Project Summary Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV strains in vitro, and infusion of bNAbs is protective against HCV infection in animal models. In contrast to some other chronic viral infections like HIV- 1 where bNAbs do not appear to influence disease outcome, early development of high plasma bNAb titers is associated with spontaneous clearance of primary HCV infection in humans. Although it is clear that bNAbs can play a critical role in clearance of primary HCV infection, detailed analysis of antibody titers, epitopes targeted, and B cell phenotypes associated with clearance of infection are still lacking. Individuals who clear multiple reinfections may be the ideal study subjects to further define protective antibody responses. Of those who clear their first infection, 80% clear subsequent reinfections with a rapid rise in neutralizing antibody (NAb) titers, shorter duration of infection, and lower peak viremia, demonstrating protective adaptive immunity that can serve as a model for a desired vaccine response. It is not known which parameters of the B cell response are most critical for repeated clearance of infection, or what antigenic stimuli are necessary for induction of these responses. In Aim 1 of this proposal, we will define plasma anti-HCV antibody binding and neutralizing activity associated with repeated clearance of reinfection. In Aim 2, we will determine the mechanistic basis for changes in neutralizing activity by characterizing the dynamic interplay between the circulating B cell repertoire and HCV sequence changes during reinfection. In Aim 3, we will define phenotypes of HCV-specific B cells associated with repeated clearance of reinfection. Because reinfections are generally cleared very efficiently, these immune responses can serve as a model for responses that should be induced by a vaccine. By characterizing plasma antibody responses, B cell repertoires, viral antigenic variation, and B cell phenotypes in human subjects with repeated spontaneous clearance of infection, we will inform HCV vaccine development.