ABSTRACT - PROJECT 1 An effective HCV vaccine to prevent chronic infection will need to induce robust T and B cell responses shortly after exposure in the susceptible host, the overall goal of this U19. Project 1 focuses on a structure-guided approach to develop an immunogen that increases the neutralization potency/affinity of elicited broadly neutralizing antibodies (bNAbs) to conserved epitopes and particularly to those epitopes associated with neutralization synergy. It will be an optimized mutant (mt) E1E2 heterodimer that maintains native conformational structures for multiple bNAb epitopes to minimize viral escape. The increasing information on E2 crystal structures made possible by human monoclonal antibodies (HMAbs) that we and others have isolated and characterized as bNAbs, provides a strong foundation for the molecular approaches in rational design. A critical and complementary element is the availability of a high-resolution, functional map of both bNAb and non-neutralizing epitopes in E2 and E1E2. We have developed a large database on immunogenic domains on E2 that is derived from epitope mapping studies with a large panel of E2 bNAbs. We have also developed a large panel of bNAbs to E1E2 that will contribute to structural studies on E1E2 heterodimer in Project 4. In addition, Project 1 is built on substantial achievements with native recombinant wild-type (wt) E1E2 heterodimer as a vaccine against HCV. We have developed the necessary production and purification processes and tested to show that our wt E1E2 expresses many bNAbs epitopes and that antisera generated from vaccinated mice, guinea pigs, chimpanzees and humans contains bNAbs. However, there are gaps in the breadth of protection against some genotypes 2, 3 and 7 HCV isolates. Thus, a benchmark is to design a mt E1E2 that elicits antisera more able to neutralize these genotype isolates while maintaining a high level of neutralization towards the other genotype isolates achieved with wt E1E2. We propose to accomplish this goal by the following Specific Aims: Aim 1. Structure-guided modeling to redesign E1E2 structures to optimize presentation of E1E2 bNAb epitopes; Aim 2. Expression, purification, and characterization of modified/mutant E1E2 heterodimers; and Aim 3. Immunological characterization of candidate E1E2 vaccines to select optimized E1E2. Finally, we aim to combine optimized E1E2 with powerful adjuvants to enhance neutralizing antibody titers (Project 3). Aim 4 is then the immunological characterization of NHP receiving an HCV vaccine optimized for bNAbs and T cell responses