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Downloaded on 2017-09-05T00:12:48Z Title In vitro infectivity of the Hepatitis C Virus in the context of humoral immunity and immune escape Author(s) Naik, Amruta S. Publication date 2017 Original citation Naik, A. S. 2017. In vitro infectivity of the Hepatitis C Virus in the context of humoral immunity and immune escape. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2017, Amruta S. Naik. http://creativecommons.org/licenses/by-nc-nd/3.0/ Embargo information No embargo required Item downloaded http://hdl.handle.net/10468/4076 from Downloaded on 2017-09-05T00:12:48Z In vitro infectivity of the Hepatitis C Virus in the context of humoral immunity and immune escape Amruta S. Naik Department of Medicine National University of Ireland, University College Cork, Cork, Ireland. 2017 Thesis submitted for the Degree of Doctor of Philosophy Supervisors Dr. Liam J. Fanning (Department of Medicine, UCC), Dr. Orla Crosbie and Dr. Elizabeth Kenny-Walsh (Department of Hepatology, Cork University Hospital) Contents Acknowledgments .............................................................................................. vi Declaration ........................................................................................................ vii Statement of Contribution ................................................................................ viii List of Publications ............................................................................................ ix List of Abbreviations.......................................................................................... xi One letter amino acid code ............................................................................... xvi Abstract ................................................................................................ xvii 1. Introduction 1.1 Hepatitis C Virus Classification ....................................................... 2 1.2 Genome Organisation of HCV ......................................................... 3 1.2.1 E1E2 envelope glycoproteins……………………………… . 7 1.2.2 Quasispecies and Hypervariable regions .............................. 12 1.3 Cellular receptors involved in HCV entry and HCV life cycle ... 15 1.3.1 HCV as lipoviro particle ...................................................... .17 1.4 Immune response to HCV infection ............................................... 19 1.4.1 Innate immune response ....................................................... 19 1.4.2 Adaptive immune response ................................................... 21 1.4.2.1 Cellular immune response ........................................... 22 1.4.2.2 Humoral immune response ...................................... 24 1.5 Epidemiology and geographical distribution…………………….28 1.6 Diagnosis ........................................................................................... 31 1.7 Signs and symptom ......................................................................... .31 1.8 HCV treatment ............................................................................... 32 1.8.1 HCV immunoglobulin therapy .............................................. 36 1.9 Laboratory models to study HCV 1.9.1 In-Vivo models ...................................................................... 42 1.9.2 In-vitro models ...................................................................... 43 1.9.2.1 Serum derived HCV ................................................... .43 1.9.2.2 HCV Replicon System ................................................ 43 1.9.2.3 Retroviral pseudoparticles ........................................... 44 1.9.2.4 Cell cultured HCV ....................................................... 46 1.10 Thesis research outline .................................................................. 48 i 2. Materials and Methods 2.1 Materials 2.1.1 Reagents ..................................................................................... 52 2.1.2 Plasmids ..................................................................................... 55 2.1.3 Antibodies .................................................................................. 56 2.1.4 HCV serum samples ..................................................................... 56 2.2 Methods 2.2.1 Fractionation of viraemic sera 2.2.1.1 Validation of Ab Spin Trap Column .............................. 58 2.2.1.2 Separation of viraemic sera into antibody ..................... 59 associated virus (AAV) and antibody free virus (AFV) fractions 2.2.1.3 Dissociation of antibody-virion complexes and ............ 59 collection of VF-Fab λ-VF-Fab and κ-VF-Fab 2.2.1.4 Antibody- Sera (non-detectable AAV) pull down assay.60 2.2.2 Molecular Cloning 2.2.2.1 Nucleic acid isolation and cDNA synthesis ................... 