Pre-Clinical Evaluation of a Vaccination Strategy to Induce Liver Resident Memory T Cells against Hepatitis C Virus Zelalem Addis Mekonnen The Virology Laboratory The Basil Hetzel Institute of Medical Research Discipline of Surgery School of Medicine Faculty of Health and Medical Sciences The University of Adelaide January 2019 Contents Contents ------------------------------------------------------------------------------------------------------ i Abstract ----------------------------------------------------------------------------------------------------- ix Declaration -------------------------------------------------------------------------------------------------- xi Acknowledgments ---------------------------------------------------------------------------------------- xii Abbreviations -------------------------------------------------------------------------------------------- xiii 1 CHapter 1: Literature Review ---------------------------------------------------------------------- 1 1.1 Hepatitis C virus (HCV) ------------------------------------------------------------------------- 1 1.1.1 Biology and Epidemiology of HCV ------------------------------------------------------ 1 1.1.2 HCV genome organisation and replication ---------------------------------------------- 3 1.1.2.1 Genome organisation ------------------------------------------------------------------ 3 1.1.2.2 HCV attachment and entry to the cell ----------------------------------------------- 4 1.1.2.3 RNA replication and protein translation -------------------------------------------- 6 1.1.3 Pathogenesis of HCV ----------------------------------------------------------------------- 8 1.1.4 Immune response to HCV ---------------------------------------------------------------- 10 1.1.4.1 Innate immune response to HCV -------------------------------------------------- 10 1.1.4.1.1 Innate immune recognition of PAMPs by PRRs ------------------------------ 12 1.1.4.1.2 Interaction of PRRs with HCV PAMPs ---------------------------------------- 13 1.1.4.1.3 HCV and Natural killer (NK) cells ---------------------------------------------- 14 1.1.4.1.4 Innate immune evasion by HCV ------------------------------------------------ 15 1.1.4.2 The adaptive immune response ---------------------------------------------------- 16 1.1.4.2.1 Humoral immune response ------------------------------------------------------- 16 1.1.4.2.2 Cell mediated immune response (CMI) ---------------------------------------- 17 i 1.1.5 Model systems for HCV study ---------------------------------------------------------- 19 1.1.5.1 In vitro systems ---------------------------------------------------------------------- 19 1.1.5.1.1 Primary hepatocytes --------------------------------------------------------------- 19 1.1.5.1.2 HCV Replicon --------------------------------------------------------------------- 20 1.1.5.1.3 Cell culture grown HCV (HCVcc) ---------------------------------------------- 21 1.1.5.1.4 HCV Pseudoparticles (HCVpp) ------------------------------------------------- 21 1.1.5.2 Animal models to study HCV ------------------------------------------------------ 22 1.1.5.2.1 Chimpanzees ----------------------------------------------------------------------- 22 1.1.5.2.2 Tupaia ------------------------------------------------------------------------------- 22 1.1.5.2.3 Mouse models for HCV ---------------------------------------------------------- 23 1.1.6 Treatment and prevention of HCV infection ------------------------------------------ 26 1.1.6.1 Treatment ----------------------------------------------------------------------------- 26 1.1.6.2 Vaccines ------------------------------------------------------------------------------- 29 1.1.6.2.1 Live attenuated vaccines (LAV) ------------------------------------------------- 30 1.1.6.2.2 Inactivated virus vaccines -------------------------------------------------------- 30 1.1.6.2.3 Subunit vaccines ------------------------------------------------------------------- 30 1.1.6.2.4 Virus like particles (VLP) -------------------------------------------------------- 31 1.1.6.3 HCV vaccines ------------------------------------------------------------------------ 32 1.1.6.3.1 Vaccine approaches to elicit NAbs --------------------------------------------- 32 1.1.6.3.2 Vaccine approaches to elicit CMI ----------------------------------------------- 33 1.2 Adeno associated virus (AAV) ---------------------------------------------------------------- 36 1.2.1 Virus structure and genome organization ---------------------------------------------- 36 1.2.1.1 Cap gene ------------------------------------------------------------------------------ 37 1.2.1.2 Rep gene ------------------------------------------------------------------------------ 