Project 2. B Cell-Mediated Protection Against HCV Reinfection Prevention of HCV infection remains an important public health objective even with the recent adoption of highly effective antiviral therapies. A vaccine to prevent HCV persistence is needed to stem an emerging epidemic in susceptible populations. Recall responses to secondary viral infections naturally emulate the protective immune mechanisms associated with vaccination against viral antigens. Our findings suggest that spontaneous resolvers may possess antigen-specific B cells that become readily activated by T follicular helper (Tfh) cells and expand rapidly following HCV antigenic exposure. However, resolution of secondary HCV infection is low in individuals cured of persistent HCV infection by DAAs, despite an initial burst of HCV-specific CD4+ T cells at the start of DAA treatment. Such contrasting recall responses between these two scenarios implicate profound phenotypic and functional differences in antigen-specific memory T and B cell responses between DAA-treated versus untreated, resolving individuals. We propose to elucidate the differences in antibody recall responses between individuals who spontaneously resolve primary infection and subsequently either clear their secondary HCV reinfection (SR/SR) or develop persistent infection (SR/CI). We will also compare these responses to those of DAA-treated individuals who are cured of persistent HCV infection. We will analyze longitudinal samples from two separate cohorts of HCV infected individuals for these studies. One cohort consists of people who inject drugs (PWIDs) recruited from Montreal, Canada, who spontaneously resolve primary HCV infection but have different secondary reinfection outcomes. The other cohort consists of individuals from Egypt who cleared persistent HCV infection following DAA treatment (HCV-cured). We hypothesize that SR/SR PWID mount a more accelerated and sustained memory B cell response that produces early, broadly neutralizing antibodies (bNAbs) that contribute to faster clearance, while delayed antibody responses from SR/CI PWID or HCV-cured individuals fail to suppress viremia early, facilitating viral escape from neutralization. We propose the following aims: Aim 1. Determine the phenotypic and transcriptional profiles of bulk and antigen-specific memory B cells from primary resolvers who were reinfected but experienced divergent infection sequelae. Aim 2. Evaluate neutralizing efficacy and breadth of HCV-specific antibodies from resolvers with contrasting HCV reinfection outcomes. Aim 3. Determine phenotypic and functional changes in B cells of DAA-treated individuals before and after viral clearance.