Pre-Clinical Evaluation of a Vaccination Strategy to Induce Liver Resident Memory T Cells against Hepatitis C Virus

Hepatitis C virus (HCV) is a significant contributor to the global burden of disease with at least 71 million individuals persistently infected with the virus. Although effective anti-viral therapies are available, they are prohibitive in cost and at least 80% of infected individuals are undiagnosed for HCV making the development of an effective vaccine a crucial requirement to eliminate HCV infections. There is much conjecture about how to design an effective HCV vaccine, nonetheless, there is evidence to suggest that a vaccine which can elicit T cell immunity in the liver will be protective. Firstly, tissue-resident memory CD8⁺ T (TRM) cells have been reported to patrol the liver sinusoids for the lifespan of the individual and play vital roles in the elimination of malaria parasites (Plasmodium berghei) following invasion of hepatocytes. Secondly, CD4⁺ and CD8⁺ T cell-mediated immunity against non-structural (NS) proteins of HCV correlate with recovery from acute infections in patients. DNA and adeno-associated virus (AAV) have an excellent safety profile and have been effective in gene therapy and vaccinations in humans. Our laboratory has also shown that a DNA vaccine encoding HCV NS proteins and a cytolytic protein, perforin (PRF), is more effective than a canonical DNA vaccine in eliciting HCV-specific T cell responses against NS proteins of HCV. Thus, using a DNA vaccine encoding NS5B from HCV genotype 3a and PRF (pVAX-NS5B-PRF) and recombinant AAV (rAAV) encoding the vaccine antigen (rAAV-NS5B-2A-eGFP) vaccination regimens designed to elicit HCV-specific T cell mediated immunity in the liver were developed and evaluated in this thesis. The vaccination strategy involved intradermal (ID) vaccination of mice with pVAX-NS5B-PRF to prime T cells followed by intravenous (IV) vaccination with rAAV-NS5B-2A-eGFP to trap the primed cells. To achieve the aims of this thesis, an established fluorescent target array (FTA) technology was modified to identify immunodominant CD4⁺ T cell and CD8⁺ T cell epitopes of NS5B in vivo to thoroughly analyse NS5B-specific T cell responses in vaccinated mice in vivo. Based on the in vivo epitope mapping analysis, a NS5B451-459 tetramer was developed that was crucial for isolating NS5B-specific CD8⁺ T cells in vaccinated BALB/c mice and determining whether cells had differentiated into TRM (i.e. CD69⁺ CD62L⁻) cells in the liver. rAAV-NS5B-2A-eGFP capable of transducing hepatocytes in vivo was also developed and used in vaccination studies. The vaccination regimen described in this thesis elicited robust NS5B-specific killing responses by CD8⁺ T cells and helper CD4⁺ T cell responses. Importantly, the heightened immunogenicity of the vaccination regimen also correlated with the formation of NS5B-specific CD8⁺ TRM cells in the liver. Although, the protective efficacy of this regimen still needs to be evaluated following an authentic HCV challenge, this study highlights that the prime/trap regimen is a promising strategy that can be used to develop a protective vaccination regimen against HCV in the future. Moreover, the results bridge an important gap in the HCV vaccine development especially for vaccines that aim to elicit protective T cell responses in the liver to eliminate the virus shortly after infection and before a divergent quasi-species emerges.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201