n Alotaibi, Sultan Merja T. (2016) The effect of the HLA B27 allele on the immune response to acute HCV in HIV infected patients. PhD thesis. http://theses.gla.ac.uk/7709/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] The effect of the HLA B27 allele on the immune response to acute HCV in HIV infected patients Sultan Merja T. Alotaibi BSc, MSc Submitted for the fulfilment of the requirements of degree of Doctor of Philosophy School of Medicine College of Medical, Veterinary and Life Sciences University of Glasgow May 2016 Abstract In mono-infected individuals, the HLA-B27 allele is strongly associated with spontaneous clearance of HCV in association with a strong CD8+ response targeted against a single epitope within the HCV RNA-dependent RNA polymerase (NS5B). We studied variation across the whole HCV genome and T cell responses over time in a rare cohort of HLA-B27+ patients with acute HCV and HIV co-infection, the majority of whom progressed to chronicity. We used next generation sequencing to detect changes within and outwith the immuno-dominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B (ARMILMTHF) during evolving progression of early HCV infection. 2841-2849 Within the Acute HCV UK cohort, 10 patients carried the HLA B27 allele. Of these, 3/8 patients (37.5%) with HIV infection and 2/2 (100%) without HIV spontaneously cleared HCV (p=0.44). Sequential samples from nine HLA-B27+ patients (2 with monoinfection and 7 with HIV co-infection) were available for analysis (four spontaneous clearers and five evolving progressors). Mutations identified using NGS were assessed using a replicon genotype 1a system to evaluate viral fitness. Multiple mutations within the HLA-B27 restricted NS5B 2841- epitope were associated with progression to chroncity whereas patients who 2849 cleared the HCV infection spontaneously had no or only one mutation at this site (p=0.03). A triple NS5B mutant observed during progression to chronicity 2841-2849 was associated with restored replication when compared to wild-type virus while single or double mutants were significantly associated with impaired replication (p=0.0495). T cell responses measured in these patients using ELISpot and flow cytometry. HLA-B27+ patients had significantly higher IFN-γ responses than patients who were HLA-B27- (p=0.0014). Those who progressed to chronicity had lower IFN-γ responses than those who cleared HCV (p=0.0011). Mono-infected patients had higher IFN-γ responses compared to co-infected patients (p=0.0015). HIV co-infection is associated with a lower likelihood of spontaneous clearance of HCV in HLA B27+ patients and this is associated with impaired T cell ii | Pa g e function in this group. Table of contents ABSTRACT ................................................................................................................................................ II TABLE OF CONTENTS .......................................................................................................................... III LIST OF TABLES ................................................................................................................................... VII LIST OF FIGURES ................................................................................................................................... IX ACKNOWLEDGEMENTS ..................................................................................................................... XII AUTHOR’S DECLARATION ............................................................................................................... XIV DEFINITIONS/ ABBREVIATIONS ..................................................................................................... XV PUBLICATIONS................................................................................................................................. XVIII CHAPTER 1: INTRODUCTION – OVERVIEW OF HEPATITIS C VIRUS INFECTION ............... 19 1.1 DISCOVERY OF THE HEPATITIS C VIRUS ........................................................................................................... 19 1.1.1 Classification and genotypes of HCV ................................................................................................. 20 1.1.2 Global prevalence and incidence of HCV ......................................................................................... 21 1.2 CLINICAL FEATURES OF HCV INFECTION .......................................................................................................... 25 1.2.1 Acute HCV infection .................................................................................................................................. 26 1.2.2 Chronic HCV infection .............................................................................................................................. 26 1.3 DIAGNOSIS OF HCV INFECTION........................................................................................................................... 27 1.3.1 Serological Assays ..................................................................................................................................... 27 1.3.2 Molecular virological assays: ............................................................................................................... 29 1.4 TREATMENT OF CO-INFECTED INDIVIDUALS .................................................................................................... 30 1.5 MOLECULAR FEATURES OF HCV ......................................................................................................................... 32 1.5.1 Virion morphology .................................................................................................................................... 32 1.5.2 The viral genome ....................................................................................................................................... 32 1.5.2.1 The 5’ untranslated region (5’UTR) ................................................................................................................................ 33 1.5.2.2 Core ................................................................................................................................................................................................ 34 1.5.2.3 E1 and E2 ..................................................................................................................................................................................... 35 1.5.2.4 p7 ..................................................................................................................................................................................................... 36 1.5.2.5 NS2 .................................................................................................................................................................................................. 37 1.5.2.6 NS3-4A .......................................................................................................................................................................................... 