PROJECT SUMMARY Hepatitis C virus (HCV) infects more than 71 million people and causes liver scarring and cancer. An effective vaccine to prevent infection is urgently required. This should be an achievable goal, since 25% of individuals spontaneously resolve (SR) primary infection generating anti-viral immunity. An effective vaccine may need to mimic the immune responses associated with SR including the induction of anti-HCV neutralizing antibodies (NAbs) and HCV-specific T cells. One method to achieve this, is through the use of viral vectored vaccines (both chimpanzee Adenoviral [ChAd] and modified vaccinia Ankara [MVA]), which have emerged as powerful tools to generate immune responses against an encoded immunogen, including HCV. However, viral genetic diversity is a major challenge for the development of HCV vaccines, with at least eight distinct genotypes (gt) and multiple subtypes between and within HCV infected people. Recently, a viral vectored vaccine encoding a single gt-1b sequence failed to prevent chronic infection, highlighting the need to develop new approaches with novel HCV immunogens that generate NAbs and T cells against multiple gts. Project 5 tests the hypothesis that an optimized viral vectored HCV vaccine strategy can generate anti-HCV nAbs and cross-reactive T cells. To test this hypothesis, we will pursue three specific aims. Aim 1 uses bioinformatic analysis and generates novel T cell immunogens encoded in ChAd and MVA, designed to generate broad T cell coverage to all HCV gts assessed in inbred, outbred and (HLA-2) humanised mice. The most promising T cell immunogen(s) will then progress to aim 2. Aim 2 will increase viral vectored vaccine generated T cell responses using genetic adjuvants (including variants of class II invariant chain) and assess alternative adenovirus vectors to overcome potential anti-vector immunity, that may be present in humans. Aim 3 will develop new vaccine strategies to generate both anti-HCV Abs and also T cells. This will be achieved through assessing: i) Different B cell immunogens (identified in project 2 and 3) encoded in viral vectored vaccines; ii) Combining promising T cell vaccine candidates with strategies designed to generate bNAbs including Viral Like Particles (VLP) or nanoparticles that present the HCV envelope (E2) from Project 3 or with a ChAd expressing E proteins; and iii) developing a single ‘bivalent’ viral vectored vaccine that encodes the optimal B cell and T cell immunogens together. Importantly, the project will also take account of findings in partner projects should T cell profiles (project 1) or viral sequence motifs (project 4) linked to SR be identified. Together the aims will contribute to developing an optimized viral vectored HCV vaccine strategy for assessment in Rhesus monkeys with a view to future human clinical trials.