61 2.2.2.1a RNA isolation from serum .......................... 61 2.2.2.1b cDNA synthesis ........................................... 62 2.2.2.2 Amplification of the E1E2 region encompassing ......... 62 HVR1 and full length E1/E2 gene 2.2.2.3 Site directed mutagenesis ............................................. .66 2.2.2.4 Agarose gel electrophoresis .......................................... 67 2.2.2.5 Purification of PCR products ........................................ 67 2.2.2.6 Cloning of PCR purified products and transformation 2.2.2.6a Cloning of 318 base pair product in .............. 68 Clone JET PCR cloning Kit 2.2.2.6bTransformation of pJET1.2 in One Shot........... 69 Top 10 Chemically Competent E.Coli 2.2.2.6c Cloning of full length E1E2 glycoprotein ...... 69 in pcDNA3.1D/V5-His-TOPO vector 2.2.2.6d Transformation of pcDNA3.1D/V5-His-....... 70 TOPO vector in SURE2 SuperComp Cells 2.2.2.7 Restriction Digestion ..................................................... 71 ii 2.2.2.8 Miniprep ......................................................................... 72 2.2.2.9 Maxiprep ........................................................................ 73 2.2.2.10 Sequencing .................................................................... .74 2.2.2 HCVpp Based Work 2.2.3.1 Cell lines ........................................................................ 74 2.2.3.2 Expression of E1E2 glycoprotein in HEK-293T cells ... 75 2.2.3.3 Analysis of expressed E1E2 glycoproteins .................... 75 2.2.3.4 Sodium Dodecyl Sulphate-Polyacrylamide gel ............. 75 electrophoresis (SDS-PAGE) and Western Blotting 2.2.3.5 Generation of HCV pseudotyped particles .................... 76 2.2.3.6 Infectivity assay ............................................................ .81 2.2.3.7 VF-Fab mediated neutralisation of HCVpp ................... 81 2.2.3.8 GNA capture ELISA ...................................................... 82 2.2.4 Colorimetric ELISA assay ......................................................... 83 2.2.5 Epitope Mapping 2.2.5.1 Epitope mapping of amino acid region 364-430 ........... 83 2.2.5.2 Conformational epitope mapping of ............................. 85 E2 glycoprotein (residues 384-619) 3. Humoral immune system targets clonotypic antibody associated Hepatitis C Virus 3.1 Introduction ...................................................................................... 90 3.2 Methods ........................................................................................... 92 3.3 Results 3.3.1 Validation of Ab Spin TrapTM column ................................. .93 3.3.2 Separation of viraemic sera into antibody associated ........... 95 virus and antibody free virus fraction 3.3.3 Antibodies from AAV positive sera capture viral .............. 100 variants from unrelated patients 3.3.4 Dissociation of antibody-virion complex ........................... 105 3.3.5 Patient derived VF-Fab selectively targets homologous .... 108 genotype 3.3.6 Source of VF-Fab does not affect the ................................. 112 selective binding to viral variants iii 3.4 Discussion ...................................................................................... .115 4. Amplification, cloning and expression of full length E1E2 glycoprotein from antibody associated HCV 4.1 Introduction .................................................................................... 122 4.2 Methods .......................................................................................... 124 4.3 Results 4.3.1 Cloning and expression of E1E2 glycoprotein ................... 126 sequence derived from AAV 4.3.2 Difference in the infectivity of HCVpp .............................. 129 4.3.3 Analysis of E1E2 glycoprotein from the cell extracts ........ 131 4.3.4 Mutations at 292 in the E1 and 388 in the HVR1 of .......... 135 glycoprotein related to infectivity of HCVpp1b-1-3 4.3.4 HCVpp entry is CD81 dependant ....................................... 139 4.4 Discussion ................................................................................... 141 5. Neutralisation and epitope mapping using patient derived VF-Fab 5.1 Introduction .................................................................................... 147 5.2 Method ............................................................................................ 150 5.3 Results 5.3.1 Neutralisation of antibody associated E1E2 HCVpp .......... 151 5.3.2 Total IgG derived from sera without detectable ................. 157 AAV shows neutralisation activity 5.3.3 Potential epitopes targeted by VF-Fab1b-5 ........................ 160 and VF-Fab1b-10 5.3.4 Conformational epitope mapping of E2 ............................. 164 5.3.5 Combination
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