38 1.5.2.7 NS4A............................................................................................................................................................................................... 40 1.5.2.8 NS4B............................................................................................................................................................................................... 40 1.5.2.9 NS5A............................................................................................................................................................................................... 41 1.5.2.10 NS5B ............................................................................................................................................................................................ 43 1.5.2.11 The 3’ untranslated region (3’ UTR). ........................................................................................................................... 45 1.6 THE IMMUNE RESPONSE TO HCV ....................................................................................................................... 46 1.6.1 Innate immunity ........................................................................................................................................ 46 1.6.2 Natural killer cells (NK). ....................................................................................................................... 47 1.6.3 Dendritic cells (DC) ................................................................................................................................... 48 1.6.4 Adaptive immune responses to HCV .................................................................................................. 49 1.6.4.1 The CD8+ T cell response during acute HCV infection .......................................................................................... 49 1.6.4.2 CD4+ T cell responses during acute HCV ..................................................................................................................... 52 1.6.4.3 Humoral responses during acute HCV infection ...................................................................................................... 54 1.6.5 Summary of immune responses and immune evasion during acute HCV infection ...... 56 1.6.6 The HLA-B27 allele and disease .......................................................................................................... 57 1.6.6.1 Peptide Presentation by HLA-B27 to CD8+ Cytotoxic T Cells ............................................................................ 57 1.6.6.2 HLA-B27 Epitopes ................................................................................................................................................................... 59 1.6.6.3 The protective role of HLA-B27 in HIV infection ..................................................................................................... 59 iii | Pa g e 1.6.6.4 The protective role of HLA-B27 in HCV infection .................................................................................................... 60 1.6.7 HIV and hepatitis C virus co-infection .............................................................................................. 61 1.6.7.1 The influence of HCV on HIV infection .......................................................................................................................... 62 1.6.7.2 The influence of HIV on HCV infection .......................................................................................................................... 63 1.7 METHODS USED TO STUDY HCV ......................................................................................................................... 64 1.7.1 Cell culture of HCV .................................................................................................................................... 64 1.7.1.1 Chimeric JFH-1 genomes ...................................................................................................................................................... 66 1.7.2 The HCV replicon system ........................................................................................................................ 67 1.7.3 Permissive cell lines for HCV replication ......................................................................................... 69 1.7.4 Next-generation sequencing (NGS) ................................................................................................... 69 1.7.4.1 NGS using the Illumina platform ...................................................................................................................................... 70 1.7.4.2 Errors and limitations of NGS ............................................................................................................................................ 71 1.7.4.3 Mapping........................................................................................................................................................................................ 72 1.7.5 Enzyme-Linked ImmunoSpot (ELISPOT) assays .......................................................................... 73 CHAPTER 2: MATERIALS AND METHODS ..................................................................................... 75 2.1 MATERIALS .............................................................................................................................................................. 75 2.2 METHODS ................................................................................................................................................................. 79 2.2.1 Patient cohort ............................................................................................................................................. 79 2.2.2 Diagnosis of acute HCV ........................................................................................................................... 79 2.2.3 Patient treatment outcome ................................................................................................................... 80 2.2.4 Storage of blood samples: ...................................................................................................................... 80 2.2.5 RNA Extraction ........................................................................................................................................... 80 2.2.5.1 QIAamp® Viral RNA kit (Qiagen) ..................................................................................................................................... 80 2.2.5.2 EasyMAG® NucliSENS Extractor....................................................................................................................................... 81 2.2.6 cDNA Synthesis ........................................................................................................................................... 81 2.2.6.1 Superscript III ® reverse transcriptase kit .................................................................................................................. 