DEVELOPMENT AND ASSESSMENT OF NOVEL VACCINATION STRATEGIES FOR HEPATITIS B VIRUS INFECTION Devin TEOH (Chee Quin TEOH) B.Sc. (Biotechnology), B.Sc. (Hons) A thesis submitted to the University of Adelaide in fulfilment of the requirements for the degree of Doctor of Philosophy Discipline of Microbiology and Immunology School of Molecular and Biomedical Science The University of Adelaide South Australia Australia November 2014 To my loving family for your boundless love, care, support and always being there for me & To Jean Ang who stood by me when things looked bleak and supported me each step of the way Table of Contents Abstract-----------------------------------------------------------------------------------------------------i Declaration of originality and consent---------------------------------------------------------------v Publications and presentations resulting from this thesis---------------------------------------vi Acknowledgements--------------------------------------------------------------------------------------ix Abbreviations--------------------------------------------------------------------------------------------xi Chapter 1 Introduction 1.1 Background: Epidemiology of HBV infection--------------------------------------1 1.2 Hepadnaviruses--------------------------------------------------------------------------2 1.2.1 Hepadnavirus Family--------------------------------------------------------------------2 1.2.2 B virus structure and genome organisation-------------------------------------------2 1.2.3 Duck hepatitis B virus structure and genome organisation------------------------9 1.3 Hepadnavirus Replication-------------------------------------------------------------10 1.3.1 Viral entry, transcription and translation to generate new virus-----------------10 1.3.2 An important reservoir of hepadnavirus infection – cccDNA--------------------13 1.4 Outcomes of HBV infection----------------------------------------------------------13 1.4.1 Acute HBV infection------------------------------------------------------------------17 1.4.1.1 Resolution of acute HBV infection--------------------------------------------------17 1.4.2 Chronic HBV infection----------------------------------------------------------------20 1.5 Animal Model – Duck hepatitis B virus--------------------------------------------23 1.6 Outcomes of DHBV infection -------------------------------------------------------23 1.6.1 Acute DHBV infection ---------------------------------------------------------------25 1.6.2 Persistent DHBV infection------------------------------------------------------------25 1.6.3 CMI responses to DHBV infection -------------------------------------------------25 1.7 Treatment of HBV infection ---------------------------------------------------------27 1.7.1 Immunomodulators--------------------------------------------------------------------27 1.7.2 Antiviral agents-------------------------------------------------------------------------28 1.7.2.1 Entecavir--------------------------------------------------------------------------------29 1.7.2.2 Antiviral resistance to Nucleotide/nucleoside analogues-------------------------30 1.8 Hepadnavirus vaccines----------------------------------------------------------------31 1.8.1 The prophylactic HBV vaccines-----------------------------------------------------31 1.8.2 DNA vaccines--------------------------------------------------------------------------32 1.8.2.1 Mechanism of antigen presentation following DNA vaccination---------------34 1.8.2.2 Enhancement of immunogenicity of DNA vaccines------------------------------35 1.8.2.3 Costimulatory molecules as genetic adjuvants ------------------------------------36 1.8.2.4 The co-stimulatory molecule CD40L------------------------------------------------38 1.8.2.5 Current hepadnaviral vaccine studies------------------------------------------------42 1.9 Poxviruses ------------------------------------------------------------------------------43 1.9.1 Family Poxviridae----------------------------------------------------------------------43 1.9.2 Structure and genome of Avipoxviruses--------------------------------------------43 1.9.3 Replication of Avipoxviruses---------------------------------------------------------44 1.9.4 FPV as vaccine vectors----------------------------------------------------------------45 1.9.5 Poxvirus vaccine developed to treat hepadnavirus infection---------------------46 1.10 Research aims and study outline-----------------------------------------------------47 Chapter 2 Materials and Methods 2.1 Experimental animals------------------------------------------------------------------50 2.2 Cell culture------------------------------------------------------------------------------50 2.2.1 Preparation