Microbiology & Immunology - Theses
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ItemThe viral glycoproteins of the Hepaciviruses. Structural and functional studies to inform vaccine design( 2022)Hepatitis C virus (HCV) infection is a major global health burden, with an estimated 71 million people infected worldwide. Vaccine design for HCV is challenging for multiple reasons, including high sequence variability of the glycoprotein E2, as well as the lack of a small animal challenge model in which to test vaccine candidates. This thesis addresses aspects of both challenges. Glycoprotein E2 is present on the virion surface and is a major target of neutralizing antibodies which can prevent infection. The N-terminal hypervariable region 1, HVR1 (residues 384-411), of E2 is an immunodominant region within E2 and elicits neutralizing antibodies that are usually isolate specific. We previously identified a novel murine monoclonal antibody, MAb33, which binds to an unusual epitope bridging HVR1 and the adjacent target of broadly neutralizing antibodies referred to as epitope I (residues 412-423). MAb33 potently neutralizes genotype 1a viruses and can cross-neutralize 3 different HCV genotypes. This study defined the epitope of MAb33 to include residues within the E2 region 401-415 and resolved its structure in complex with its epitope. The epitope adopts an alpha-helical conformation with residues G406, A407 and N410 involved in direct polar interaction with the antibody. The helical structure of the epitope differs from the extended conformation of other E2 crystal structures that include this region, suggesting that it could be conformationally flexible. Sero-surveys of HCV positive individuals have identified significant reactivity to a peptide encompassing the MAb33 epitope, indicating a role for MAb33-like antibodies in natural infection. To address the need for an immune competent model for HCV vaccine development, a Rodent hepacivirus (RHV) was investigated as it shows close evolutionary relatedness and virological similarities with HCV and represents an important potential model of HCV pathogenesis and host responses. While the T cell response to RHV has been characterized, detailed studies characterizing the RHV E2 glycoprotein, the development of the humoral immune response after infection and in vaccination studies are lacking. This thesis characterized the antigenic and immunogenic properties of RHV E2. A minimal ectodomain expressed as a soluble protein was defined (residues 418-603), which has 4 sites for N-linked glycans and 12 cysteine residues. The development of the anti-E2 antibody response in outbred rats infected with RHV was analysed and the appearance of anti-E2 antibodies observed at 28 days post-infection. RHV E2 was assessed as a potential vaccine antigen in immunisation/challenge studies in rats. Rats received a E2 protein prime followed by a protein boost, or a combined vaccination of E2 protein and a simian adenovirus (ChAdOx1) encoding the non-structural proteins NS3-NS5B from RHV. Both groups failed to generate anti-E2 antibody prior to challenge at 6 weeks. Following challenge with RHV, anti-E2 antibodies appeared 14 days later, with most animals seroconverting by 28 days post challenge. These studies are the first of their kind to define a soluble ectodomain of the RHV E2 protein and explore the development of anti-E2 antibodies in infection.