81 2.2.6.2 Maxima H Minus Reverse Transcriptase® ................................................................................................................... 82 2.2.7 DNA second strand synthesis ................................................................................................................ 82 2.2.8 Quantitative RT-PCR assays.................................................................................................................. 82 2.2.9 Gel electrophoresis .................................................................................................................................... 83 2.2.10 DNA purification ..................................................................................................................................... 83 2.2.10.1 Purification of DNA from agarose gels ....................................................................................................................... 83 2.2.10.2 Agencourt AMPure XP® beads ........................................................................................................................................ 84 2.2.11 Measuring DNA concentration ......................................................................................................... 85 2.2.11.1 Measurement of nucleic acid concentration using Qubit® ............................................................................... 85 2.2.11.2 Nucleic acid QC using the 2200 TapeStation ........................................................................................................... 85 2.2.12 Bacterial cloning ..................................................................................................................................... 86 2.2.12.1 TOPO-TA® Cloning kit ........................................................................................................................................................ 86 2.2.12.2 CloneJET® PCR cloning kit ................................................................................................................................................ 86 2.2.13 High efficiency transformation protocol ...................................................................................... 87 2.2.13.1 One shot® Top 10 cells ....................................................................................................................................................... 87 2.2.13.2 NEB 10-beta Competent E. coli....................................................................................................................................... 87 2.2.14 Preparation of DNA following bacterial cloning ...................................................................... 88 2.2.14.1 Small scale plasmid preparation from transformed bacteria ......................................................................... 88 2.2.14.2 Large scale plasmid preparation from transformed bacteria ......................................................................... 89 2.2.14.3 Alignment of NGS Data ....................................................................................................................................................... 90 2.2.15 Nucleotide sequencing and analysis ............................................................................................... 90 2.2.16 Illumina bioinformatic analysis ....................................................................................................... 90 2.2.17 Human hepatoma cells (Huh7.5) ..................................................................................................... 91 2.2.18 In vitro transcription............................................................................................................................. 91 2.2.18.1 Site-Directed Mutagenesis ................................................................................................................................................ 91 2.2.18.1 Introduction of mutations into the HCV-1a subgenomic replicon ................................................................ 92 2.2.18.2 Oligonucleotide Synthesis for Site-Direct Mutagenesis ..................................................................................... 94 2.2.19 DNA digestion ........................................................................................................................................... 94 2.2.20 DNA ligation.............................................................................................................................................. 96 2.2.20.1 Linearisation of HCV genomic plasmid DNA and sub-genomic replicon (SGR) DNA .......................... 96 2.2.20.2 Transcription procedure ................................................................................................................................................... 97 2.2.20.3 Electroporation of RNA into Huh 7.5 mammalian cells ..................................................................................... 97 2.2.20.4 Firefly luciferase activity assay ...................................................................................................................................... 97 2.2.21 Enzyme-linked immunospot (ELISPOT) assay ........................................................................... 98 iv | Pa g e 2.2.22 Peptides:.................................................................................................................................................... 101 2.2.23 Flow cytometry ...................................................................................................................................... 101 2.2.24 Extraction of DNA from whole blood ........................................................................................... 103 2.2.25 HLA Typing .............................................................................................................................................. 103 2.2.26 Sanger sequencing and analysis..................................................................................................... 103 2.2.27 Nextera XT® DNA Sample Preparation ....................................................................................... 103 2.2.28 Illumina sequencing ............................................................................................................................ 104 2.2.29 Statistical analysis ................................................................................................................................ 108 CHAPTER 3: RESULTS ...................................................................................................................... 109 3.1.1 Patient cohort ........................................................................................................................................... 109 3.1.2 DNA library quality ................................................................................................................................ 113 3.1.3 Sequencing quality scores .................................................................................................................... 