Studies of HIV and HCV co-infection in the context of haemophilia A thesis submitted to the University of London for the degree of Doctor of Philosophy in the Faculty of Medicine by Esteban Herrero-Martinez B.Sc. Department of Virology and Haemophilia and Haemostasis Centre Royal Free and University College Medical School University of London October 2002 ProQuest Number: U643503 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 U643503 Published by ProQuest LLC(2016). 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 Dedicated to my family Angel, Celia and Erica 11 Abstract HIV and HCV co-infection is highly prevalent in intravenous drug users and individuals with inherited bleeding disorders treated with clotting factor concentrates prior to the introduction of heat sterilisation in 1985. The presence of HIV accelerates HCV disease progression and the presence of HCV impairs CD4+ T-cell recovery post-HAART, although the underlying mechanisms for these effects remain to be elucidated. The aim of this thesis was to investigate HIV and HCV co-infection in the context of haemophilia. Initially, a quantitative competitive RT-PCR assay for HCV was developed. This assay was used in a retrospective cohort study of 96 men with haemophilia to determine the prognostic value of a single HCV RNA load measured early post HIV seroconversion. This study showed for the first time, that HCV RNA level early post HIV seroconversion is associated with progression to both AIDS and all-cause mortality over a period of at least 15 years. The effect of HAART on HCV replication is controversial, with some studies reporting no effect and others increases, reductions and clearances of HCV RNA post­ therapy. I have investigated the effect of HAART on the titre of anti-HCV specific antibodies and on the relationship between these antibodies and HCV RNA level in a cohort of 24 patients with inherited bleeding disorders. A significant inverse correlation between anti-HCV antibodies was observed pre-HAART that disappeared or was obscured post-therapy. I have therefore shown that HAART affects HCV specific humoral immune responses without affecting HCV RNA level. Finally, I have investigated the effect of HCV co-infection on the expression of a range of memory markers on total CD8+ T-cells in HIV positive and negative patients. Significant differences in memory marker expression were observed in all four cohorts investigated; HIV mono-infected, HCV mono-infected, HIV and HCV co-infected and HIV and HCV negative controls. I ll Acknowledgements I would like to express my thanks to my supervisors Professors Vince Emery and Christine Lee for all their help, support and advice throughout my PhD and the writing of this thesis. I would also like to thank Dr Caroline Sabin for her help in the analysis of viral load and immunofluorescence data, Professor Paul Griffiths for giving me the opportunity to write for Reviews in Medical Virology, Dr Helen Devereux for her help and encouragement during the early stages of my PhD and Drs Paul Klenerman, Michaela Lucas and Ana Vargas for their contributions to my work on CD8+ T-cell memory markers. I would also like to thank the Katharine Dormandy Trust and the Royal Free Hospital Virology Department for funding my PhD. Many thanks to Ms Joyce Beo for the HLA typing of patient samples, to all the staff at the Ian Charleson Day Care Centre and especially to all my colleagues at the Royal Free Virology and Haemophilia Departments for all their help and friendship over the last four years; Aycan, Carol, Chris D, Claire A, Claire SL, Donna, Duncan, Ed, Elaine, Emma, Eva, Frank, Gill, Jacqueline, Jane, Jeremy, Kali, Keirissa, Kuba, Matt, Nigel, Ruth, Shelley, Sly, Ting, Toyin, Ann H, Anne R, Anja, Chris H, Debra, Gillian, Jacqui, Pura, Sarah and Thynn. I would finally like to thank all my friends outside the Royal Free Hospital (you know who you are!) and my family for always being there when I needed a pint of beer or a bottle of wine. IV Contents Contents Abstract iii Acknowledgements iv Contents v List of Figures xii List of Tables xvi Abbreviations xviii Chapter 1 : General introduction 1 1.1 Discovery of HCV 2 1.1 Epidemiology of HCV 3 1.2 Natural History of HCV 4 1.3 HCV Genome organisation 5 1.4.1 The 5’untranslated region 5 1.4.2 Core and F proteins 9 1.4.3 Envelope glycoproteins and p7 protein 10 1.4.4 Non-structural protein 2 (NS2) 11 1.4.5 NS3 and NS4 11 1.4.6 NS5 12 1.4.7 The 3’untranslated region 13 1.5 HCV replication 14 1.6 Genetic variability