Overall Project Summary Hepatitis C virus (HCV) infects ~70 million people worldwide and is a major cause of hepatocellular carcinoma and liver failure. Even with highly effective HCV treatment, recent data show that 80% of high-income countries are not on target to meet the WHO goals of elimination of HCV. In most countries, the annual number of new infections remains higher than the number cured by treatment. A vaccine for HCV should be possible because 25% of people resolve primary infection with effective anti-viral T cells and the generation of broadly neutralizing antibodies (bNAbs). Recently, we have identified people who are repeatedly exposed to HCV with reinfection and control of up to six distinct HCV infections, often of more than one HCV genotype. To date, one candidate vaccine has been tested in an at-risk human population. This vaccine was designed to induce robust T cell responses and was evaluated by proposal investigators for immunogenicity in healthy volunteers and for efficacy in the prevention of HCV persistence in people at high risk of HCV infection. Although not protective against chronic infection, vaccinated participants had significantly reduced mean peak HCV RNA compared to placebo recipients. Our overarching hypothesis is that defining HCV-specific T cell and antibody mediated immunity in effective control of HCV infection can be directly translated into more effective vaccine strategies. Therefore, we plan an integrated analysis of T cell and B-cell/Ab mediated immunity alongside an assessment of viral antigen sequences in resolved infection and vaccinees. This will inform the design and pre-clinical assessment of novel vaccine strategies. In Project 1, CD4 and CD8 T cell responses will be compared between people who are repeatedly exposed to and spontaneously control HCV and HCV clinical trial participants, both to define T cell properties associated with HCV control, but also to identify the reasons for vaccine failure. NAbs also contribute to successful control of repeated HCV exposure and work across several projects will identify and test novel vaccine antigens and platforms with potential to induce anti-HCV bNAbs. Binding (Project 2) and structural (Project 3) studies of bNAbs in complex with envelope proteins (E2 or E1E2) selected through a collaboration between projects 2 and 4 will identify a panel of potential vaccine antigens with unique structural characteristics that favor bNAb induction and maturation. Project 3 will develop nanoparticle (NP) and virus-like particle (VLP)- based vaccines to present these E2 or E1E2 antigens and test them in mice. Project 5 will assess new T cell immunogens in viral vectored vaccines, with viral vectored E2 or E1E2, NPs, or VLPs in mice, aiming to generate both anti-E1E2 antibodies and the effective, genotype cross-reactive T cell responses defined in Project 1. We will then test the two most successful vaccine candidates in non-human primates. The proposed integrative r...