113 3.1.4 Raw data ..................................................................................................................................................... 116 3.1.5 Sequence alignments ............................................................................................................................. 117 3.1.6 Quality control for NGS data: ............................................................................................................. 123 3.1.6.1 Cross-contamination during next-generation sequencing:.............................................................................. 123 3.1.6.2 Alignment bias during mapping to closely related HCV references sequences ..................................... 124 3.1.7 Phylogenetic analysis............................................................................................................................. 127 3.1.8 Best model calculation .......................................................................................................................... 128 3.1.9 Target enrichment versus metagenomic (unenriched) sequencing .................................. 130 3.1.10 Viral load dynamics ............................................................................................................................. 132 3.1.11 Mutations located within HLA-B27 restricted HCV epitopes ............................................. 137 3.1.11.1 Spontaneous clearer patients ...................................................................................................................................... 137 3.1.11.2 Progressor patients........................................................................................................................................................... 138 3.1.12 Structural analysis ............................................................................................................................... 153 3.1.12.1 Predicted structure ........................................................................................................................................................... 153 3.1.13 Selection pressure across the HCV genome. .............................................................................. 158 3.2 FUNCTIONAL ANALYSES ..................................................................................................................................... 166 3.2.1 Design of a genotype 1a replicon containing a luciferase reporter gene ....................... 166 3.2.1.1 Introduction of adaptive mutations and a luciferase reporter gene into the genotype 1a replicon ....................................................................................................................................................................................................................... 166 3.2.2 Strategy for the construction of a replication-competent genotype 1a sub-genomic replicon. .................................................................................................................................................................. 167 3.2.3 Introduction of a luciferase reporter gene into the genotype 1a sub-genomic replicon .................................................................................................................................................................................... 167 3.2.4 Introduction of mutations within the NS5B restricted epitope ............................ 171 2841-2849 3.2.5 Assessing the replication assay ......................................................................................................... 176 3.2.6 Replicative fitness of mutations within the NS5B restricted epitope................. 177 2841-2849 3.2.7 The effect of single and multiple clustered mutations on replication .............................. 178 3.3 DETECTING VIRUS SPECIFIC CD8+ T-CELL RESPONSE USING ELISPOT ................................................ 191 3.3.1 Subject characteristics .......................................................................................................................... 191 3.3.1.1 Measuring production of IFN-γ in HLA-B27+ spontaneous clearers and progressors ...................... 191 3.3.1.2 T cell responses in HLA-B27+ and HLA-B27- patients. ...................................................................................... 192 3.3.2 T- cell response in HIV/HCV co-infected and HCV mono-infected patients .................... 194 3.3.3 Individual patient responses .............................................................................................................. 197 3.3.4 Flow cytometry ......................................................................................................................................... 202 CHAPTER 4: DISCUSSION ................................................................................................................ 207 4.1 THE T CELL MEDIATED IMMUNE RESPONSE TO ACUTE HCV .................................................................... 208 4.2 FAILURE OF THE T CELL RESPONSE IN ACUTE HCV INFECTION ............................................................... 208 4.3 VIRAL ESCAPE ....................................................................................................................................................... 209 4.4 CD8+ T-CELL DYSFUNCTION ............................................................................................................................ 212 4.5 LACK OF CD4+ HELP .......................................................................................................................................... 213 4.5.1 Importance of the HLA-B27 allele and the association with spontaneous clearance of HCV ........................................................................................................................................................................... 213 4.5.2 Mutations detected within HLA-B27 epitopes using NGS ...................................................... 216 4.5.3 Functional analysis of observed mutations in the NS5B epitope ......................... 221 2841-2849 v | Pa g e 4.5.3.1 Single mutations.................................................................................................................................................................... 221 4.5.3.2 Multiple mutations ............................................................................................................................................................... 222 4.5.4 CD8+ T-cell responses in evolving spontaneous clearance and progression to chronicity. .............................................................................................................................................................. 225 4.5.5 Conclusions and future work .............................................................................................................. 227 BIBLIOGRAPHY .................................................................................................................................. 230 vi | Pa g e List of Tables Table 1-1: Sensitivity and positive predictive values of EIA for HCV detection ----------- 29 Table 1-2: Recommendation on treatment of HCV (EASL guidelines 2016) ---------------- 31 Table 2-1 DNA Manipulation & Purification ------------------------------------------------------ 75 Table 2-2: Cell lines --------------------------------------------------------------------------------- 76 Table 2-3: Enzymes ---------------------------------------------------------------------------------- 76 Table 2-4 Cell culture medium --------------------------------------------------------------------- 77 Table 2-5 Commonly Used Chemicals ------------------------------------------------------------- 77 Table 2-6 Bacterial Expression 