of primary duck embryonic fibroblasts (PDEF)---------------------50 2.2.2 Collection and purification of duck peripheral blood mononuclear cells (PBMC)----------------------------------------------------------------------------------51 2.2.3 Cell culture and PHA and ConA stimulation of spleen cells---------------------51 2.2.4 Culture of 293T, LMH and PDEF cells---------------------------------------------53 2.2.5 Cell transfection------------------------------------------------------------------------53 2.2.6 Detection of DHBV antigens using Immuno-fluorescence (IMF) assay-------54 2.2.7 Detection of DHBV antigens using western blot----------------------------------55 2.3 Production of polyclonal and mAB to recombinant DHBV core antigen (rDHBcAg)------------------------------------------------------------------------------60 2.3.1 Preparation of rDHBcAg particles---------------------------------------------------60 2.3.1.1 Growth and transformed E.coli strains and partial purification of rDHBcAg particles----------------------------------------------------------------------------------60 2.3.1.2 Separation and isolation of rDHBcAg particles by caesium chloride isopycnic density gradient centrifugation-------------------------------------------------------61 2.3.1.3 Detection of rDHBcAg particles by ELISA----------------------------------------62 2.3.1.4 Precipitation of rDHBcAg using trichloroacetic acid (TCA)--------------------63 2.3.1.5 To concentrate rDHBcAg particles and to determine the concentration of rDHBcAg particles --------------------------------------------------------------------63 2.3.2 Production of polyclonal rabbit antibodies to rDHBcAg-------------------------64 2.3.2.1 Testing antibody responses by ELISA following immunisation with rDHBcAg--------------------------------------------------------------------------------64 2.3.2.2 Absorption of polyclonal antisera with normal duck liver (NDL) and NDS---65 2.3.3 Production of mABs to rDHBcAg---------------------------------------------------65 2.3.3.1 Hybridoma production ----------------------------------------------------------------66 2.3.3.2 Hybridoma screening for anti-DHBc antibodies ----------------------------------69 2.4 Procedure involving Duck CD40L (DuCD40L) ----------------------------------69 2.4.1 Sub cloning of DuCD40L gene into pcSNA3 expression vector----------------69 2.4.1.1 Plasmid vector and primer design----------------------------------------------------69 2.4.1.2 RNA extraction-------------------------------------------------------------------------70 2.4.1.3 The cDNA synthesis and cloning by PCR------------------------------------------70 2.4.1.4 Restriction Endonuclease (RE) digestion-------------------------------------------72 2.4.1.5 Ligation----------------------------------------------------------------------------------72 2.4.1.6 Transformation-------------------------------------------------------------------------73 2.4.1.7 Preparation of plasmid DNA---------------------------------------------------------73 2.4.2 Nitrate assay----------------------------------------------------------------------------74 2.4.3 Statistical analysis----------------------------------------------------------------------75 2.4.4 Quantitative reverse transcription PCR (qRT-PCR) to detect iNOS mRNA---75 2.5 Procedures involving ducks-----------------------------------------------------------75 2.5.1 DHBV stock and intravenous inoculation of ducks-------------------------------75 2.5.2 DHBV vaccine and vaccination protocol-------------------------------------------76 2.5.2.1 DNA vaccine----------------------------------------------------------------------------76 2.5.2.2 Vaccination protocols------------------------------------------------------------------77 2.5.3 Administration of antiviral drugs----------------------------------------------------77 2.5.4 Venipuncture----------------------------------------------------------------------------78 2.5.5 Liver Biopsy----------------------------------------------------------------------------78 2.5.6 Autopsy----------------------------------------------------------------------------------79 2.5.7 Tissue fixation, embedding and sectioning-----------------------------------------79 2.6 Serological Assays --------------------------------------------------------------------79 2.6.1 Quantitative detection of DHBsAg by ELISA-------------------------------------80 2.6.2 Quantitative detection of anti-DHBs antibodies by ELISA --------------------- 80 2.6.3 Detection of anti-DHBc antibodies by ELISA-------------------------------------81 2.7 Histology and immuno-staining of tissue sections--------------------------------82 2.7.1 Immuno-staining to detect DHBsAg------------------------------------------------82