and genotype 16 1.6.1 Geographical distribution and disease associations 17 1.6.2 HCV genotype and disease associations 20 1.6.3 HCV quasispecies 21 1.6.4 Assigning HCV genotype 22 1.7 HCV diagnosis 22 1.7.1 Serological assays 23 1.7.2 Qualitative and quantitative nucleic acid amplification 23 1.7.3 Chiron TMA qualitative assay 24 1.7.4 Roche AMPLICOR (manual and COBAS assays) 25 V Contents 1.7.5 HCV quantification by branched chain DNA 25 1.7.6 NucliSense quantification of HCV 26 1.8 Associations of HCV RNA load with disease and treatment outcomes 26 1.8.1 Response to treatment 26 1.8.2 Disease progression 27 1.9 HCV therapy 28 1.9.1 Interferon-oj monotherapy 28 1.9.2 Interferon-a and ribavirin combination therapy 29 1.9.3 Pegylated interferon therapy 29 1.9.4 Future therapies for HCV 30 1.9.5 Progress towards the development of a vaccine for HCV 31 1.10 Pathogenesis of HCV 32 1.10.1 Humoral immunity 3 2 1.10.2 Cellular immunity (CD4+ T-lymphocytes) 33 1.10.3 Cellular immunity (CD8+T-lymphocytes) 35 1.10.4 Innate immunity and additional factors 37 1.11 HIV epidemiology 39 1.12 HIV natural history 40 1.13 HIV genome organisation and replication 42 1.14 HIV and HCV co-infection 51 1.14.1 Effect of HIV co-infection on liver disease 51 1.14.2 Mechanisms by which HIV may influence HCV disease progression 52 1.14.3 Effect of HCV co-infection on HIV disease 53 1.15 HIV therapy 54 1.15.1 Novel approaches to HIV therapy 56 1.15.2 HIV therapy in HCV co-infected patients 56 1.15.3 Effect of HIV therapy of HCV 58 1.16 Immune response to HIV 58 1.16.1 Humoral immunity 5 8 1.16.2 Cellular immunity (CD4+T-lymphocytes) 59 1.16.3 Cellular immunity (CD8 + T-lymphocytes) 60 1.18 Aims of thesis 61 VI Contents Chapter 2: Materials and methods 62 2.1 Development of a quantitative-competitive RT PCR assay (QC RT-PCR) 63 for HCV 2.1.1 Cloning of HCV 5’UTR into pCR2.1 TA cloning vector 63 2.1.2 Mutagenic PCR reactions 64 2.1.3 Agarose gel electrophoresis 66 2.1.4 Annealing and ‘Klenow fill’ of PCR products 66 2.1.5 PCR reactions 67 2.1.6 Purification of the 5’UTR wild type and control inserts 67 2.1.7 Ligation into pGEM T-Easy vector 68 2.1.8 Preparation of transformation competent E.coli JMl 09 cells 68 2.1.9 Transformation into competent JM l09 cells 69 2.1.10 Mini-preparation of plasmid DNA from bacterial cultures 70 2.1.11 Screening of plasmid colonies by restriction enzyme digestion 70 2.1.12 Plasmid sequencing reactions 71 2.1.13 Analysis of sequencing reactions by 7M polyacrylamide urea gel 73 2.1.14 Large-scale preparation of plasmid DNA 74 2.2.15 Linearisation of plasmid prior to transcription 75 2.2.16 In-vitro transcription using T7 RNA polymerase 75 2.1.17 Removal of DNA template with RNase free DNase 1 76 2.1.18 Clean up of RNA 76 2.1.19 MOPS/ formaldehyde gel electrophoresis of RNA transcripts 76 2.2 Optimisation of QC RT-PCR using internal standard RNA transcripts 78 2.2.1 Reverse transcription and PCR amplification of cloned 5’UTR 78 2.2.2 Generation of standard curve for QC RT-PCR assay 79 2.2.3 Hind III digests of amplified DNA 79 2.2.4 Polyacrylamide gel electrophoresis (PAGE) of RT-PCR products 79 2.2.5 Gel scanning and quantification 80 2.2.6 Viral RNA extractions 80 2.3 Routine use of the QC RT-PCR assay for HCV 81 2.4 HCV genotyping assay 84 2.4.1 Reverse transcription of HCV RNA 84 2.4.2 First round PCR 84 V ll Contents 2.4.3 Second round PCR 85 2.4.4 Restriction digests of second round PCR products 86 2.4.5 15% polyacrylamide gel electrophoresis of digested DNA 86 2.5 Immunofluorescence assays for determining HIV and HCV specific 87 antibody titres 2.5.1 Sf-21 cell culture 87 2.5.2 Generation of high titre baculovirus stocks 88 2.5.3 Sf-21 cell plaque assay 88 2.5.4 Baculovirus protein expression time course in Sf-21 cells 90 2.5.5 SDS polyacrylamide gel electrophoresis 91 2.5.6 Western blot analysis 91 2.5.7 Immunofluorescence assay 94 2.6 Effect of HCV co-infection on expression of memory markers on CD8+ 99 T-cells in HIV positive patients 2.6.1 PBMC isolation 100 2.6.2 HLA typing 101 2.6.3 Thawing of frozen PBMCs 101 2.6.4 Phenotyping of CD8+ T-cells using directly conjugated antibodies 101 2.6.5 Unconjugated antibody staining of CCR7 on PBMCs 103 2.6.6 Acquisition of data and flow cytometric analysis 103 2.7 Statistical methods 106 2.7.1 Sign test 106 2.7.2 Wilcoxon signed ranks test 106 2.7.3 Chi squared test 107 2.7.4 Analysis of variance (ANOVA) 107 2.7.5 Pearson’s product moment correlation coefficient 108 2.7.6 Multivariate linear regression model 108