 ------------------------------------------------------------------- 78 Table 2-7 PCR cycle for Site-Direct mutagenesis ---------------------------------------------- 92 Table 2-8: Oligonucleotide sequences designed for site direct mutagenesis: ------------- 94 Table 2-9: Flow cytometry panels --------------------------------------------------------------- 102 Table 2-10: Flow cytometry antibodies--------------------------------------------------------- 102 Table 3-1 General Cohort description ---------------------------------------------------------- 110 Table 3-2 Characteristics of the HLA-B27 patient cohort ----------------------------------- 112 Table 3-3 Patients characteristics – controls -------------------------------------------------- 113 Table 3-4: HLA-B27 restricted epitopes within the HCV genome. --------------------- 119 Table 3-5: HLA-B27 restricted epitope within the HIV genome. --------------------------- 120 Table 3-6: HCV read depth and coverage of samples from HLA-B27+ patients (spontaneous clearers) with acute HCV ------------------------------------------ 121 Table 3-7: HCV read depth and coverage of samples from HLA-B27+ patients (progressors) with acute HCV. ----------------------------------------------------- 122 Table 3-8: Calculated enrichment in P45 HCV reads mapped to genotype 1a and 1b reference sequences. ---------------------------------------------------------- 126 Table 3-9: Mutations observed within HLA-B27 restricted epitope within patient G5 -- 140 Table 3-10: Mutations observed over time within HLA-B27 restricted epitopes in patient G18 (G18) -------------------------------------------------------------------- 141 Table 3-11: Mutations observed over time within HLA-B27 restricted epitopes in patient 49 (P49) ---------------------------------------------------------------------- 142 Table 3-12: Mutations observed over time within HLA-B27 epitopes in patient 110 (P110) ---------------------------------------------------------------------------------- 143 Table 3-13: Mutations observed in HLA-B27+ restricted epitopes in patient 10 (P10) -- 144 Table 3-14: Mutations observed over time within HLA-B27 restricted epitopes in patient 28 (P28) ---------------------------------------------------------------------- 145 vii | Pa g e Table 3-15 Mutations observed over time within HLA-B27 restricted epitopes in patient 45 ----------------------------------------------------------------------------- 148 Table 3-16: Mutations observed over time within HLA-B27 restricted epitopes in patient 113 ---------------------------------------------------------------------------- 148 Table 3-17: Mutations detected within the HLA-B27-restricted HIV gag epitope in patient 49 ----------------------------------------------------------------------------- 149 Table 3-18: Mutations detected within HLA-B27 HIV epitope in patient 28 (P28) ------- 150 Table 3-19: Mutations detect within HLA-B27 epitope in patient 45 (P45). -------------- 151 Table 3-20 Summary of the median number of mutations within different epitopes in spontaneous clearer and progressor patients. ---------------------------------- 152 Table 3-21: Summary of the immune response in the HLA B27+ cohort ------------------ 206 viii | Pa g e List of Figures Figure 1-1: Prevalence of hepatitis C virus genotypes based on the NCBI HCV database. -------------------------------------------------------------------------------- 23 Figure 1-2: Global distribution of hepatitis C virus genotypes.------------------------------- 24 Figure 1-3: HCV Genome ---------------------------------------------------------------------------- 33 Figure 2-1: Agencourt AMPure XP beads workflow --------------------------------------------- 84 Figure 2-2: Site-direct mutagensis ---------------------------------------------------------------- 93 Figure 2-3 Subgenomic replicon (APP 238 pH/SG-Neo (L+I) ----------------------------------- 95 Figure 2-4 The ELISpot procedure --------------------------------------------------------------- 100 Figure 2-5: Nextera XT workflow ---------------------------------------------------------------- 105 Figure 2-6: Overview of Illumina sequencing -------------------------------------------------- 107 Figure 3-1: DNA fragment size assessed using a 4200 Tapestation® (Agilent) ----------- 114 Figure 3-2: Illustrative example of Phred score data obtained from fastq file ---------- 115 Figure 3-3: An example of Fastq file sequence format -------------------------------------- 116 Figure 3-4: Read coverage across the whole HCV genome using Tanoti ------------------ 118 Figure 3-5: Phylogenetic tree illustrating bias when the mapping reference sequence used comes from a different HCV subgenotype (P45).------------------------- 125 Figure 3-6: Phylogenetic analysis of samples sequenced using metagenomic (MG) or target enrichment (TE). ------------------------------------------------------------- 130 Figure 3-7: Phylogenetic tree calculated using consensus sequences from all samples sequenced. --------------------------------------------------------------- 131 Figure 3-8: Viral load dynamics and sampling from patient G5 ---------------------------- 132 Figure 3-9: Viral load dynamics and sampling from patient G17 --------------------------- 132 Figure 3-10: Viral load dynamics and sampling from patient G18 ------------------------- 133 Figure 3-11: Viral load dynamics and sampling from patient P49 -------------------------- 133 Figure 3-12: Viral load dynamics and sampling from patient P110 – episode 1 (E1) ---- 134 Figure 3-13: Viral load dynamics and sampling from patient P110 – episode 2 (E2) ---- 134 Figure 3-14: Viral load dynamics and sampling from patient P28 -------------------------- 135 Figure 3-15: Viral load dynamics and sampling from patient P45 -------------------------- 135 Figure 3-16: Viral load dynamics and sampling from patient P113 ------------------------ 136 Figure 3-17: Viral load dynamics and sampling from patient P10 -------------------------- 136 Figure 3-18: Number of mapped reads to genotype 1a and 4d referenced at different time points in patient 28 ------------------------------------------------ 146 Figure 3-19: Single mutation (VRMILMTHF) compared to wild type sequence ----------- 153 Figure 3-20: Two mutations (VRVILMTHF) compared to wild type sequence ------------- 154 Figure 3-21: Three mutations (VRVVLMTHF) compared to wild type sequence ---------- 154 ix | Pa g e