39 1.2.2 Replication of AAV ----------------------------------------------------------------------- 39 ii 1.2.2.1 Viral entry and trafficking ---------------------------------------------------------- 39 1.2.2.2 DNA replication, gene expression and virus assembly ------------------------- 43 1.2.2.3 Helper viruses and cellular proteins required for AAV replication ----------- 44 1.2.3 Site specific integration of AAV to the human genome ----------------------------- 47 1.2.4 AAV serotypes and human infection --------------------------------------------------- 48 1.2.5 Recombinant AAV (rAAV) as a viral vector: Developments and its features ---- 49 1.2.6 Production of rAAV ---------------------------------------------------------------------- 51 1.2.7 Purification of rAAV --------------------------------------------------------------------- 52 1.3 Tissue Resident Memory T (TRM) cells ------------------------------------------------------ 52 1.3.1 Introduction to memory T cells ---------------------------------------------------------- 52 1.3.2 Tissue Resident Memory T (TRM) cells ------------------------------------------------ 55 1.3.2.1 Maintenance of TRM cells ----------------------------------------------------------- 56 1.3.2.2 Role of TRM cells in bacterial and parasitic infection --------------------------- 59 1.3.2.3 Role of TRM cells in viral infections ----------------------------------------------- 60 1.3.2.4 Targeting TRM cells in vaccination ------------------------------------------------ 61 1.3.2.5 Prime/Trap vaccination strategy to induce TRM cells ---------------------------- 63 1.4 Experimental Aims ----------------------------------------------------------------------------- 65 2 CHapter 2: Materials and Methods -------------------------------------------------------------- 66 2.1 Bacterial strains, supplements and storage -------------------------------------------------- 66 2.1.1 Bacterial strains ---------------------------------------------------------------------------- 66 2.1.1.1 Escherichia coli DH5α cells -------------------------------------------------------- 66 2.1.1.2 SURE-2 super-competent cells ---------------------------------------------------- 66 2.1.2 Preparation of heat competent E. coli -------------------------------------------------- 66 2.1.3 Supplement --------------------------------------------------------------------------------- 67 2.1.4 Glycerol stocks of bacteria --------------------------------------------------------------- 67 iii 2.2 In vitro techniques ------------------------------------------------------------------------------ 67 2.2.1 pAM2AA-eGFP plasmid ----------------------------------------------------------------- 67 2.2.2 PCR primer design and gene amplification -------------------------------------------- 68 2.2.3 Gel electrophoresis and DNA purification --------------------------------------------- 69 2.2.4 Digestion and Ligation ------------------------------------------------------------------- 70 2.2.5 DNA Sequencing -------------------------------------------------------------------------- 71 2.2.6 Plasmid DNA amplification and purification ------------------------------------------ 71 2.2.7 Cell culture and transfection ------------------------------------------------------------- 72 2.2.7.1 Cell culture ---------------------------------------------------------------------------- 72 2.2.7.2 Cell storage and retrieval ----------------------------------------------------------- 72 2.2.7.3 Transient transfection --------------------------------------------------------------- 72 2.2.8 Immunofluorescence staining ------------------------------------------------------------ 73 2.2.9 rAAV-NS5B-2A-GFP production ------------------------------------------------------ 73 2.2.9.1 Preparation of HEK293 T cells ---------------------------------------------------- 73 2.2.9.2 Preparation of polyethyleneimine (PEI) ------------------------------------------ 73 2.2.9.3 Triple transfection ------------------------------------------------------------------- 74 2.2.9.4 Virus harvest and purification ------------------------------------------------------ 74 2.2.9.5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ------------------------ 75 2.2.9.6 Titration of rAAV-NS5B-2A-GFP using quantitative real time-PCR -------- 75 2.2.9.7 In vitro transduction of HT1080 Cells with rAAV-NS5B-2A-GFP ---------- 76 2.2.9.8 RT-PCR for the detection of packaged viral genome --------------------------- 76 2.2.9.8.1 RNA extraction -------------------------------------------------------------------- 76 2.2.9.8.2 cDNA synthesis -------------------------------------------------------------------- 77 2.2.9.8.3 PCR ---------------------------------------------------------------------------------