Characterization of HCV vaccine induced-neutralizing antibody response in non-human primates

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PROJECT SUMMARY Chronic HCV infection is a leading cause of liver disease and hepatocellular carcinoma, yet no vaccine has been developed against HCV due to its extremely high genetic diversity. Despite the high variability of the virus, 30% of HCV-infected individuals clear the viral infection by developing broadly neutralizing antibodies (bNAbs) that bind to conserved epitopes in the receptor binding site of HCV glycoprotein E2 and conformational epitopes spanning the E1E2 heterodimer. If we could stimulate the production of such HCV bNAbs along with protective T cell responses by vaccination, a rising epidemic of HCV might be stopped. We recently identified several E2 proteins from different HCV isolates that bind to germline precursors of human bNAbs, suggesting vaccine development using these variants. However, it is not yet clear which would make a more effective vaccine candidate: E2-based or E1E2-based immunogens. In this proposal, we aim to evaluate both sets of vaccine candidates along with different methods of immunogen delivery in non-human primates that include germline gene segments capable of targeting E1E2 neutralizing epitopes. The work is organized into three major Specific Aims. Aim #1: Develop immunization strategies that elicit bNAbs to conserved HCV E1E2 epitopes. Working together with Project 5, we will test different immunization platforms in mice, evaluating both, the development of neutralizing antibodies to E1E2 conformational epitopes (this Project), and multi-specific T cell responses (Project 1). Next, the best vaccine candidates with respect to both T cell and antibody induction will each be tested in non-human primates, which utilize an ortholog of an antibody gene segment frequently used by the most potent human HCV-specific bNAbs. Aim #2: Generate high-resolution structures of E2-specific bNAbs elicited after vaccination in non-human primates and after clearance of natural HCV infection in humans. In addition to analyzing serum responses from immunized animals in Aim 1, we will generate high-resolution crystal structures of vaccine-induced bNAbs or human antibodies isolated from individuals who cleared their infection (Project 2). We will sort and sequence antibody genes in E2-specific macaque B cells after immunization and express antibodies from these sequences. We will then determine the structures of vaccine-induced bNAbs in complex with immunogens and compare them to structures of human bNAbs bound to E2 proteins (Project 4) to elucidate the structural determinants of broad neutralization of HCV. Aim #3: Determine a structure of the full- length E1E2 heterodimer in a membrane environment using cryo-electron tomography (cryo-ET). To expand upon bNAb-E2 structures from Aim 2, we will use cryo-ET with sub-tomogram averaging to examine bNAb recognition of the E1E2 heterodimer in its intact membrane form. Together, the results of these aims will lead to the comprehensive characterization of novel vaccine candidates that elicit HCV-specific bNAbs and strong T- cell responses, facilitating the development of an effective HCV vaccine.