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dc.contributor.advisorJilbert, Allison Raeen
dc.contributor.advisorFeng, Fengen
dc.contributor.authorTeoh, Chee Quin (Devin)en
dc.description.abstractThe current hepatitis B virus (HBV) vaccine is of no benefit in the treatment of patients with chronic HBV infection, and current antiviral therapies which inhibit the virus polymerase are not highly effective. The ultimate aim of this Ph.D. project was to develop and assess therapeutic vaccination strategies that induce immune responses that target virus infected hepatocytes and allow successful control of chronic HBV infection. To this end, a number of vaccination strategies were tested using the duck hepatitis B virus (DHBV) model. The DHBV model provides a versatile and reproducible experimental system for testing vaccination strategies as the outcomes of DHBV infection in ducks of different ages infected with different doses of virus have been well characterised. In initial studies described in Chapter 3, recombinant DHBV core antigen (rDHBcAg) expressed in E. coli, was purified and used to immunise rabbits and mice to produce specific polyclonal and monoclonal antibodies for the detection of DHBV core antigen (DHBcAg). Immuno-staining techniques using these anti-DHBcAg-specific antibodies were then optimised. Immunoperoxidase detection of DHBcAg in duck liver sections was an essential part of this analysis as it allowed comparison with detection of DHBV surface antigen (DHBsAg) and confirmation of the percentage of DHBV-infected hepatocytes. Western Blot and immunofluorescent detection of DHBcAg were also developed and optimised and then all 3 immuno-staining techniques were used in subsequent Chapters to assess the efficacy of the different vaccination strategies. As described in Chapter 4, duck CD40L (DuCD40L), was assessed as an immunological adjuvant in a protective DNA vaccine study. In humans, CD40L acts as a co-stimulatory molecule in the CD40-signalling pathway that is involved in the activation of antigen presenting cells (APC) and the generation of humoral and cell mediated immune (CMI) responses. In the current studies the DuCD40L cDNA sequence was cloned using mRNA from duck peripheral blood mononuclear cells (PBMC) into the expression construct, pcDNA3, to yield pcDNA3-DuCD40L. Cells and supernatants of cells transfected with pcDNA3-DuCD40L were subsequently tested for bioactivity using in vitro assays. The cloning and analysis of expression of the DuCD40L was performed in our laboratory by Dr Feng Feng. The DuCD40L expression construct was then co-administered intramuscularly with DNA vaccines expressing the DHBV surface and core antigens to ducks at 14 and 28 days of age. Two weeks after the second vaccination, ducks were challenged intravenously (i.v.) with 4.5x10¹⁰ DHBV genomes. Administration of the DuCD40L expression construct and the DHBV DNA vaccines in combination resulted in a 10-fold greater anti-DHBs antibody response and a significant decrease in the number of DHBV-infected hepatocytes at day 4 post-challenge (p.c.) compared to ducks that received DHBV DNA vaccines alone. Ultimately, as expected, all of the ducks successfully cleared their DHBV infection. Nevertheless, we determined that DuCD40L enhanced humoral immune responses and lead to reductions in the percentage of DHBV-infected hepatocytes following challenge. As described in Chapter 5, the expression of DuCD40L was further assessed in a 2nd protective DNA vaccine study in 14-day-old ducks which are more susceptible to the development of persistent DHBV infection. The DuCD40L expression construct and DNA vaccines expressing the DHBV surface proteins were administered to ducks at 4 and 14 days of age. On the same day as the second vaccination, ducks were challenged i.v. with 5x10⁸ DHBV genomes, a dose of DHBV that is 500-times higher than the dose known to result in persistent DHBV infection. Unexpectedly, following DHBV challenge, no significant differences in the percentage of DHBV-infected hepatocytes or anti-DHBs antibody titres were observed between ducks receiving DHBV DNA vaccines with DuCD40L expression construct and ducks receiving DHBV DNA vaccines alone. At day 21 p.c., all ducks vaccinated with DHBV DNA vaccines with or without the DuCD40L expression construct had successfully cleared their DHBV infection while five out of five vector control ducks developed persistent DHBV infection. Interestingly, two out of five ducks that received the DuCD40L expression construct alone also cleared their DHBV infection. The studies described in Chapters 4 and 5 suggest DuCD40L may enhance immune responses in ducks and that DuCD40L should be further investigated as an immunological adjuvant in future vaccine studies. Finally as described in Chapter 6, a post-exposure vaccination study was performed that combined treatment with the Bristol-Myers Squibb nucleoside analogue, Entecavir (ETV), and “prime-boost” vaccination using DNA vaccines and recombinant fowl-pox virus (rFPV) strains that express DHBV surface or core alone. Previous “prime-boost” vaccination studies in the laboratory had used DNA vaccines and rFPV strains that expressed both DHBV surface and DHBV core in combination. We aimed to determine if DHBV surface antigen which generates neutralising anti-DHBs antibodies, or DHBV core antigen which generates non-neutralising anti-DHBc antibodies, could provide the essential epitopes in a DNA vaccine and “prime-boost” protocol to enable the resolution of DHBV infection. 14 day-old ducks were inoculated i.v with 5x10⁸ DHBV genomes and immediately treated with ETV (1.0mg/kg/day) for 14 days. At the same time, ducks received the “priming” DHBV DNA vaccines and 7 days later received the “boosting” vaccination with the rFPV- DHBV vaccines. The findings showed that protective humoral and CMI responses generated by “prime - boost” vaccination strategies with either DHBV surface or DHBV core alone blocked virus spread and replication and resulted in the targeting the destruction of infected hepatocytes and the resolution of DHBV infection. In contrast, ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection in the liver during ETV treatment, but DHBV infection become widespread in four out of five ducks after ETV treatment was withdrawn. These findings indicate that DHBV surface and core antigen as measured in our studies are equally effective as components of our post - exposure “prime - boost” protocol. Since anti DHBc antibodies are non-neutralising this suggests that our “prime-boost” protocol may be generating effective CMI that were able to control DHBV infection. The studies described in this thesis show that increasing levels of expression of DuCD40L may increase the efficacy of DNA vaccination protocols. Our novel vaccination strategy using ETV treatment in combination with “prime-boost” vaccination also suggests that DHBV surface and core antigen are both able to provide sufficient immunity to allow clearance of DHBV infection. Future studies are warranted to test other types of immunotherapy and antiviral agents to provide new directions for future therapeutic vaccination strategies for chronic HBV infection.en
dc.subjectHepatitis B; Vaccinationen
dc.titleDevelopment and assessment of novel vaccination strategies for hepatitis B virus infection.en
dc.contributor.schoolSchool of Molecular and Biomedical Scienceen
dc.provenanceThis electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
dc.description.dissertationThesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014en
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