immunol 158,1231-1237. Gerlach, T., Diepolder, H., Jung, M. C., Greuner, N., Schraut, W., Zachoval, R., Hoffmann, R., Schirren, C. A., Santantonio, T., and Pape, G. (1999). Recurrence of HCV after loss of virus specific CD4+ T cell response in acute hepatitis 0. Gasteroenterol 117, 933-941. Germi, R., Crance, J. M., Garin, D., Guimet, J., Lorat-Jacob, H., Ruigrok, R. W., Zarski, J. P., and Drouet, E. (2002). Cellular glycosaminoglycans and low density lipoprotein receptor are involved in hepatitis C virus adsorption. J Med Virol 68, 206-215. Gerosa, P., Baldani-Guerra, B., Nisii, C., Marchesini, V., Carra, G., and Trinchieri, G. (2002). Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med 195, 327-333. Ghosh, A. K., Majumder, M., Steele, R., Ray, R., and Ray, R. B. (2003). Modulation of interferon expression by hepatitis C virus NS5A protein and human homeodomain protein ptxi. Virology 306, 51-59. Giannelli, G., Antonelli, G., and Fera, G. (1993). 2'5'-OAS activity as a repense marker during interferon therapy for chronic hepatitis C virus. J Interferon Res 13, 57-60. Giannini, C., Caini, P., Giannelli, P., Fontana, P., Kremsdorf, D., Brechot, C., and Zignego, A. L. (2002). Hepatitis C virus core protein expression in human B-cell line does not significantly modify main proliferative and apoptosis pathways. J Gen Virol 83,1665-1671. Goeser, T., Muller, H. M., Solbach, C., Gmelin, K., Kurzen, P., and Kommerell, B. (1994). Prevalence and serological manifestation of hepatitis C virus infection in patients with non- A, non-B: A follow-up study. Presse Med 23, 793-796. Goh, P. Y., Tan, Y. J., Lim, S. G., Tan, Y. H., and Hong, W. J. (2001). The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolyic cleavage. Virology 290, 224-236. Gomez, J., Martell, M., Quer, J., Cabot, B., and Esteban, J. I. (1999). Hepatitis C viral quasispecies. J Viral Hep 6, 3-16. Gong, G. Z., Lai, L. Y., Jiang, Y. P., He, Y., and Su, X. 8. (2003). HCV replication in PBMC and its influence on interferon therapy. World J Gastroenterol 9, 291-294. Gonzalez-Peralta, R. P., Pang, J. W. S., and Davis, G. L. (1994). Immunopathobiology of chronic hepatitis C virus infection. Hepatology 20, 232A. Goodbourne, S., Didcock, L., and Randall, R. E. (2000). Interferons: cell signalling, immune modulation, antiviral responses and virus countermeasures. J Gen Virol 81, 2341- 2364. 296 Gooding, L. R., Elmore, L. W., Tollefson, A. E., Brady, H. A., and Wold, W. S. (1988). A 14,700 MW protein from the E3 region of adenovirus inhibits cytolysis by tumor necrosis factor. Cell 53, 341-346. Gooding, L. R., Ranheim, T. S., Tollefson, A. E., Aquino, L., Duerksen-Hughes, P., Horton, T. M., and Wold, W. S. (1991). The 10,400- and 14,500-dalton proteins encoded by region E3 in adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor. J Virol 65, 4114-4123. Gottlieb, R. A., Klieinerman, E. S., O'Brien, C. A., Tsujimoto, S., Cianciolo, G. J., and Lennarz, W. J. (1990). Inhibition of protein kinase C by a peptide conjugate homologous to a domain of the retroviral protein p15E. J Immunol 145, 2566-2570. Goutagny, N., Fatmi, A., Ledinghen, V. D., Penin, P., Couzigou, P., Inchauspe, G., and Bain, C. (2003). Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection. J Infect Dis 187, 1951-1958. Graham, F. L. (1984). Covalently closed circles of human adenovirus DNA are infectious. EMBO J 3, 2917-2922. Graham, F. L. (2000). Adenovirus vectors for high-efficiency gene transfer into mammalian cells. Trends in Immunology 21, 426-428. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993a). Characterisation of the hepatitis C virus-encoded serine protease: determination of proteinase-dependant polyprotein cleavage sites. J Virol 67, 2832-2843. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993b). A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci USA 90,10583-10587. Grakoui, A., Wychowski, C., Lin, C. H., Feinstone, S. M., and Rice, C. M. (1993c). Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol 67, 1385-1395. Grant, M. D., Smaill, F. M., and Rosenthal, K. L. (1993). Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic lymphocytes from HIV-infected individuals. Clin Exp Immunol 93, 356-362. Gregory, C. D., Murray, R. J., Edwards, C. F., and Rickinson, A. B. (1988). Downregulation of cell adhesion molecules LFA-3 and ICAM-1 in Epstein-Barr virus-positive Burkitt's lymphoma underlies tumor cell escape from virus-specific T cell surveillance. 1988 167, 1811-1824. Griffin, S. D., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., Harris, M. P., and Rowlands, D. J. (2003). The p7 protein of hepatitis C virus forms an ion channel that blocked by the antiviral drug. Amantadine. FEBS Lett 535, 34-38. Grobler, J. A., Markel, E. J., Fay, J. F., Graham, D. J., Simcoe, A. L., Ludmerer, 8. W., Murray, E. M., Migliaccio, G., and Flores, O. A. (2003). Identification of a key determinant of hepatitis C virus cell-culture adaptation in domain II of NS3 helicase. J Biol Chem 278, 16741-16746. 297 Gruner, N. H., Gerlach, T. J., Jung, M. C., Diepolder, H. M., Albrecht Schirren, C., Schraut, W. W., Hoffmann, R., Zachoval, R., Sanantonio, T., Cucchiarini, M., et al. (2000). Association of hepatitis C virus-specific CDS"" T cells with viral clearance in acute hepatitis C. J Infect Dis 181, 1528-1536. Gu, B., Johnston, V. K., Gutshall, L. L., Nguyen, T. T., Gontarek, R. R., Darcy, M. G., Tedesco, R., Dhanak, D., Duffy, K. J., Kao, C. C., and Sarisky, R. T. (2003). Arresting initiation of HCV RNA synthesis using heterocyclic derivatives. J Biol Chem 278, 16602- 16607. Guidotti, L. G., and Chisari, F. V. (1996). To kill or cure: options in host defense against viral infection. Curr Opin Immunol 8, 478-483. Guidotti, L. G., and Chisari, F. V. (2000). Cytokine-mediated control of viral infections. Virology 273, 221-227. Guidotti, L. G., Morris, A., Mendez, H., Koch, R., Silverman, R. H., Williams, B. R. G., and Chisari, F. V. (2002). Interferon-regulated pathways that control hepatitis B virus replication in transgenic mice. J Virol 76, 2617-2621. Haber, M. M., West, A. B., Haber, A. D., and Reuben, A. (1995). Relationship of aminotransferases to liver histological status in chronic hepatitis C virus. Am J Gasteroenterol 90, 1250-1257. Haddad, J., Deny, P., Munz-Gotheil, C., Ambrosini, J. C., Trinchet, J., Paterson, D., Mai, F., Callard, P., and Beaugrand, M. (1992). Lymphocytic sialadenitis of Sjogren's syndrome associated with chronic hepatitis C virus liver disease. Lancet 339, 321-323. Hahn, C. S., Cho, Y. G., Kang, B. S., Lester, I. M., and Hahn, Y. S. (2000). The HCV core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology 276, 127-137. Hanke, T., Graham, F. L., Lulitanond, V., and Johnson, D. C. (1990). Herpes simplex virus IgG Fc receptors induced using recombinant adenovirus vectors expressing glycoproteins E and I. Virology 177, 437-444. Harada, S., Watanabe, Y., Takeeuchi, K., Suzuki, T., Katayama, T., Takebe, Y., Saito, I., and Miyamura, T. (1991). Expression of processed core protein of hepatitis C virus in mammalian cells. J Virol 65, 3015-3021. Harada, T., Tautz, N., and Theil, H. J. (2000). E2-p7 region of the bovine diarrhea virus polyprotein: processing and functional studies. J Virol 74, 9498-9506. Haraguchi, S., Good, R. A., Cianciolo, G. J., Engleman, R. W., and Day, N. K. (1997). Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections. J Leukoc Biol 61, 654-666. Harcourt, G., Hellier, S., Bunce, M., Satsangi, J., Collier, J., Chapman, R., Phillips, R. E., and Klenerman, P. (2001). Effect of HLA class II genotype on T helper lymphocyte responses and viral control in HCV infection. J Viral Hepat 8, 174-179. 298 Hardy, R. W., Marcotrigiano, J., Blight, K. J., Majors, J. E., and Rice, C. M. (2003). Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J Virol 77, 2029-2037. Haufater, P., Rosenthal, E., and Cocoub, P. (2000). Lymphoproliferative diseases and hepatitis C virus infection. Ann Med Interne (Paris) 151, 53-57. He, L. P., Ailing, D,, Shapiro, M., Alter, M., and Purcell, R. H. (1987). Determining the size of non-A non-B hepatitis virus by filtration. J Infect Dis 156, 636-640. He, T.-C., Zhou, S., Da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci 95, 2509- 2514. He, X.-S., and Greenberg, H. B. (2002). CD8+ T-cell response against Hepatitis 0 virus. Viral Immunol 15, 121-131. He, X. S., Rehermann, B., Lopez-Labrador, F. X., Boisvert, J., Cheung, R., and Mumm, J. (1999). Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers. Proc Natl Acad Sci USA 96, 5692-5697. He, Y., Nakao, H., Tan, S. L., Polyak, S. J., Neddermann, P., Vijaysri, S., Jacobs, B. L., and Katze, M. G. (2002). Subversion of cell signalling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase. J Virol 76, 9270-9217. He, Y., Yan, W., Coito, C., Li, Y., Gale, M. J., and Katze, M. G. (2003). The regulation of hepatitis C virus (HCV) internal ribosome-entry site-mediated translation by HCV replicons and nonstructural proteins. J Gen Virol 84, 535-543. Healy, C. J., Chapmann, R. W. G., and Fleming, K. A. (1995). Liver histology in hepatitis C infection: a comparison between patients with persistently normal or abnormal transaminases. Gut 37, 274-278. Heathcote, E. J., Shiftman, M. C., Cooksley, W. G. E., DuSheiko, G. M., Lee, S. S., Balort, L., Reindouor, R., Reddy, R. K., Wright, T. L., Lin, A., ef a/. (2000). Peginterferon alpha2a in patients with chronic hepatitis C and cirrhosis. New Eng J Med 343,1673-1680. Heinrich, D., Kotitschke, R., and Berthold, H. (1982). Clinical evaluation of the hepatitis safety of a beta-propriolactone/UV treated factor IX concentrate (PPSB). Thromb Res 28, 75-83. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G., and Rickinson, A. B. (1993). Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci USA 90, 8479-8483. Hermans, I. F., Ritchie, D. S., Yang, J., Roberts, J. M., and Ronchese, F. (2000). CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity. J Immunol 164, 3095-3101. 299 Herzer, K., Falk, C. S., Encke, J., Eichhorst, S. T., Ulsenheimer, A., Selinger, B., and Krammer, P. H. (2003). Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity. J Virol 77, 8299-8309. Hiasa, Y., Horiike, N., Akbar, S. M. P., Saito, I., Miyamura, T., Matsuura, Y., and Onji, M. (1998). Low stimulatory capacity of lymphoid dendritic cells expressing hepatitis C virus genes. Biochem Biophys Res Comm249, 90-95. Hidaka, 0., Milano, E., Leopold, P. L., Bergelson, J. M., Hackett, N. R., Finberg, R. W., Wickham, T. J., Kovesdi, I., Roelvink, P., and Crystal, R. G. (1999). CAR-dependent and CAR-independent pathways of adenovirus-vector mediated gene transfer and expression in human fibroblasts. J Clin Invest 103, 579-587. Higashi, Y., Kakumu, S., Yoshioka, K., Wakita, T., Mizokami, M., Ohba, K., Ito, Y., Ishikawa, T., Takayangi, M., and Nagai, Y. (1993). Dynamics of genome change in the E2/NS1 region of hepatitis C virus in vivo. Virology 197, 659-668. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., and Shimotohno, K. (1991a). Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175, 220-228. Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., and Shimotohno, K. (1991b). Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc Natl Acad Sci USA 88, 5547-5551. Hijikata, M., Mizushima, H., Tanji, Y., Komodo, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K., and Shimotohno, K. (1993). Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci USA 90, 10773- 10777. Hill, A., Jugovic, P., York, I. A., Russ, G., Bennink, J., Yewdell, J., Ploegh, H., and Johnson, D. C. (1995). Herpes simplex virus turns off the TAP to evade host immune responses. Nature 375, 411-415. Hino, K., Sainokami, S., Shimoda, K., Niwa, H., and lino, S. (1994). Clinical course of acute hepatitis C virus and changes in hepatitis C virus markers. Dig Dis Sci 3 919-27. , Hiramatsu, N., Hayashi, N., and Katayama, K. (1994). Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 1 91354-1359. , Hiranuma, K., Tamaki, S., Nishimura, Y., Kusaki, J., Igogawa, M., Kim, G., Kaito, M., Kuribayashi, K., Adachi, Y., and Yasutimi, Y. (1999). Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against HCV. J Gen Virol 8 0 ,187-193. Hirschowitz, E. A., Weaver, J. D., Hidalgo, G. E., and Doherty, D. E. (2000). Murine dendritic cells infected with adenovirus vectors show signs of activation. Gene Ther 7, 1112- 1120. 300 Hoffmann, R., Diepolder, H., Zachoval, R., Zwiebel, F. M., Jung, M. C., and Scholz, S. (1995). Mapping of immunodominant CD4+ T lymphocyte epitopes of hepatitis C virus antigens and their relevance during the course of chronic infection. Hepatology21, 632- 638. Hollinger, F. B., Gitnick, G., Aach, R. D., Semuness, W., Mosely, J. W., Stevens, C. E., Peters, R. L., Weiner, J. M., Welch, J. B., and Lander, J. J. (1978). Non-A non-B hepatitis tranmission in chimpanzees: A project of the transfusion-transmited viruses study group. Intervirology 10, 60-68. Honda, A., Hatano, M., Kohara, M., Arai, Y., Hartatik, T., Moriyama, T., Imawari, M., Koike, K., Yokosuka, O., Shimotohno, K., and Tokuhisa, T. (2000). HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice. J Hepatol 33, 440-447. Hong, S. S., Karayan, L., Tournier, J., Curiel, D. T., and Boulanger, P. A. (1997). Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoma cells. EMBO J 16, 2294-2306. Hong, Z., Beadet-Miller, M., Lanford, R. E., Guerra, B., Wright-Minogue, J., Skelton, A., Boroudy, B. M., Reyes, G. R., and Lau, J. Y. (1999). Generation of transmissible HCV virions from a molecular clone in chimpanzees. Virology 256, 36-44. Hoofnagle, J., Mullen, K. D., Jones, D. B., Rustgi, V., Di Bisceglie, A., Peters, M., Waggoner, J. G., Park, Y., and Jones, E. A. (1986). Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon . A preliminary report. N Eng J Med 315, 1575-1578. Hope, R. G., and McLauchlan, J. (2000). Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis 0 virus core protein. J Gen Virol 81, 1913-1925. Horton, I . M., Ranheim, I . S., Aquino, L., Kusher, D. I., Shah, S. K., Ware, 0. F., Wold, W. S., and Gooding, L. R. (1991). Adenovirus E3 14.7K protein functions in the absence of other adenovirus protein to protect transfected cells from tumor necrosis factor cytolysis. J Virol 65, 2629-2639. Hsieh, T.-Y., Matsumoto, M., Chou, H.-C., Schneider, R., Hwang, S. B., Lee, A. S., and Lai, M. M. (1998). Hepatitis C virus core protein interacts with heterogeneous ribonuclearprotein K. J Biol Chem273, 17651-17659. Hsu, H. H., Donets, M., Greenberg, H. B., and Feinstone, S. M. (1993). Characterisation of hepatitis C virus structural proteins with a recombinant baculovirus expression system. Hepatology 17, 763-771. Hu, G. J., Wang, R. Y., Han, D. S., Alter, H. J., and Shih, J. W. (1999). Characterisation of the humoral and cellular responses against HCV core induced by DNA-based immunisation. Vaccine 17, 3160-3170. 301 Hu, Y., Shahidi, A., Park, S., Guilfoyle, D., and Hirshfield, I. (2003). Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction. Am J Clin Pathol 119, 95-100. Huang, S., Kamata, T., Takada, Y., Ruggeri, Z. M., and Nemerow, G. R. (1996). Adenovirus interaction with distinct integrins mediated separate events in cell entry and gene delivery to hematopoietic cells. J Virol70, 4502-4508. Hugle, T., Fehrmann, P., Bieck, E., Kohara, M., Krausslich, H. G., Rice, 0. M., Blum, H. E., and Moradapour, D. (2001). The hepatitis 0 virus nonstructural protein 4B is an integral endoplasmic reticulum protein. Virology284, 70-81. Hung, 0. H., Lee, 0. M., Lu, S. N., Lee, J. P., Wang, J. H., Tung, H. □., Chen, T. M., Hu, T. H., Chen, W. J., and Changchien, C. S. (2003). Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin. J Viral Hepat 10, 87-94. Hunziker, I. P., Zurbriggen, R., Glueck, R., Engler, O. B., Reichen, J., Dai, W. J., Pichler, W. J., and Cerny, A. (2001). Perspectives: towards a peptide-based vaccine against hepatitis C virus. Mol Immunol 38, 475-484. Hunzulker, I., Grabsheld, B., Zurbriggen, R., Gluck, R., Pichler, W. J., and Cerny, A. (2002). In vitro studies of core peptide-bearing immunopotentiating reconstituted influenza virosomes as a non-live prototype vaccine against hepatitis C virus. Int Immunol 14, 615- 526. Hussy, P., Langen, H., Mous, J., and Jacobsen, H. (1996). Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology224, 93-104. Hwang, S. B., Lo, S.-Y., Ou, J.-H., and Lai, M. M. (1995). Detection of cellular proteins and viral core protein interacting with the 5' untranslated region of hepatitis C virus RNA. J Biomed Sci 2, 93-104. Ikeda, K., Saitoh, S., Koida, I., Arase, Y., Tsubota, A., Chayama, K., Kumao, H., and Kawanishi, M. (1993). A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology 18, 47-53. Inchauspe, G., Major, M. E., Nakano, I., Vivitski, L., Maisonnas, M., and Trepo, C. (1998). Immune responses against hepatitis C virus structural proteins following genetic immunisation. Dev Biol Stand 9 2163-168. , Ishido, S., Pujita, T., and Hotta, H. (1998). Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus. Biochem Biophys Res Commun 244, 35-40. Ishii, K., Rosa, D., Y., W., Katayama, T., Harada, H., Wyatt, C., Kiyosawa, K., Aizaki, H., Matusuura, Y., Houghton, M., et a/. (1998). High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of hepatitis C. Hepatology28, 1117-1120. 302 Isoyama, T., Kuge, S., and Nomoto, A. (2002). The core protein of hepatitis C virus is imported into the nucleus by transport receptor Kap123p but inhibits Kap123p-dependent nuclear import of yeast AP1-like transcription factor in yeast cells. J Biol Chem 277, 39634-39641. James, P. (2000). Measurement of proliferative responses of cultured lymphocytes. In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. (John Wiley & Sons, Inc.), pp. 7.10. Janssen, E. M., Lemmens, E. E., Wolfe, T., Christen, U., von Herrath, M. G., and Schoenberger, S. P. (2003). CD4+ I cells are required for secondary expansion and memory in CD8+ I lymphoc^es. Nature 421, 852-856. Jarvis, J. N., Kaplan, J., and Fine, N. (1992). Increase in CD5+ B-cells in juvenile rheumatoid arthritis - Relationship to IgM rheumatoid factor expression and disease activity. Arthritis Rheum 35, 204. Jiao, X., Wang, R. Y., Feng, Z., Alter, H., and Shih, J. W. (2003). Modulation of cellular immune responses against hepatitis C virus nonstructural protein 3 by cationic liposome encapsulated DMA immunisation. Hepatology 37, 452-460. Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E., Sarfrit, J. T., Mittler, J., Weinberger, et L., ai (1999). Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodefciency virus-infected macaques. J Exp Med 189, 991-998. Jonuleit, H., luting. T., Steitz, J., Bruck, J., Giesecke, A., Steinbrink, K., Knop, J., and Enk, A. H. (2000). Efficient transduction of mature CDOO'" dendritic cells using recombinant adenovirus suppressed I cell stimulatory capacity. Gene Ther 7, 249-254. Joseph, B. S., Lampert, P. W., and Oldstone, M. B. A. (1975). Replication and persistence of measles virus in defined subpopulations of human leukocytes. J Virol 16, 1638-1649. Kagawa, S., Gu, J., Honda, T., McDonnell, T. J., Swisher, S. G., Roth, J. A., and Fang, B. (2001 ). Deficiency of caspase-3 in MCF7 cells blocks Bax-mediated nuclear fragmentation but not cell death. Clin Cancer Res 7,1474-1480. Kakimi, K., Guidotti, L. G., Koezuka, Y., and Chisari, F. V. (2000). Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 192, 921-930. Kakumu, S., Ito, S., Ishikawa, T., Mita, Y., Tagaya, T., Fukuzawa, Y., and Yoshioka, K. (2000). Decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis B and C virus infection. J Gastroenterol Hepatol 15, 431-436. Kalkeri, G., Khalap, N., Garry, R. F., Fermin, C. D., and Dash, 8. (2001). Hepatitis C virus protein expression induces apoptosis in HepG2 cells. Virology 282, 26-37. Kamei, A., Tamaki, S., Taniyama, H., Takamura, S., Nishimura, Y., Kagawa, Y., Uno- Furuta, S., Kaito, M., Kim, G., Toda, M., at si (2000). Induction of hepatitis C virus-specific 303 cytotoxic T lymphocytes in mice by an intrahepatic innoculation with an expression plasmid. Virology 273,120-126. Kaneko, T., Tanji, Y., Satoh, S., Hijikata, M., Asabe, S., Kimura, K., and Shimotohno, K. (1994). Production of two phosphoproteins from the NS5A region of the hepatitis C virus genome. Biochem Biophys Res Comm205, 320-326. Kaneko, Y. B., Harada, M., Kawano, T., Yamashita, M., Shibata, Y., Gejco, F., Nakayama, T., and Taniguchi, M. (2000). Augmentation of Valpha14 NKT cell-mediated cytotoxicity by interleukin-4 in an autocrine mechanism resulting in the development of concanavalin A- induced hepatitis. J Exp Med 191, 105-114. Kanto, T., Hayashi, N., Takehara, T., Tatsumi, T., Kuzushita, N., Ito, A., Sansaki, Y., Kasahara, A., and Hori, M. (1999). Impaired immunostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis 0 virus-infected individuals. J Immunol162, 5584-5591. Kapadia, S. B., Brideau-Anderson, A., and Chisari, F. V. (2003). Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA 100, 2014-2018. Karayiannis, P., Scheur, P. J., Bamber, M., Cohn, D., Hum, B. A., and Thomas, H. C. (1983). Experimental infection of tamarins with human NANB hepatitis virus. J Med Virol fY, 251-256. Kato, J., Kato, N., Yoshida, H., Ono-Nita, S. K., Shiratori, Y., and Omata, M. (2002). Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo. J Med Virol 66, 187-199. Kato, N., Ootsuyama, Y., Tanaka, T., Nakagawa, M., Nakazawa, T., Muraiso, K., Okoshi, S., Hijikata, M., and Shimotohno, K. (1992). Marked sequence diversity in the putative envelope proteins of hepatitis C virus. Virus Res 22,107-123. Kato, N., Sekiya, H., Ootsuyama, Y., Nakazawa, T., Hijikata, M., Ohkoshi, S., and Shimotohno, K. (1993). Humoral response to hypervariable region 1 of the putative envelope glycoprotein (gp70) of hepatitis C virus. J Virol 67, 3923-3930. Kato, T., Miyamoto, M., Furusaka, A., Date, T., Yasui, K., Kato, J., Matsushima, S., and Wakita, T. (2003). Processing of hepatitis C virus core protein is regulated by its C- terminal sequence. J Med Virol69, 357-366. Katsoulidou, A., Paraskevis, D., Kalapothaki, V., Arvantiti, S. D., Karayiannis, P., Hadjiconstantiou, V., and Hatzakis, A. (1999). Molecular epidemiology of a hepatitis C virus outbreak in a haemodialysis unit: Multi-centre haemodialysis cohort study on viral hepatitis. Nephrol Dial Transplant 14,1188-1194. Kawamura, T., Furusaka, A., M.J., K., Chung, R. T., Wong, T. C., Schmidt, E. V., and Liang, T. J. (1997). Transgenic expression of the hepatitis C virus structural proteins in the mouse. Hepatology 2 51014-1021. , 304 Kien, F., Abraham, J. D., Schuster, C., and Kieny, M. P. (2003). Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins. J Gen Virol 84, 561-566. Kilinman, D. M., and Nutman, T. B. (2000). ELISPOT assay to detect cytokine secreting murine and human cells. In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. (John Wiley & Sons, Inc.), pp. 6.19. Kim, H. S., Lee, J. K., Yang, I. H., Ahn, J. K., Oh, Y.-l., Kim, C. J., Kim, Y. S., and Lee, C.- K. (2002a). Identification of Hepatitis C Virus Core Domain Inducing Suppression of Allostimulatory Capacity of Dendritic Cells. Arch Pharm Res 25, 364-369. Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., et al. (1996). Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell87, 343-355. Kim, Y. K., Kim, C. S., Lee, S. H., and Jang, S. K. (2002b). Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication. Biochem Biophys Res Commun 290, 105-112. Kinkhabwala, M., Sehajpal, P., and Skolnik, E. (1990). A novel addition to the T cell repertory: Cell surface expression of tumor necrosis factor/cachectin by activated normal human T cells. J Exp Med 171, 941-946. Kittlesen, D. J., Chianese-Bullock, K. A., Yao, Z. Q., Braciale, T. J., and Hahn, Y. S. (2000). Interaction between complement receptor gClqR and hepatitis C virus core protein inhibits T-lymphocyte proliferation. J Clin Invest106, 1239-1249. Kiyosawa, K., Sodeyama, T., Tanka, E., Gibo, Y., Yoshizawa, K., Nankano, Y., Furuta, S., Ahkahane, Y., Nishioka, K., and Purcell, R. H. (1990). Inter-relationship of blood transfusion, non-A non-B hepatitis and hepatocellular carcinome: Analysis by detection of antibody to hepatitis C virus. Hepatology 12, 671-675. Knight, S. C., Askonas, B. A., and Macatonia, S. E. (1997). Dendritic cells as targets for cytotoxic T lymphocytes. Adv Exp Med Biol 417, 389-394. Koch, N., and Bartenschlager, R. (1999). Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A and NS4B. J Virol 73, 7138- 7146. Kolykhalov, A. A., Agapov, E. V., Blight, K. J., Mihalik, K., Feinstone, S. M., and Rice, C. M. (1997). Transmission of Hepatitis C by intrahepatic innoculation with transcribed RNA. Science277, 570-574. Kolykhalov, A. A., Mihalik, K., Feinstone, 8. M., and Rice, C. M. (2000). HCV-encoded enzymatic activities and conserved RNA element in the 3' nontranslated region are essential for virus replication in vivo. J Virol 74, 2046-2051. 305 Kondo, Y., Suda, T., Fukuyama, H., Adachi, M., and Nagata, S. (1997). Essential roles of the Fas ligand in the development of hepatitis. Nat Med 3, 409-413. Korst, R. J., Mahtabifard, A., Yamada, R., and Crystal, R. G. (2002). Effect of adenovirus gene transfer vectors on the immunological functions of mouse dendritic cells. Mol Ther 5, 307-315. Koshy, R., and Inchauspe, G. (1996). Evaluation of hepatitis 0 virus protein epitopes for vaccine development. TIBTECH14, 364-369. Koup, R. A., and Ho, D. D. (1994). Shutting down HIV. Nature 370, 416. Koziel, M. J., Dudley, D., Afdhal, N., Choo, Q. L., Houghton, M., Ralston, R., and Walker, B. D. (1993). Hepatitis 0 virus specific cytotoxic T lymphocytes recognise epitopes in the core and envelope proteins of hepatitis 0 virus. J Virol151, 4189-4199. Koziel, M. J., Dudley, D., Afdhal, N., Grakoui, A., Rice, 0. M., Choo, Q. L, Houghton, M., and Walker, B. D. (1995). HLA class-1 restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterisation of patterns of cytokine release. J Clin Invest96, 2311-2321. Koziel, M. J., Dudley, D., Wong, J. T., Dienstag, J., Houghton, M., Ralston, R., and Walker, B. D. (1992). Intrahepatic cytotoxic I lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. J Immunol 149, 3339-3344. Koziel, M. J., Wong, D. K., Dudley, D., Houghton, M., and Walker, B. D. (1997). Hepatitis C virus-specific cytolytic I lymphocytes and I helper cell responses in seronegative persons. J Infect Dis 176, 859-866. Kroemer, G. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3, 614-620. Kurosaki, M., Enomoto, N., Marumo, F., and Sato, C. (1993). Rapid sequence variation of the hypervariable region of hepatitis C virus during the course of chronic infection. Hepatology 18, 1293-1299. Kwun, H. J., and Jang, K. L. (2003). Dual effects of hepatitis C virus core protein on the transcription of cyclin-dependant kinase inhibitor p21 gene. J Viral Hepat 10, 249-255. Lai, M. M., and Ware, C. F. (2000). Hepatitis C virus core protein: possible roles in viral pathogenesis. Curr Top Microbiol Immunol 2 42117-134. , Lanzavecchia, A. (1998). Immunology. License to kill. Nature 393, 413-414. Large, M. K., Kittlesen, D. J., and Hahn, Y. 8. (1999). Suppression of host immune response by the core protein of hepatitis C virus: Possible implications for hepatitis 0 virus persistence. J Immunol162, 931-938. Lasarte, J. J., Corrales, F. J., Cabares, N., Lopez-Diaz de Cerio, A., Qian, C., Xie, X., Borras-Qesta, F., and Prieto, J. (1999). Different doses of adenoviral vector expressing IL- 12 enhance or depress the immune response to a co-administered antigen: The role of nitric oxide. J Immunol 162, 5270-5277. 306 Laskus, T., Radkowski, M., Wang, L-F., Vargas, H., and Rakela, J. (1998). Search for HCV extrahepatic replication sites in patients with acquired immune deficiency syndrome: specific detection of negative strand viral RNA in various tissues. Hepatology 28, 1398- 1401. Lauer, G. M., Nguyen, T. N., Day, 0. L, Robbins, G. K., Flynn, T., and McGowan, K. (2002). Human immunodeficiency virus type 1 -hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses. J Virol 76, 2817- 2826. Lauer, G. M., and Walker, B. (2001). Hepatitis 0 Virus Infection. N Engl J Med 345, 41-52. Le Bon, A., and Tough, D. (2002). Links between innate and adaptive immunity via type 1 interferon. Curr Opin Immunol14, 432-436. Lechmann, M., Murata, K., Satoi, J., Vergalla, J., Baumert, I . F., and Liang, I . J. (2001). Hepatitis C virus-like particles induce virus-specific humoral cellular immune responses in mice. Hepatology34, 417-423. Lechner, F., Gruener, N. H., Urbani, S., Uggeri, J., Santantonio, T., Cerny, A., Phillips, R. E., Ferrari, C., Pape, G., and Klenerman, P. (2000a). CTL responses are induced during acute HCV infection but are not sustained. Eur J Immunol 30, 2479-2487. Lechner, F., Wong, D. K. H., Dunbar, P. R., Chapman, R., Chung, R. T., Dohrenwend, P., Robbins, G., Phillips, R., Klenerman, P., and Walker, B. D. (2000b). Analysis of sucessful immune responses in persons infected with Hepatitis C Virus. J Exp Med 191, 1499-1512. Lee, C. H., Choi, Y. H., Yang, S.-H., Lee, C. W., Ha, S. J., and Sung, Y. C. (2001). Hepatitis C Virus Core Protein Inhibits Interleukin 12 and Nitric Oxide Production from Activated Macrophages. Virology 279, 271-279. Lee, 8. W., Cho, J. H., and Sung, Y. C. (1998). Optimal induction of hepatitis C virus envelope-specific immunity by bicistronic plasmid DNA innoculation with the GM-CSF gene. J Virol 72, 8430-8436. Lemon, S. M., and Honda, M. (1997). Internal ribosome entry sites within the RNA genomes of hepatitis C virus and othe flaviviruses. Semin Virol 8, 274-288. Lerat, H., Rumin, S., Habersetzer, F., Berby, F., Trauaud, M. A., Trepo, C., and Inchauspe, G. (1998). In Vivo tropism of Hepatitis C virus genomic sequences in hematopoietic cells: Influence of viral load, viral genotype and cell phenotype. Blood91, 3841-3849. Levitskaya, J., Coram, M., Levitsky, V., Imreh, S., Steigerwald-Mullen, P. M., Klein,G., Kurilla, M. G., and Masucci, M. G. (1995). Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375, 685-688. Levy, S., Todd, S. C., and Maecker, H. T. (1998). CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Ann Rev Immunol 16, 89- 109. 307 Li, D., Takyar, S. T., Lott, W. B., and Gowans, E. J. (2003a). Amino acids 1-20 of the hepatitis C virus (HCV) core protein specifically inhibit HCV IRES-dependant translation in HepG2 cells, and inhibit both HCV 1RES- and cap-dependant translation in Huh-7 and CV- 1 cells. J Gen Virol84, 815-825. Li, K., Wang, L., Cheng, J., Lu, Y. Y., Zhang, L. X., Mu, J. S., Hong, Y., Liu, Y., Duan, H. J., Wang, G., et al. (2003b). Interaction between hepatitis C virus core protein and translin protein - a possible mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus. World J Gasteroenterol 9, 300-303. Lieber, A., He, C. Y., Polyak, S. J., G retch, D. R., Barr, D., and Kay, M. A. (1996). Elimination of HCV RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes. J Virol 70, 8782-8791. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W., and Rice, C. M. (1994). Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2- specific prodcuts with different C termini. J Virol86, 5063-5073. Lin, C., Wu, J. W., Hsiao, K., and Su, M. S. (1997). The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins. J Virol 71, 6465-6471. Littaua, R. A., Oldstone, M. B. A., Takeda, A., and Ennis, F. A. (1992). A CD4+ cytotoxic T- lymphocyte clone to a conserved epitope in human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6. J Virol66, 608-611. Liu, Q., Tackney, C., Bhat, R. A., Prince, A. M., and Zhang, P. (1997). Regulated processing of hepatitis C virus core protein is linked to subcellular localisation. J Virol 71, 657-662. Liu, Z.-X., Nishida, H., He, J.-W., Lai, M. M. C., Feng, N., and Dennert, G. (2002). Hepatitis C virus genotype 1b core protein does not exert immunomodulatory effects on virus- induced cellular immunity. J Virol76, 990-997. Lo, S., Masiarz, F., Hwang, S. B., Lai, M. M., and Ou, J.-H. (1995). Differential subcellular localisation of hepatitis C virus core protein gene products. Virology 213, 455-461. Lo, S.-Y., Selby, M., and Ou, J.-H. (1996). Interaction between hepatitis 0 virus core protein and El envelope proteins. J Virol70, S I77-182. Lo, S.-Y., Selby, M., Tong, M., and Ou, J.-H. (1994). Comparative studies of the core gene products of two different hepatitis G virus isolates: Two alternative forms determined by a single amino acid substitution. Virology 199, 124-131. Locatelli, G. A., Spadari, S., and Maga, G. (2002). Hepatitis 0 virus NS3 ATPase/helicase: an ATP switch regulates the cooperativity among the different substrate binding sites. Biochemistry 4 1 ,10332-10342. Lohman, B. L., Razvi, E. S., and Welsh, R. M. (1996). T-lymphocyte downregulation after acute viral infection is not dependant on CD95 (Fas) receptor-ligand interactions. J Virol 70, 8199-8203. 308 Lohmann, V., Hoffmann, S., Herian, U., Penin, F., and Bartenschlager, R. (2003). Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 77, 3007- 3019. Lohmann, V., Korner, P., Dobierzewska, A., and Bartenschlarger, R. (2001). Mutations in HCV RNAs conferring cell culture adaptation. J Virol 75,1437-1449. Lohmann, V., Korner, P., Koch, J. O., Herian, U., Theilmann, L., and Bartenschlager, R. (1999). Replication of subgenomic hepatitis 0 virus RNAs in a hepatoma cell line. Science 285, 110-113. Loyer, V., Fontaine, P., Pion, S., Hetu, P., Roy, D. 0., and Perreault, 0. (1999). The in vivo fate of APCs displaying minor H antigen and/or MHO differences is regulated by CTLs specific for immunodominant class-1 associated epitopes. J Immunol 163, 6462-6467. Lozach, P. Y., Lortat-Jacob, H., De Lacroix De Lavalette, A., Staropoli, I., Poung, S., Amara, A., Houles, 0., Pieschi, P., Schwartz, O., Virelizier, J. L., et al. (2003). DC-SIGN and L-SIGN are high-affinity binding receptors for hepatitis 0 virus glycoprotein E2. J Biol Chem 278, 20358-20366. Lucas, M., Gadola, S., Meier, U., Young, N. T., Harcourt, G., Karadimitris, A., Coumi, N., Brown, D., Dusheiko, G., Cerundolo, V., and Klenerman, P. (2003). Frequency and phenotype of circulating Valpha24/Vbeta11 double-positive natural killer T cells during hepatitis C infection. J Virol 77, 2251-2257. Luo, G., Xin, S., and Cai, Z. (2003). Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of the hepatitis C virus RNA. J Virol 77, 3312-3318. Lusida, M. I., Nagano-Pujii, M., Nidom, C. A., Soetjipto, Handajani, R., Pujita, T., Oka, K., and Hotta, H. (2001). Correlation between mutations in the interferon sensitivity- determining region of NS5A protein and viral load of hepatitis C virus subtypes 1b, 1c and 2a. J Clin Microbiol 39, 3858-3864. MacDonald, A., Crowder, K., Street, A., McCormick, C., Saksela, K., and Harris, M. (2003). The hepatitis C virus NS5A protein inhibits activation protein 1 (API ) function by perturbing Ras-ERK pathway signalling. J Biol Chem 2 78 ,17775-17784. Macejak, D. G., Jensen, K. L., Pavco, P. A., Phipps, K. M., Heinz, B. A., Colacinal, M., and Blatt, L. M. (2001). Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNA. J Viral Hepatol 8 ,400-405. Machida, K., Tsukiyama-Kohara, K., Seike, E., Tone, S., Shibasaki, P., Shimizu, M., Takahashi, H., Hayashi, Y., Punata, N., Taya, C., et al. (2001). Inhibition of cytochrome c release in Pas-mediated signalling pathway in transgenic mice induced to express hepatitis C viral proteins. J Biol Chem 276, 12140-12146. Mahr, J. A., and Gooding, L. R. (1999). Immune evasion by adenoviruses. Immunol Rev 168, 121-130. 309 Major, M. E., Vitvitski, L., Mink, M. A., Schleef, M., Whalen, R. G., Trepo, G., and Inchauspe, G. (1995). DNA based immunisation with chimeric vectors for the induction of immune responses against HCV nucleocapsid. J Virol 69, 5798-5805. Majumder, M., Ghosh, A. K., Steele, R., Zhou, X. Y., Phillips, N. J., Ray, R., and Ray, R. B. (2002). Hepatitis 0 virus NS5A protein impairs TNF-mediated hepatic apoptosis, but not by an anti-Fas antibody, in transgenic mice. Virology 294, 94-105. Makimura, M., Miyake, S., Akino, N., Takamori, K., Matsuura, Y., Miyamura, T., and Saito, I. (1996). Induction of antibodies against structural proteins of hepatitis 0 virus in mice using recombinant adenovirus. Vaccine 14, 28-34. Makino, M., Wakamatsu, S., Shimokubo, S., Arima, N., and Baba, M. (2000). Production of functionally deficient dendritic cells from HTLV-1 infected monocytes: implications for the dendritic cell defect in adult I cell leukemia. Virology2 74 ,140-148. Mamane Y., Heylbroeck, 0., Genin, P., Algarte, M., Servant, M.J., LePage, 0., DeLuca, 0., Kwon, H., Lin, R., Hiscott, J., (1999), Interferon regulatory factors: the next generation.. Gene, 237(1), 1-14. Manzini, P., Dusheiko, G., Brown, D. J. 0., Khakoo, S. I., and Owen, J. S. (1998). Hepatitis C virus (HCV) tends to associate preferentially with high-density lipoproteins by standard ultracentifugal fractionation of plasma from patients with chronic HCV infection. Hepatol Res 11i 158-165. Marcellin, P. (1999). Hepatitis C: The clinical spectrum of disease. Journal of Hepatology 31, 9-16. Marcellin, P., Levy, S., and Erlinger, S. (1997). Therapy of Hepatitis C: Patients with Normal Aminotransferase Levels. Hepatol 26, SI 33-136. Marrogi, A. J., Cheles, M. K., and Gerber, M. A. (1995). Chronic hepatitis C. Analysis of host immune response in liver-infiltrating lymphocytes in chronic hepatitis C. Arch Pathol Lab Med 119, 232-237. Martell, M., Esteban, J. L., Quer, J., J., G., Weiner, A., Esteban, R., Guardia, J., and Gomez, J. (1992). Hepatitis C virus circulates as a population of different but closely related genomes: Quasispecies nature of a HCV genome distribution. J Virol66, 3225- 3229. Marusawa, H., Hijikata, M., Chiba, T., and Shimotohno, K. (1999). Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-kB activation. J Virol 73, 4713-4720. Mathurin, P., Moussalli, J., Cadranel, J. F., Thibault, V., Charlote, F., Dumouchel, P., Cazier, A., Huraux, J. M., Devergie, B., Vidaud, M., et aL (1998). Slow progression rate of fibrosis in hepatitis C patients with persistently normal ALT activity. Hepatology 27, 868- 872. 310 Matsuda, J., Suzuki, M., Nozaki, C., Shlnya, N., Tashiro, K., Mizuno, K., Uchinuno, Y., and Yamamura, K. (1998). Transgenic mouse expressing a full-length hepatitis C virus cDNA. Jp J Cancer Res8 9 ,150-158. Matsumoto, M., Hsieh, T.-Y., Zhu, N., Van Arsdale, T., Hwang, S. B., Jeng, K.-S., Gorbalenya, A. E., Lo, S.-Y., Ou, J.-H., Ware, 0. F., and Lai, M. M. (1997). Hepatitis 0 virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta-receptor. J Virol 71, 1301-1309. Matsumoto, M., Hwang, S. B., Jeng, K.-S., Zhu, N., and Lai, M. M. (1996). Homotypic interactions and multimerisation of hepatitis C virus core protein. Virology 218, 43-51. McGory, W. J., Bautista, D. S., and Graham, F. L. (1988). A simple technique for the rescue of early region 1 mutations into human adenovirus type 5. Virology 163, 614-617. McGuinness, P. H., Painter, D., Davies, S., and McCaughan, G. (2000). Increases in intrahepatic CD68 positive cells, MAC387 positive cells, and proinflammatory cytokines (particularly interleukin 18) in chronic hepatitis 0 infection. Gut 46, 260-269. McHutchinson, J. G., Gordon, S. 0., Schiff, E. R., Schiffman, M. L., Lee, W. M., Rustgi, V. K., Goodman, Z. D., Ling, M. H., Oort, S., and Albrecht, J. K. (1998). IFNapha2b alone or in combination with ribavirin as initial treatment for chronic hepatitis 0 virus. Hepatitis Intervention Therapy Group. New Eng J Med339 1485-1492. , McLellan, A., Heldmann, M., Terbeck, G., Weih, F., Linden, 0., Brocker, E., Leverkus, M., and Kampgen, E. (2000). MHC class II and CD40 play opposing roles in dendritic cell survival. Eur J Immunol 30, 2612-2619. McOmish, F., Yap, P. L., Dow, B. C., Follett, A. C., Seed, C., Keller, A. J., Cobain, T. J., Krusius, T., Koloho, E., Naukkarinen, R., et al. (1994). Geographical distribution of Hepatitis C Virus genotypes in blood donors: an international collaborative study. J Clin Microbiol 32, 884-892. Medema, J. P., Schuuhuis, D. H., Rea, D., Van Tongeren, J., de Jong, J., Bres, S. A., Laban, S., Toes, R. E. M., Toebes, M., Schumacher, T. N. M., et al. (2001). Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte- induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J Exp Med 194, 657-667. Mendenhall, C. L., Seef, L., Diehl, A., Ghosn, S. J., French, S. W., Gartside, P. S., Rouster, S. D., Buskell-Bales, Z., Grossman, C. J., and Roselle, G. A. (1991). Antibodies to hepatitis B and hepatitis C in alcoholic hepatitis and cirrhosis: Their prevalence and clinical relevance. Hepatology14, 581-589. Meng Soo, H., Garzino-Demo, A., Hong, W., Hwee Tan, Y., Joo Tan, Y., Goh, P. Y., Gee Lim, S., and Pheng Lim, S. (2002). Expression of a full-length hepatitits C virus cDNA upregulates the expression of CC chemokines MCP-1 and RANTES. Virology 303, 253- 277. Meola, A., Sbardellati, A., Bruni Ercole, B., Cerretani, M., Pezzanera, M., Ceccacci, A., Vitelli, A., Levy, S., Nicosia, A., Traboni, C., et ai. (2000). Binding of hepatitis C virus E2 311 glycoprotein to CD81 does not correlate with species permissiveness to infection. J Virol 74, 5933-5938. Mercer, D. F., Schiller, D. E., Elliott, J. P., Douglas, D. N., Hao, C., Rinfret, A., Addison, W. R., Fischer, K. P., Churchill, T. A., Lakey, J. R. T., et al. (2001). Hepatitis 0 virus replication in mice with chimeric human livers. Nat Med 7, 927-933. Michalak, J. P., Wychowski, 0., Choukhi, A., Meunier, J. 0., Ling, S.,Rice, 0. M., and Dubuisson, J. (1997). Characterization of truncated forms of hepatitis C virus glycoproteins. J Gen Virol 78, 2299-2306. Miller, G., Lahrs, S., Pillarisetty, V. G., Shah, A. B., and DeMatteo, R. P. (2002). Adenovirus infection enhances dendritic cell immunostimulatory properties and induces natural killer T-cell mediated tumour protection. Cancer Res 62, 5260-5266. Missale, G., Bertoni, R., Lamonaca, V., Valli, A., Massar, M., Mori, C., Rumi, M. G., Houghton, M., Fiaccadori, P., and Ferrari, C. (1996). Different clinical behaviours of acute hepatitis C infection are associated with different vigour of the anti-viral cell mediated immune response. J Clin Invest98, 706-714. Missale, G., Cariani, E., Lamonaca, V., Ravaggi, A., Rossini, A., and Bertoni, R. (1997). Effects of interferon treatment on the antiviral T cell response in hepatitis C virus genotype 1 b- and genotype 2c-infected patients. Hepatology 26, 792-797. Mita, E., Hayashi, N., and lio, S. (1994). Role of Fas-ligand in apoptosis induced by hepatitis C virus infection. Biochem Biophys Res Comm 204, 468-474. Miyamoto, H., Okamoto, H., Sato, K., Tanaka, T., and Mishiro, S. (1992). Extraordinarily low density of hepatitis C virus estimated by sucrose density gradient centrifugation and the polymerase chain reaction. J Gen Virol 73, 715-718. Modlin, J. (2000). Poliovirus. In Principles and Practice of Infectious Diseases., G. L. Mandell, and R. Dolin, eds. (Philadelphia, Churchill Livingstone), pp. 1895-1903. Moennig, V., and Plagemann, G. W. (1992). The Pestiviruses. Adv Virus Res 41, 53-98. Molenkamp, R., Kooi, E. A., Lucassen, M. A., Greve, S., Thijssen, J. C., Spaan, W. J., and Bredenbeek, P. J. (2003). Yellow fever virus replicons as an expression system for hepatitis C virus structural proteins. J Virol 77, 1644-1648. Monazahian, M., Bohme, I., Bonk, S., Koch, A., Scholz, C., Grethe, S., and Thomssen, R. (1999). Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol 57, 223-229. Moorman, J. P., Joo, M., and Hahn, Y. S. (2001). Evasion of host immune surveillance by hepatitis C virus: Potential roles in viral persistence. Arch Immunol Ther Exp 4 9 ,189-194. Moorman, J. P., Prayther, D., McVey, D., Hahn, Y. S., and Hahn, C. S. (2003). The C- terminal region of hepatitis C core protein is required for Fas-ligand independent apoptosis in Jurkat cells by facilitating Fas oligomerisation. Virology 312, 320-329. 312 Morita, M., Watanabe, Y., and Akaike, T. (1995). Protective effect of hepatocyte growth factor on interferon-gamma- induced cytotoxicity in mouse hepatocytes. Hepatology 188, 223-232. Moriya, K., Fujie, H., Shintani, Y., Yotsuyanagi, H., Tsutsumi, T., Ishibashi, K., Matsuura, Y., Kimura, S., Miyamura, T., and Koike, K. (1998). The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 4 ,1065-1067. Moriya, K., and Yotsuganagi, H. (1997). Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J Gen Virol 78, 1527-1531. Morrelli, A. E., Larregina, A. T., Ganster, R. W., Zahorchak, A. F., Plowey, J. M., Takayama, T., Logar, A. J., Robbins, P. D., Falo, L. D., and Thomson, A. W. (2000). Recombinant adenovirus induces maturation of dendritic cells via NF-k-B-dependant pathway. J Virol 74, 9617-9628. Moskophidis, D., Lechner, F., Pircher, H., and Zinkernagel, R. M. (1993). Virus persistence in acutely infected immunocompetent mice: exhaustion of antiviral cytotoxic effector T cells. Nature 362, 758-761. Mosnier, J. F., Scoaze, J. Y., Marcellin, P., Degott, C., Benahmou, J. P., and Feldmann, G. (1994). Expression of cytokine-dependant immune adhesion molecules by hepatocytes. Gasteroenterol 107, 1467-1468. Mottola, G., Cardinali, G., Ceccacci, A., Trozzi, 0., Bartholomew, L., Torrisi, M. R., Pedrazzini, E., Bonatti, S., and Migliaccio, G. (2002). Hepatitis 0 virus nonstructural proteins are localised in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons. Virology 293, 31-43. Muchmore, E., Popper, H., Peterson, D. A., Miller, M. F., and Lieberman, H. M. (1988). NANB hepatitis-related hepatocellular carcinoma in a chimpanzee. J Med Primatol 17, 235-246. Muller, H. M., Pfaff, E., Goeser, T., Kallinowski, B., Solbach, 0., and Theilmann, L. (1993). Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication. J Gen Virol74, 669-676. Murakawa, K., Esumi, M., Kato, T., Kambara, H., and Shikata, T. (1992). Heterogeneity within the NS5-encoding region of hepatitis 0 virus from a single patient. Gene117, 229- 232. Murata, K., Lechmann, M., Qiao, M., Gunji, T., Alter, H., and Liang, T. J. (2003). Immunization with hepatitis 0 virus-like particles protects mice from recombinant hepatitis 0 virus-vaccinia infection. Proc Natl Acad Sci USA 100, 6753-6758. Murray, E. M., Grobler, J. A., Markel, E. J., Pagoni, M. F., Paonessa, G., Simon, A. J., and Flores, O. A. (2003). Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh-7 cells. J Virol 77, 2928-2935. 313 Nakamoto, Y., Kaneko, S., Handa, M., Unoura, M., Cheong, J., Harada, A., Matsushima, K., Kobayashi, K., and Murakami, S. (1994). Detection of the putative E2 protein of HCV in human liver. J Med Virol 42, 374-379. Napoli, J., Bishop, G. A., McGuinness, P. H., Painter, D., and McCaughan, G. (1996). Progressive liver injury in chronic hepatitis C infection correlates with increased intrahepatic expression of Thi associated cytokines. Hepatology 24, 759-765. Navas, M. C., Fuchs, A., Schvoerer, E., Bohbot, A., Aubertin, A. M., and Stoll-Keller, F. (2002). Dendritic cell susceptibility to hepatitis C virus genotype 1 infection. J Med Virol 67, 152-161. Neddermann, P., Clementi, A., and De Francesco, R. (1999). Hyperphosphorylation of the hepatitis C virus NS5A protein requires and active NS3 protease, NS4A, NS4B and NS5A encoded on the same polyprotein. J Virol73, 9984-9991. Negro, F., Krawczynski, K., Quadri, R., Rubbia-Brandt, L., Mondelli, M., Zarski, J. P., and Hadengue, A. (1999). Detection of genomic and minus-strand HCV RNA in the liver of chronic HCV patients by strand-specific semiquantitative RT-PCR. Hepatology 29, 536- 542. Nelson, D. R., Marousis, C. G., Davis, G. L., Rice, C. M., Wong, J., and Houghton, M. (1998). Intrahepatic hepatitis C virus-specific cytotoxic T lymphocyte activity and response to interferon alfa therapy in chronic hepatitis C. Hepatology 28, 225-230. Nemchinov, L. G., Liang, T. J., Rifaat, M. M., Mazyad, H. M., Hadidi, A., and Keith, J. M. (2000). Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch Virol 145, 2557-2573. Neumann, A. U., Lam, N. P., and Dahari, H. (1998). Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science282, 103-107. Nguyen, H., MudryJ, M., Guadalupe, M., and Dandekar, 8. (2003). Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle. Virology312, 245-253. Nobile, 0., Moris, A., Porrot, F., Sol-Foulon, N., and Schwartz, O. (2003). Inhibition of human immunodeficiency virus type 1 Env-mediated fusion by DC-SIGN. J Virol 77, 5313- 5323. Noguchi, T., Satoh, S., Noshi, T., Hatada, E., Fukeda, R., Kawai, A., Ikeda, S., Hijikata, M., and Shimotohno, K. (2001 ). Effects of mutation in hepatitis 0 virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells. Microbiol Immunol 45, 829-840. Nolandt, O., Kern, V., Mulle, H., Pfaff, E., Theilman, L., Welker, R., and Krausslich, H. (1997). Analysis of hepatitis 0 virus core protein interaction domains. J Gen Virol 78, 1331-1340. 314 Nozaki, A., Ikeda, M., Naganuma, A., Inudoh, M., Tanaka, K., and Kato, N. (2003). identification of a iactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein. J Biol Chem 278, 10162-10173. Nuti, S., Rosa, D., Valiante, N. M., Saletti, G., Caratozzolo, M., Dellabona, P., Barnaba, V., and Abrignani, S. (1998). Dynamics of intra-hepatic lymphocytes in chronic hepatitis 0 enrichment for Valpha24+ T cells and rapid elimination of effector cells by apoptosis. Eur J Immunol 28, 3448-3455. O'Connell, J. F. (1997). Mechanisms of action of interferon: Potential role in hepatitis C. Viral Hepatitis Reviews 3, 121-128. Ogata, N., Alter, H. J., Miller, R. H., and Purcell, R. H. (1991). Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci USA 88, 3392-3396. Ogata, S., Nagano-Fujii, M., Ku, Y., Yoon, S., and Hotta, H. (2002). Comparative sequence analysis of the core protein and its frameshift product, the F protein, of hepatitis C virus subtype 1b strains obtained from patients with and without hepatocellular carcinoma. J Clin Microbiol 40, 3625-3630. Ohkawa, K., Ishida, H., Nakanishi, F., Hosui, A., Sato, A., Ueda, K., Takehara, T., Kasahara, A., Sasaki, Y., Hori, M., and Hayashi, N. (2003). Changes in gene expression profile by HCV core protein in cultured liver cells: analysis by DNA array assay. Hepatol Res 25, 396-408. Ohoto, H., Terazawa, S., Sasaki, N., Sasaki, N., Hino, K., Ishiwata, C., Kako, M., Ujiie, N., Endo, C., Matsui, A., et al. (1994). Transmission of hepatitis C virus from mothers to infants. N Eng J Med 330, 744-750. Oka, K., Nagano-Fujii, M., Yoshida, I., Hidajat, R., Deng, L, Akutsu, M., and Hotta, H. (2003). Hepatitis C virus core protein selectively inhibits synthesis and accumulation of p21A/Vaf1 and certain nuclear proteins. Microbiol Immunol 47, 429-438. Okamoto, H., Kojima, M., Okada, S., Yoshizawa, H., lizuka, H., Tanaka, T., Muchmore, E. E., Peterson, D. A., Ito, Y., and Mishiro, S. (1992). Genetic drift of hepatitis C virus during an 8.2 year infection in a chimpanzee. Proc Natl Acad Sci USA 88, 3392-3396. Okamoto, H., and Mishiro, S. (1994). Genetic heterogenity of hepatitis C virus. Intervirology43, 68-76. Omata, N., Yasutomi, M., Yamada, A., Iwasaki, H., Mayumi, M., Oshima, Y., (2002), Monocyte chemoattractant protein-1 selectively inhibits the acquisition of CD40 ligand- dependent IL-12-producing capacity of monocyte-derived dendritic cells and modulates Thi immune response., J. Immunol.,169 , 4861-6. Onji, M., Kikuchi, T., and Kumon, I. (1992). Intrahepatic lymphocyte subpopulations and HI_A class I antigen expression by hepatocytes in chronic hepatitis 0. Hepatogasteroenterology39, 340-343. 315 Otsuka, M., Kato, N., Taniguchi, H., Yoshida, H., Goto, T., Shiratori, Y., and Omata, M. (2002). Hepatitis C virus core protein inhibits apoptosis via enhanced Bcl-xL expression. Virology 296, 84-93. Ou-Yang, P., Hwang, L-H., Tao, M.-H., Chiang, B.-L., and Chen, D.-S. (2002). Co-delivery of GM-CSF enhances the immune responses of hepatitis C viral core protein-expressing DNA vaccine: Role of dendritic cells. J Med Virol66, 320-328. Oxenius, A., Zinkernagel, R. M., and Hengartner, H. (1998). Comparison of activated versus induction of unresponsive virus-specific CD4+ and CD8+ T cells upon acute versus persistent viral infection. Immunity 9, 449-457. Padgett, G. A., Reiquam, C. W., Henson, J. B., and Gorham, J. R. (1968). Comparative studies of susceptibility to infection in the Chediak-Higashi syndrome. J Pathol Bacteriol 95, 509-522. Pancholi, P., Liu, Q., Tricoche, N., Zhang, P., Perkus, M. E., and Prince, A. M. (2000). DNA prime-canarypox boost with polycistronic hepatitis C virus (HCV) genes generates potent immune responses to structural and nonstructural proteins. J Infect Dis 162, 18-27. Pancholi, P., Perkus, M., Tricoche, N., Liu, Q., and Prince, A. (2003). DNA immunisation with hepatitis C virus (HCV) polycistronic genes or immunisation by HCV DNA priming- recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1 transgenic mice. J Virol 77, 382- 390. Pang, P. S., Jankowsky, E., Planet, P. J., and Pyle, A. M. (2002). The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J 21, 1168-1176. Paoletti, E., Taylor, J., Meignier, B., Meric, C., and Tartaglia, J. (1995). Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC. Dev Biol Stand 84, 159-163. Par, A., Paal, M., Gog I, A., Gervain, J., Szekeres-Bartho, J., and Sipos, J. (1995). Chronic hepatitis C: clinical and immunological features and the effect of interferon treatment. Int J Immunother 1 1 ,115-127. Par, G., Rukavina, D., Podack, E., Horanyi, M., Szekeres-Bartho, J., Hegedus, G., Paal, M., Szereday, L., Mozsik, G., and Par, A. (2002). Decrease in CD3-negative-CD8dim(+) and Vdelta2/Vgamma9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection. J Hepatol 37, 514. Pardoll, D. M. (2002). Spinning molecular immunology into successful immunotherapy. Nat Rev Immunol 2, 227-238. Pares, A., Barrera, J. M., Caballeria, J., Ercilla, G., Bruguera, M., Caballeria, L., Castillo, R., and Rodes, J. (1990). Hepatitis C virus antibodies in chronic alcoholic patients: Association with severity of liver injury. Hepatology 1 2 ,1295-1299. 316 Park, K.-J., Choi, S.-H., Koh, M. S., Kim, D. J., Yie, S. W., Lee, S. Y., and Hwang, 8. B. (2001). Hepatitis 0 virus core protein potentiates c-Jun N-terminal kinase activation through a signalling complex involving TRADD and TRAF2. Virus Res 74, 89-98. Pasquinelli, 0., Schoenberger, J. M., Chung, J., Chang, K. M., Guidotti, L. G., Selby, M., Berger, K., Lesniewski, R., Houghton, M., and Chisari, F. V. (1997). Hepatitis C virus core and E2 protein expression in transgenic mice. Hepatology 25, 719-727. Patel, J., Patel, A. H., and McLauchlan, J. (2001). The transmembrane domain of the hepatitis C virus E2 glycoprotein is required for correct folding of the El glycoprotein and native complex formation. Virology 279, 58-68. Pavio, N., and Lai, M. M. (2003). The hepatitis C virus persistence: how to evade the immune system? J Biosci 28, 287-304. Pavio, N., Taylor, D. R., and Lai, M. M. (2002). Detection of a novel unglycosylated form of hepatitis C virus E2 envelope protein that is located in the cytosol and interacts with PKR. J Virol 76, 1265-1272. Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B., and Zitzmann, N. (2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci USA 100, 6140-6148. Pawlotsky, J.-M., Hovanessian, A., and Roudot-Thoraval, F. (1995). Activity of the interferon induced 2'5'OAS in patients with chronic hepatitis C virus. J Interferon Cytokine Res 15, 857-862. Penin, F., Combet, C., Germanidis, G., Frainais, P. O., Deleage, G., and Pawlotsky, J. M. (2001 ). Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment. J Virol 75, 5703-5710. Penna, A., Missale, G., Lamonaca, V., Pilli, M., Mori, C., and Zanelli, P. (2002). Intrahepatic and circulating HLA class ll-restricted, hepatitis C virus-specific T cells: Functional characterisation in patients with chronic hepatitis C. Hepatology 35, 1225-1236. Peter, M. E., Dhein, J., Ehret, A., Hellbardt, S., Walczak, H., Moldenhauer, G., and Krammer, P. H. (1995). APO-1 (CD95)-dependent and -independent antigen receptor induced apoptosis in human T and B cell lines. Int Immunol 7,1873-1877. Petracca, R., Falugi, F., Galli, G., Norais, N., Rosa, □., Campagnoli, S., Burgio, V., Di Stasio, E., Giardina, B., Houghton, M., et al. (2000). Structure-function analysis of hepatitis C virus envelope-CD81 binding. J Virol74, 4824-4830. Pfiugheber, J., Fredericksen, B., Sumpter, R. J., Wang, C., Ware, F., Sodo, D. L, and Gale, M. J. J. (2002). Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. Proc Natl Acad Sci USA 99, 4650-4655. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K. D., and McCarth, J. E. (2002). Modulation of the hepatitis C virus RNA-dependant RNA polymerase activity by the 317 non-structural (NS) 3 helicase and the NS4B membrane protein. J Biol Chem 277, 45670- 45679. Piccioli, D., Sbrana, S., Melandri, E., and Valiante, N. M. (2002). Contact-dependant stimulation and inhibition of dendritic cells by natural killer cells. J Exp Med 195, 335-341. Pietschmann, T., Lohmann, V., Kaul, A., Kreiger, N., Rinck, G., Rutter, G., Strand, D., and Bartenschlager, R. (2002). Persistent and transient replication of full-length hepatitis 0 virus genomes in cell culture. J Virol 76, 4008-4021. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, P., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, 8. (1998). Binding of hepatitis C virus to CD81. Science 282, 938-941. Pircher, H., Burki, K., Lang, R., Hengartner, H., and Zinkernagel, R. M. (1989). Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature 342, 559-561. Planz, O., Ehl, S., Furrer, E., Horvath, E., Brundler, M. A., Hengartner, H., and Zinkernagel, R. M. (1997). A critical role for neutralising-antibody producing B cells, CD8(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers. Proc Natl Acad Sci USA 94, 6874-6879. Poccia, P., and Agrati, C. (2003). Intrahepatic natural immunity and HCV immunopathogenesis. Cell Death and Differentiation 10, S9-12. Pohlmann, S., Zhang, J., Baribaud, P., Chen, Z., Leslie, G. J., Lin, G., Granell, Piperno, A., Doms, R. W., Rice, C. M., and McKeating, J. (2003). Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR. J Virol 77, 4070-4080. Polakos, N. K., Drane, D., Cox, J., Ng, P., Selby, M. J., Chein, D., O'Hagen, D. T., Houghton, M., and Paliard, X. (2001). Characterisation of hepatitis C virus core-specific immune responses primed in rhesus macaques by a nonclassical ISCOM vaccine. J Immunol 166, 3589-3598. Polyak, S. J., Khaber, K. S., Paschal, D. M., Ezelle, H. J., Duverlie, G., Barber, G. N., Levy, D. E., Mukaida, N., and G retch, D. R. (2001). Hepatitis C virus nonstructural protein 5A induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J Virol 75, 6095-6106. Poynard, T., Marcellin, P., Lee, S. S., Niederau, C., Minuk, G. S., Ideo, G., Bain, V., Heathcote, J., Zeuzum, S., Trepo, C., and Albrecht, J. (1998). Randomised trial of IPN2alpha2b plus ribavirin for 48 weeks or for 24 weeks versus IPN2b plus placebo for 48 weeks for treatment of chronic hepatitis C virus. Lancet 3521426-1432. , Pozzato, G., and Moretti, M. (1994). Lymphocyte subsets in HCV-positive chronic liver disease. Immunol Today 15,137-138. Prieto, M., Olaso, V., Verdu, C., Cordoba, J., Gisbert, C., Rayon, M., Carrasco, D., Berenguer, M., Hignon, M. D., and Berenguer, J. (1995). Does the Healthy Hepatitis C 318 Virus Carrier State Really Exist? An Analysis Using Polymerase Chain Reaction. Hepatol 22, 413-417. Prince, A., Brotman, B., Huima, T., Pascual, D., Jaffery, M., and Inchauspe, G. (1992). Immunity in hepatitis C infection. J Infect Dis 165, 438-443. Prince, A. M., Huima-Byron, T., Parker, T. S., and Levine, D. M. (1996). Visualisation of hepatitis C virus virions and putative defective interfering particles isolated from low- density lipoproteins. J Viral Hepat 3,11-17. Puoti, M., Bonacini, M., Spinetti, A., Putzolu, V., Govindarajan, S., and Zaltron, 8. (2001). Liver fibrosis progression is related to CD4 cell depletion in patients co-infected with hepatitis C virus and human immunodeficiency virus. J Infect Dis 183,134-137. Puoti, M., Zanaro, A., Ravaggi, A., Marin, M. G., Castelnuovo, F., and Cariani, E. (1992). Hepatitis C virus RNA and antibody response in the clinical course of hepatitis C virus infection. Hepatology16, 877-881. Purcell, R. H., Gerin, J. L., Popper, H., London, W. T., Ciemone, J., Eichberg, J. W., Neuman, J., and Hrinda, M. E. (1985). Hepatitis B virus, hepatitis non-A, non-B virus and hepatitis delta virus in lyophilised anti-haemophilic factor: Relative sensitivity to heat. Hepatology 5, 1091-1099. Qiao, M., Murata, K., Davis, A. R., Jeong, 8. H., and Liang, T. J. (2003). Hepatitis C virus like particles combined with novel adjuvant systems enhance virus-specific immune responses. Hepatology 37, 52-59. Randall, G., Grakoui, A., and Rice, C. M. (2003). Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad 8ci Ü8A 100, 235- 240. Ray, R. B., Meyer, K., and Ray, R. (1996). 8uppression of apoptotic cell death by hepatitis C virus core protein. Virology226, 176-182. Ray, R. B., Meyer, K., 8teele, R., 8hrivastava, A., Aggarwal, B. B., and Ray, R. (1998a). Inhibition of tumor necrosis factor (TNF-alpha)-mediated apoptosis by hepatitis C virus core protein. J Biol Chem 273, 2256-2259. Ray, R. B., 8teele, R., Meyer, K., and Ray, R. (1997). Transcriptional repression of p53 promoter by hepatitis C virus core protein. J Biol Chem 272, 10983-10986. Ray, R. B., 8teele, R., Meyer, K., and Ray, R. (1998b). Hepatitis C virus core protein represses p21 wAFi/cipi/sidi promoter activity. Gene208, 331-336. Ray, 8. C., Mao, 0., Lanford, R. E., Bassett, 8. E., Laeyendecker, O., Wang, Y. M., and Thomas, D. L. (2000). Hypervariable region 1 sequence stability during hepatitis C virus replication in chimpanzees. J Virol 74, 3058-3066. Rea, D., 8chagen, F. H. E., Hoeben, R. C., Mehtali, M., Havenga, M. J. E., Toes, R. E. M., Melief, C. J. M., and Offringa, R. (1999). Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset. J Virol 73, 10245-10253. 319 Reddehase, M. J., Jonjic, S., Weiland, F., Mutter, W., and Koszinowski, U. H. (1988). Adoptive immunotherapy of murine cytomegalovirus adrenal in the immunocompromised host: CD4-helper-independant antiviral function of CD8-positive memory T lymphocytes derived from latently infected donors. J Virol6 2 ,1061-1065. Reed, and Rice (1998). Molecular characterisation of HCV. In Hepatitis 0 Virus, H. W. Reesink, ed. (Basel, Karger), pp. 1-37. Reed, K. E., and Rice, 0. M. (2000). Overview of Hepatitis 0 virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol & Immunol 242, 55-84. Reichard, O., Anderson, J., and Schovarcz, R. (1991). Ribavirin treatment for chronic hepatitis C. Lancet 337, 1058-1061. Reigadas, S., Ventura, M., Sarih-Cottin, L., Castroviejo, M., Litvak, S., and Astier-Gin, T. (2001). HCV RNA-dependant RNA polymerase replicates in vitro the S' terminal region of the minus-strand viral RNA more efficiently than the S' terminal region of the plus RNA. Eur J Biochem268, 5857-5867. Reignat, S., Webster, G. L., Brown, D., Ogg, G. S., King, A., Seneviratne, S. L., Dusheiko, G., Williams, R., Maini, M. K., and Bertoletti, A. (2002). Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B infection. J Exp Med 195, 1089-1101. Rice, C. M. (1996). Flaviviridae: The Viruses and their Replication. In Fields Virology, D. K. BN Fields, PM Howley et al, ed. (Philadelphia, Lippincott-Raven Publishers), pp. 9S1-959. Ridge, J. P., Di Rosa, F., and Matzinger, P. (1998). A conditioned dendritic cell can be a temporal bridge between CD4+ T-helper and a T-killer cell. Nature393, 474-478. Rispeter, K., Lu, M., Behrens, S. E., Fumiko, C., Yoshida, T., and Roggendorf, M. (2000). Hepatitis C virus variability: sequence analysis of an isolate after 10 years of chronic infection. Virus Genes2 1 ,179-188. Roccasecca, R., Ansuini, H., Vitelli, A., Meola, A., Scarselli, E., Acali, S., Pezzanera, M., Ercole, B. B., McKeating, J., Yagnik, A., et ai (200S). Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2. J Virol 77,1856-1867. Rosa, D., Campagnoli, S., Moretto, C., Guenzi, E., Cousens, L., Chin, M., Dong, C., Weiner, A. J., Lau, J. Y. N., Choo, Q. L.,et al. (1996). A quantitative test to estimate neutralising antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 93, 1759-176S. Rosen, H. R., Hinrichs, D. J., G retch, D. R., Koziel, M. J., Chou, S., Houghton, M., Rabkin, J., Corless, C. L., and Bouwer, H. G. (1999). Association of multispecific CD4(+) response to hepatitis C and severity of recurrence after liver transplantation. Gasteroenterol 117, 926-9S2. 320 Rosenberg, E. S., Billingsley, J. M., Caliendo, A. M., Boswell, S. L, Sax, P. E., Kalams, S. A., and Walker, B. D. (1997). Vigourous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science278, 1447-1450. Ross, R. S., Viazov, S., Gross, T., Hoffmann, F., Sneipp, H.-M., and Roggendorf, M. (2000). Transmission of hepatitis C virus from a patient to an anesthesiology assistant to five patients. N Eng J Med3 43 ,1851-1854. Rossol, S., Marinos, G., and Carucci, P. (1997). Interleukin-12 induction of Thi cytokines is important for viral clearance in chronic hepatitis B. J Clin Invest 99, 3025-3033. Roth, D. (1995). Hepatitis 0 virus: The nephrologist's view. Am J kidney Dis 25, 3-16. Rouard, H., Leon, A., Klonjikowski, B., Marquet, J., Tenneze, L., Plonquet, A., Agrawal, S. G., Abastado, J. P., Eloit, M., Farcet, J. P., and Delfau-Larue, M.-H. (2000). Adenoviral transduction of human 'clinical grade' immature dendritic cells enhances costimuulatory molecule expression and T- cell stimulatory capacity. J Immunol Methods 241, 69-81. Roussel, J., Pillez, A., Montpellier, 0., Duverlie, G., Cahour, A., Dubuisson, J., and Wychowski, 0. (2003). Characterisation of the expression of the hepatitis C virus F protein. J Gen Virol84, 1751-1759. Rouvier, E., Luciani, M. F., and Golstein, P. (1993). Fas involvement in Ca(2+)- independent T cell-mediated cytotoxicity. J Exp Med177, 195-200. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997). Sensitization to Fas- mediated apoptosis by hepatitis C virus core protein. Virology 229, 68-76. Russel, J. H., and Ley, T. J. (2002). Lymphocyte mediated cytotoxicity. Ann Rev Immunol 20, 323-370. Sagnelli, E., Gaeta, G. B., Felaco, F. M., Stroffolini, T., Conti, S., Glielmo, A., Piccinino, F., and Giusti, G. (1997). Hepatitis C virus infection in households of anti-hepatitis C virus chronic carriers in Italy: A multi-centre case-control study. Infection 25 (6), 346-9. Salazar-Mather, T. P., Orange, J. S., and Biron, C. A. (1998). Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein 1 alpha (MlP-lalpha)-dependant pathways. J Exp Med 187, 1-14. Salio, M., Celia, M., Suter, M., and Lanzavecchia, A. (1999). Inhibition of dendritic cell maturation by herpes simplex virus. Eur J Immunol 29, 3245-3253. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning. A laboratory manual., Vol 1 (New York, Cold Spring Harbour Laboratory Press). Sansonno, D., Lacobelli, A. R., Cornacchiulo, V., lodice, G., and Dammacco, F. (1996). Detection of HCV proteins by immunofluorescence and HCV RNA genomic sequences by non-isotopic in situ hybridisation in bone marrow and PBMCs of chronically HCV infected patients. Clin Exp Immunol 103, 414-421. 321 Sansonno, D., Lauletta, G., Nisi, L., Gatti, P., Pesola, F., Pansini, N., and Dammacco, F. (2003). Non-enveloped HCV core protein as constitutive antigen of coid-precipitable immune complexes in type II mixed cryoglobulinaemia. Clin Exp Immunol 133, 275-282. Sansonno, D., Lotesoriere, C., Cornacchiulo, V., Fanelli, M., Gatti, P., lodice. G., Racanelli, V., and Dammacco, F. (1998). Hepatitis C Virus infection involves CD34+ Hematopoietic cells in hepatitis C virus chronic carriers. Blood 92, 3328-3337. Santolini, E., Migliaccio, G., and La Monica, N. (1994). Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol68, 3631-3641. Santolini, E., Pacini, C., Fipaldini, C., Migliaccio, G., and Monica, N. (1995). The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 669, 7461-7471. Sarobe, P., Lasarte, J. J., Casares, N., Lopez-Diaz de Cerio, A., Baixeras, E., Labarga, P., Garcia, N., Borras-Cuesta, F., and Prieto, J. (2002). Abnormal priming of CD4'" T cells by dendritic cells expressing hepatitis C virus core and El proteins. J Virol 76, 5062-5070. Sawtell, N. M., and Thompson, R. L. (1992). Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia. J Virol66, 2150-2156. Scarselli, E., Ansuini, H., Cerino, R., Roccasecca, R. M., Acali, S., Filocamo, G., Traboni, C., Nicosia, A., Cortese, R., and Vitelli, A. (2002). The human scavenger receptor class B type 1 is a novel candidate receptor for the hepatitis C virus. The EMBO Journal21, 5017- 5025. Schmilovitz-Weiss, H., Levy, M., Thompson, N., and Dusheiko, G. (1993). Viral markers in the treatment of hepatitis B and C. Gut 34, S26-35. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., and Peter, M. E. (1998). Apoptosis signalling by death receptors. Eur J Biochem 254, 439-459. Schwartz, O., Maréchal, V., Le Gall, S., Lemonnier, F., and Heard, J. M. (1996). Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 2, 338-342. Seef, L. B. (1995). Natural history of viral hepatitis, type C. Semin, Gasterointest Dis 6, 20- 27. Sekiya, H., Kato, N., Oosuyama, Y., Nakazawa, T., Yamauchi, K., and Shimotohno, K. (1994). Genetic alterations of the putative envelope proteins encoding region of the hepatitis 0 virus in the progression to relapsed phase from acute hepatitis: Humoral immune response to hypervariable region 1. Int J Cancer 57, 664-670. Serafino, A., Valli, M. B., Andreola, F., Crema, A., Ravagnan, G., Bertolini, L., and Carloni, G. (2003). Suggested role of the Golgi apparatus and endoplasmic reticulum for crucial sites of hepatitis 0 virus replication in human lymphoblastoid cells infected in vitro. J Med Virol 70, 31-41. 322 Serfaty, L, Nousbaum, V. B., Elghouzzi, M. H., Giral, P., Legenare, C., and Poupan, R. (1995). Prevalance, severity and risk factors of liver disease in blood donors positive in a second generation anti-HCV screening test. Hepatology21, 725-729. Shakil, A. O., Conry-Cantilena, C., Alter, H. J., Hayashi, P., Kleiner, D. E., Tedeschi, V., Krawczynski, K., Conjeevaram, H. S., Sallie, R., and DiBisceglie, A. M. (1995). Volunteer blood donors with antibody to hepatitis C virus: Clinical, biochemical, virological and histological features. Ann Intern Med 123, 330-337. Shankar, P., Xu, Z., and Lieberman, J. (1999). Viral-specific cytotoxic T lymphocytes lyse human immunodeficiency virus-infected primary T lymphocytes by the granule exocytosis pathway. Blood 94, 3084-3093. Shi, S. T., Lee, K. J., Aizaki, H., Hwang, S. B., and Lai, M. M. (2003). Hepatitis 0 virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77, 4160-4168. Shields, P., Morland, 0. M., Salmon, M., Qin, S., Hubscher, S. G., and Adams, D. (1999). Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J Immunol163, 6236-6243. Shih, 0. M., Lo, S. J., Miyamura, T., Chen, S. Y., and Lee, Y. H. (1993). Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in Huh-7 cells. J Virol 676, 5823-5832. Shim, J. H., Larson, G., Wu, J. Z., and Hong, Z. (2002). Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependant RNA polymerase. J Virol 76, 7030-7039. Shimizu, Y., Igarashi, H., Kanematu, T., Fuijwara, K., Wong, D. C., Purcell, R. H., and Yoshikara, H. (1997). Sequence analysis of the hepatitis C virus genome recovered from serum, liver and peripheral blood mononuclear cells of infected chimpanzees. J Virol 77, 5769-5773. Shimizu, Y. K., Feinstone, S. M., Kohara, M., Purcell, R. H., and Yoshikura, H. (1996). Hepatitis C virus: Detection of intracellular virus particles by electron microscopy. Hepatology 23, 205-209. Shirai, M., Okada, H., Nishioka, M., Akatsuka, T., Wychowski, C., Houghton, R., Pendleton, C. D., Feinstone, S. M., and Berzofsky, J. A. (1994). An epitope in HCV core region recognised by cytotoxic T lymphocytes in mice and humans. J Virol68, 3334-3342. Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., Kobayashi, K., and Murakami, S. (2002). Hepatitis C virus (HCV) NS5A binds RNA-dependant RNA polymerase (RdRP) NS5B and modulates RNA-dependant RNA polymerase activity. J Biol Chem 277, 11149- 11155. Shrivastava, A., Manna, S. K., Ray, R., and Aggarwal, B. B. (1998). Ectopic expression of hepatitis C virus core protein differentially regulates nuclear transcription factors. J Virol 72, 9722-9728. 323 Siegal, F. P., Kadowaki, N., Shodell, M., Fitzgerald-Bocarsly, P. A., Shah, K., Ho, S., Antonenko, S., and Liu, Y.-C. (1999). The nature of the principal type 1 interferon- producing cells in human blood. Science2 84 ,1835-1837. Siler, C. A., McGettigan, J. P., Dietzshold, B., Herrine, S. K., Dubuisson, J., Pomerantz, R. J., and Schnell, M. J. (2002). Live and killed rhabdovirus-based vectors as potential hepatitis C virus vaccines. Virology 292, 24-34. Simmonds, P. (1995). Variablility of hepatitis C virus. Hepatology 21, 570-583. Simmonds, P., Holmes, E. C., Cha, I . A., Chan, S. W., McOmsih, F., Irvine, B., Beall, E., Yap, P. L., Kolberg, J., and Urdea, M. S. (1993). Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 74, 2391-2399. Smerdou, C., and Liljestrom, P. (1999). Two-helper RNA system for production of recombinant Semliki forest virus particles. J Virol 73, 1092-1098. Soilleux, E. J. (2003). DC-SIGN (dendritic cell-specific ICAM-grabbing non-integrin) and DC-SIGN-related (DC-SIGNR): friend or foe? Clin Sci (Lond)104, 437-446. Song, M. K., Lee, S. H., Suh, Y. S., Lee, K. J., and Sung, Y. C. (2000). Enhancement of lgG2a and CTL responses by a booster immunisation with recombinant HCV E2 protein in E2 DNA primed mice. J Virol74, 2920-2925. Sreenarashimhaiah, J., Jaramillo, A., Crippin, J., Lisker-Melman, M., Chapman, W. C., and Mohanakumar, T. (2003). Concommitant augmentation of type I CD4(+) and CD8(+) T-cell responses during successful interferon-alpha and ribavirin treatment for chronic hepatitis C virus infection. Hum Immumol 64,497-504. Stuyver, L. J., Whitaker, T., McBrayer, T. R., Hernandez-Santiago, B. I., Lostia, S., Tharnish, P. M., Ramesh, M., Chu, C. K., Jordan, R., Shi, J., et al. (2003). Ribonucleoside analogue that blocks replication of bovine viral diarrhoea and hepatitis C viruses in culture. Antimicrob Agents Chemother47, 244-254. Sun, J., Bodola, F., Fan, X., Irshad, H., L., S., Lemon, S. M., and Chan, T. S. (2001). Hepatitis C virus core and envelope proteins do not suppress the host's ability to clear a hepatic viral infection. J Virol 75, 11992-11998. Swingler, S., Mann, A., Jacque, J.-M., Brichacek, B., Sasseville, V. G., Williams, K., Lackner, A. A., Janoff, E. N., Wang, R., Fisher, D., and Stevenson, M. (1999). HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages. Nat Med 5, 997-1003. Tabor, E., Garety, R. J., Druker, J. A., Seeff, L. B., Hoofnagle, J. H., Jackson, D. R., April, M., Barker, L. F., and Pineda-Tomondong, G. (1978). Transmission of non-A non-B hepatitis from man to chimpanzee. Lancet 1, 463-466. Tabor, E., and Gerety, R. J. (1980). Inactivation of an agent of human non-A, non-B hepatitis by formalin. J Infect Dis 142, 767-770. 324 Takahashi, M., Yamada, G., Miyamoto, R., Doi, T., Endo, H., and Tsuji, T. (1993). Natural course of chronic hepatitis C. Amer J Gasteroenterol88, 240-243. Takaki, A., Wiese, M., Maertens, G., Depla, E., Seifert, U., Liebetrau, A., Miller, J. L, Manns, M. P., and Rehermann, B. (2000). Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis 0. Nat Med 6, 578-582. Takamatsu, M., Fujita, T., and Hotta, H. (2001). Suppression of serum starvation-induced apoptosis by hepatitis 0 virus core protein. Kobe J Med Sci 47, 97-112. Takikawa, S., Ishi, K., Aizaki, H., Suzuki, T., Asakura, H., Matsuura, Y., and Miyamura, T. (2000). Cell fusion activity of hepatitis C virus envelope proteins. J Virol74, 5066-5074. Tan, S. L, Nakao, H., He, Y., Vijaysri, S., Neddermann, P., Jacobs, B. L, May, B. J., and Katze, M. G. (1999). NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology domain/ligand-dependant manner and perturbs mitogenic signalling. Proc Natl Acad Sci USA 96, 5533-5538. Tanaka, T., Kato, N., Nakagawa, M., Ootsuyama, Y., Cho, M. J., Nakazawa, T., Hijikata, M., Ishimura, Y., and Shimotohno, K. (1992). Molecular cloning of hepatitis C virus genome from a single Japanese carrier: Sequence variation within the same individual and amoung infected individuals. Virus Res 23, 39-53. Taniguchi, S., Okamoto, H., Sakamoto, M., Kojima, M., Tsuda, P., Tanaka, S., Munekata, E., Muchmore, E., Peterson, D. A., and Mishiro, S. (1993). A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape antibody. Virology 195, 297-301. Tanji, Y., Kaneko, T., Satoh, S., and Shimotohno, K. (1995). Phosphorylation of the hepatitis C virus-encoded non-structural protein NS5A. J Virol 69, 3980-3986. Taub, D. D., Conolon, K., Lloyd, A. R., Oppenheim, J. J., and Kelvin, D. J. (1993). Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1 alpha and MIP-1 beta. Science260, 35-57. Taylor, D. R., Shi, S., Romano, P. R., Barber, G. N., and Lai, M. M. C. (1999). Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science2 8 5 107-110. , Thibeault, D., Maurice, R., Pilote, L., Lamarre, D., and Pause, A. (2001). In vitro characterisation of a purified NS2/3 protease variant of hepatitis C virus. J Biol Chem 276, 46678-46684. Thimme, R., Oldach, D., Chang, K.-M., Steiger, C., Ray, S. C., and Chisari, F. V. (2001). Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 194, 1395-1406. Thomas, D. L., Factor, S. H., Kelen, G. D., Washington, A. S., Taylor, E. J., and Quinn, T. C. (1993). Viral hepatitis in health care professionals at the John Hopkins hospital: The 325 seroprevalance of and risk factors for hepatitis B virus and hepatitis C virus infection. Arch Intern Med153, 1705-1712. Thomas, D. L, Villano, S. A., Riester, K. A., Hershow, R., Mofenson, L. M., Landesman, S. H., Hollinger, F. B., Davenny, K., Riley, L., Diaz, C.,et ai (1998). Perinatal Transmission of Hepatitis C Virus from Human Immunodeficieny Virus Type 1-infected Mothers. J Infect Dis 177, 1480-1488. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., MeinI, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J. L., Schroter, M., et ai (1997). Viral induced FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature386. Thursz, M., Yallop, R., Goldin, R., Trepo, C., and Thomas, H. C. (1999). Influences of MHC class II genotype on outcome of infection with hepatitis C virus. The HENCORE group. Hepatitis C European Network for Cooperative Research. Lancet354, 2119-2124. Tokushige, K., Wakita, T., Pachuck, 0., Moradapour, D., Weiner, D. B., Zurawski, V. R. J., and Wands, J. R. (1996). Expression and immune response to hepatitis 0 virus core DNA- based vaccine constructs. Hepatology 2 4 ,14-20. Trendelenburg, M., and Schifferli, J. A. (2003). Cryoglobulins in chronic hepatitis C virus infection. Clin Exp Immunol 133, 153-155. Triyatni, M., Saunier, B., Maruvada, P., Davis, A. R., Ulianich, L., Heller, T., Patel, A., Kohn, L. D., and Liang, T. J. (2002). Interaction of hepatitis C virus-like particles and cells: a model system for studying viral binding and entry. J Virol 76, 9335-9344. Tseng, C. T., and Klimpel, G. R. (2002). Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med 195, 43-49. Tseng, C. T., Miskovsky, E., Houghton, M., and Klimpel, G. R. (2001). Charaterisation of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections. Hepatology 33,1312-1320. Tsuchihara, K., Hijikata, M., Fukuda, T., Kuroki, T., Yamamoto, N., and Shimotohno, K. (1999). Hepatitis C virus core protein regulates cell growth and signal transduction pathways transmitting growth stimuli. Virology 258, 100-107. Tsukuma, H., Hiyama, T., and Tanaka, S. (1993). Risk factors for hepatocellular carcinoma amoung young patients with chronic liver disease. N Eng J Med 328, 1797- 1801. Tsutsumi, M., Urashima, S., Takada, A., Date, T., and Tanaka, Y. (1994). Detection of antigens related to HCV RNA encoding the NS5 region in livers of patients with chronic type C hepatitis. Hepatology 19, 265-272. Tsutsumi, T., Suzuki, T., Moriya, K., Yotsuyanagi, H., Shintani, Y., Fujie, H., Matsuura, Y., Kimura, S., Koike, K., and Miyamura, T. (2002). Alteration of intrahepatic cytokine expression and AP-1 activation in transgenic mice expressing hepatitis C virus core protein. Virology304, 415-424. 326 Urbani, S., Boni, C., Missale, G., Elia, G., Cavallo, C., Massari, M., Raimond, G., and Ferrari, 0. (2002). Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and 0. J Virol 76, 12423- 12434. Van der Poel, 0. L. (1994). Hepatitis 0 virus. Epidemiology, transmission and prevention. In Hepatitis 0 Virus, H. W. Reesink, ed. (Amsterdam, Karger), pp. 137-163. Vassalli, P. (1992). The pathophysiology of tumour necrosis factors. Ann Rev Immunol 10, 411-452. Vidalin, O., Fournillier, A., Renard, N., Chen, M., Depla, E., Boucreux, D., Brinster, 0., Baumert, I . F., Nakano, I., Fukuda, Y., et al. (2000). Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis 0 virus core and E2 antigens. Virology 276, 259-270. Wajant, H. (2002). The Fas signalling pathway: More than a paradigm. Science 296,1635- 1636. Wakita, T., Taya, 0., Katsume, A., Kato, J., Yonekawa, H., Kanegae, Y., Saito, I., Hayashi, Y., Koike, M., and Kohara, M. (1998). Efficient conditional transgene expression in HCV cDNA transgenic mice mediated by the cre/lox p system. J Biol Chem 273, 9001-9006. Walker, C. M. (1997). Comparative features of hepatitis C virus infection in humans and chimpanzees. Springer Semin Immunopathol19, 85-98. Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J., Parsons, S., Zhang, F., Rathnachalam, R., Kirkegaard, K., and Colacino, J. M. (2002). Oligomerisation and cooperative RNA synthesis activity of hepatitis C virus RNA- dependant RNA polymerase. J Virol 76, 3865-3872. Watanabe, T., Katagiri, J., Kojima, H., Kamimura, T., Ichida, F., Ashida, M., Hamada, C., and Shibayama, T. (1987). Studies on transmission of human NANB hepatitis to marmosets. J Med Virol 22, 143-156. Waxman, L., Whitney, M., Pollok, B. A., Kuo, L. C., and Darke, P. L. (2001). Host cell factor required for hepatitis C virus enzyme maturation. Proc Natl Acad Sci USA 98, 13931-13935. Weiner, A., Erikson, A. L., Kansopon, J., Crawford, K., Muchmore, E., Hughes, A. L., Houghton, M., and Walker, C. M. (1995a). Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant. Proc Natl, Acad Sci USA 92, 2755-2759. Weiner, A. J., Erickson, A. L., Kansopon, J., Crawford, K., Muchmore, E., Houghton, M., and Walker, C. M. (1995b). Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus infection. Princess Takamatsu Symp 25, 227-235. 327 Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J. E., Mason, T. J., Saracco, G., Bonino, F., Crawford, K., Marion, 0. D., Crawford, K. A., et al. (1992). Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: Potential role in chronic HCV infections. PNAS89, 3468-3472. Wejstal, R., Norkrans, R., and Weiland, O. (1992). Lymphocyte subsets and 82 microglobulin expression in chronic hepatitis C/non A-non B: effect of interferon-alpha treatment. Clin Exp Immunol 87, 340-345. Welch, P. J., Tritz, R., Yei, S., Leavitt, M., Yu, M., and Barber, J. (1996). A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for HCV infection. Gene Ther 3, 994-1001. Welch, P. J., Yei, S., and Barber, J. R. (1998). Ribozyme gene therapy for hepatitis C virus infection. Clin Diag Virol 1 0 ,163-171. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B. (1977). Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells. J Immunol 118, 355-361. Whitney, M., Stack, J. H., Darke, P. L., Zheng, W., Terzo, J., Inglese, J., Strulovici, B., Kuo, L. C., and Pollock, B. A. (2002). A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity. J Biomol Screen 7 , 149-154. Wickham, T. J., Mathias, P., Cheresh, D. A., and Nemerow, G. R. (1993). Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73, 309-319. Wieland, S., Vega, R. G., Muller, R., Evans, C. P., Hillbush, B., Guidotti, L. G., Sutcliffe, J. G., Schultz, P. G., and Chisari, F. V. (2003). Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J Virol 77, 1227-1236. Wiertz, E. J., Jones, T. R., Sun, L., Biogyo, M., Geuze, H. J., and Ploegh, H. (1996). The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell84, 769-779. Wilkinson, G. W. G., and Akrigg, A. (1992). Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. NucI Acids Res20, 2233- 2239. Willems, P., Amraoui, Z., Vanderheyde, N., Verhasselt, V., Aksoy, E., Scaffidi, C., Peter, M. E., Krammer, P. H., and Goldman, M. (2000). Expression of c-FLIP(L) and resistance to CD95-mediated apoptosis of monocyte-derived dendritic cells: inhibition by bisindolylaleimide. Blood 95, 3478-3482. Williams, B. R. G., and Sen, G. C. (2003). A viral on/off switch for interferon. Science 800, 1100- 1101. Wilson, J. A., Jayasena, S., Khvorova, A., Sabatinos, S., Rodrigue-Gervais, I. G., Arya, S., Sarangi, P., Harris-Brandts, M., Beaulieu, S., and Richardson, C. D. (2003). RNA 328 interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci USA 100, 2783-2788. Wold, W. S. M., Doronin, K., Toth, K., Kuppuswamy, M., Lichtenstein, D. L., and Tollefson, A. E. (1999). Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic. Curr Opin Immunol 11, 380-386. Wolff, S. M. (1972). The Chediak-Higashi syndrome: studies of host defenses. Ann Intern Med 76, 293-306. Wong, D. K., Dudley, D., Afdhal, N., Dienstag, J., Rice, C. M., Wang, L., Houghton, M., Walker, B. and Koziel, M. J. (1998). Liver-derived CTL in hepatitis 0 virus infection: breadth and specificity of responses in a cohort of persons with chronic infection. J Immunol 160, 147-188. Wong, D. K. H., Dudley, D. D., Dohrenwend, P. B., Lauer, G. M., Chung, R. T., Thomas, D. L., and Walker, B. D. (2001). Detection of diverse Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes in peripheral blood of infected persons by screening for responses to all translates proteins of HCV. J Virol 75, 1299-1235. Wong, J. B. (1999). Cost-effectiveness of treatments for chronic hepatitis C. Amer J Med 107, 745-785. Wong, P., and Pamer, E. G. (2003). Feedback regulation of pathogen-specific T cell priming. Immunity 18, 499-511. Wordarz, D. (2001). Helper-dependent vs. helper-independent CTL responses in HIV infection: implications for drug therapy and resistance. J Theor Biol 213, 447-459. Wordarz, D., and Jansen, V. A. (2001). The role of T cell help for anti-viral CTL responses. J Theor Biol 2f 7, 419-432. Wunderlich, J., and Shearer, G. (2000). Induction and measurement of cytotoxic T lymphocyte activity. In Current Protocols in Immunology, J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. (John Wiley & Sons, Inc.), pp. 3.11. Wyld, R., Robertson, J. R., Brettle, R. P., Mellor, J., Prescott, L., and Simmonds, P. (1997). Absence of hepatitis C virus transmission but frequent transmission of hepatitis C virus from a sexual contact with doubly-infected individuals. J Infect 35, 163-166. Xie, Z. C., Riezu-Boj, J. L., Lasarte, J. J., Guikken, J., Su, J. H., Civeira, M. P., and Prieto, J. (1998). Transmission of hepatitis C virus to tree shrews. Virology 24, 513-520. Xu, Z., Choi, J., and Ou, J. H. (2003). Hepatitis C virus F protein is a short-lived protein associated with the endoplasmic reticulum. J Virol 77, 1578-1583. Xu, Z., Choi, J., Yen, T. S., Lu, W., Strohecker, A., Govindarajan, S., Chein, D., Selby, M. J., and Ou, J. (2001). Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO 2-, 3840-3848. 329 Yamaga, A. K., and Ou, J. H. (2002). Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem 277, 33228-33234. Yamanaka, T., Kodama, T., and Doi, T. (2002). Subcellular localisation of HCV core protein regulates its ability for p53 activation and p21 suppression. Biochem Biophys Res Comm 294, 528-534. Yanagi, M., Purcell, R. H., Emerson, S. U., and Bukh, J. (1997a). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc natl Acad Sci USA 94, 8738-8743. Yanagi, M., Purcell, R. H., Emerson, S. U., and Bukh, J. (1999). Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic la and 2a chimeras. Virology 262, 250-263. Yanagi, M., St Claire, M., Emerson, S. U., Purcell, R. H., and Bukh, J. (1997b). In vivo analysis of the 3' UTR of the hepatitis C virus after in vitro mutagenesis of an infectious clone. Proc Natl Acad Sci USA 96, 2291-2295. Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H., and Bukh, J. (1998). Transcripts of a Chimeric cDNA Clone of Hepatitis C Virus Genotype 1b are Infectious in Vivo. Virology 244, 161-172. Yang, S. H., Lee, C. G., Lee, C. W., Choi, E. J., Yoon, S. K., Ahn, K. S., and Sung, Y. C. (2002). Hepatitis C virus core inhibits the , Fas-mediated p38 mitogen activated kinase signalling pathway in hepatocytes. Mol Cells 13, 452-462. Yao, Z. Q., Nguyen, D. T., Hiotellis, A. I., and Hahn, Y. S. (2001a). Hepatitis C virus core protein inhibits human T lymphocyte responses by a complement-dependant regulatory pathway. 2001 167, 5264-5272. Yao, Z. Q., Ray, S., Eisen-Vandervelde, A., Waggoner, S., and Hahn, Y. S. (2001b). Hepatitis C virus: Immunosuppression by complement regulatory pathway. Viral Immunol 14, 277-295. Yasui, K., Wakita, T., Tsukiyama-Kohara, K., Funahashi, S. I., Ichikawa, M., Kajita, T., Moradapour, D., Wands, J. R., and Kohara, M. (1998). The native form and maturation process of hepatitis C virus core protein. J Virol 72, 6048-6055. Yi, M., Kaneko, S., Yu, D. Y., and Murakami, S. (1997). Hepatitis C virus envelope protein binds lactoferrin. J Virol 71, 5997-6002. Yi, M., and Lemmon, S. M. (2003). 3' nontranslated RNA signals required for replication of hepatitis C virus RNA. J Virol 77, 3557-3568. Yi, M., and Lemon, S. M. (2003). Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA 9, 331-345. Yonekura, K., Ichida, T., Sato, K., Yamagiwa, S., Uchida, M., and Sugahara, 8. (2000). Liver-infiltrating CD56 positive T lymphocytes in hepatitis C virus infection. Liver20, 357- 365. 330 York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L, and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-535. Yoshida, H., Kato, N., Shiratori, Y., Otsuka, M., Maeda, S., Kato, J., and Omata, M. (2001 ). Hepatitis 0 virus core protein activates nuclear factor kappaB dependent signalling through tumor necrosis factor receptor-associated factor. J Biol Chem 276,16399-16405. Yoshizawa, H., Ito, Y., Iwakiri, S., Kitajima, K., Tanaka, A., Tachibana, T., Nakamura, T., Miyakawa, Y., and Mayumi, M. (1982). NANB (type 1) hepatitis agent capable of inducing tut)ular ultrastructures in the hepatocyte cytoplasm of chimpanzees: Inactivation by formalin and heat. Gasteroenterology 82, 502-506. You, L. R., Chen, C. M., Yeh, I . S., Tsai, T., Y., R.T., M., Lin, C. H., and Lee, Y. H. (1999). Hepatitis C virus core protein interacts with cellular RNA helicase. J Virol 72, 3691-3697. Yuk, K., Shimizu, M., and Aoyama, 8. (1986). Analysis of lymphocytes subsets in liver biopsy specimens with lymphoid follicle like structures. Acta Hepat Jpn 27, 720-725. Zajac, A. J., Blattman, J. N., Murali-Krishna, K., Sourdive, D. J., Suresh, M., Altman, J. D., and Ahmed, R. (1998). Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med188, 2199-2204. Zeuzem, S., Feinman, S. V., Rasenack, J., Heathcote, E. J., Lai, M. Y., Gene, E., O'Grady, J., Reichen, J., Diago, M., Lin, A.,et al. (2000). Peg interferon alpha2a in patients with chronic hepatitis C. New Engl J Med343, 1666-1672. Zhang, G., Gurtu, V., Kain, S. R., and Yan, G. (1997). Early detection of apoptosis using a fluorescent conjugate of annexin V. Biotechniques 23, 525-531. Zhong, L., Granelli-Piperno, A., Choi, Y., and Steinman, R. M. (1999). Recombinant adenovirus is an efficient and non-perturbing genetic vector for human dendritic cells. Eur J Immunol 29, 964-972. Zhu, N., Koshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C., and Lai, M. M. (1998). Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J Virol 72, 3691-3697. Zhu, N., Ware, C. F., and Lai, M. M. (2001). Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signalling of tumor necrosis factor receptor. Virology2 8 3 ,178-187. Zibert, A., Meisel, H., Kraas, W., Jung, G., and Roggendorf, M. (1997). Early antibody response against hypervariable region 1 is associated with acute self-limiting infections of hepatitis C virus. Hepatology 25, 1245-1249. 331