PROJECT SUMMARY/ABSTRACT: Treatment for chronic HCV infection is now highly effective. Direct Acting Antivirals (DAA) are generally safe and most persistent infections are cured with 2-3 months of therapy. However, a strategy to control chronic hepatitis C by antiviral therapy alone is complicated by two factors. First, although antiviral therapies have improved steadily over the past 25 years, the percentage of individuals cured of chronic hepatitis C is still remarkably low in the US and globally. Almost all untreated individuals (3 million in the US, 180 million globally) are unaware of their persistent infection; there are often no symptoms of HCV infection until progressive liver disease becomes apparent many years after exposure to the virus. Second, HCV transmission has accelerated in most regions of the world. In the US, infection rates increased over the past decade because of ongoing epidemic injection drug use amongst adolescents and young adults. New undiagnosed HCV infections will almost certainly develop at a rate that outpaces detection and antiviral cure. A vaccine could help stem HCV transmission, however a complete understanding of correlates of protection is lacking. While an early appearance of broadly neutralizing antibodies (bNAbs) generally correlates with a more favorable infection outcome, the inability to isolate HCV- specific B cells to dissect their phenotype, function, receptor repertoire, gene expression and neutralization breadth has hindered the understanding of the molecular mechanisms that determine infection outcome. We have developed a novel tetramer that detects HCV E2 glycoprotein-specific memory B cells in patients infected with HCV. The objective of this grant is to define the longitudinal changes in HCV-specific B cell clonal selection and the breadth of bNAbs resulting from the interplay between HCV-specific CD4+ T follicular helper cells (Tfh) and HCV-specific B cells. Our central hypothesis is that a rapid induction of the HCV-specific Tfh cell response accelerates the selection and expansion of HCV-specific B cell clones producing bNAbs that contribute to resolution of infection. Our specific aims will test whether: HCV-specific B cells isolated longitudinally at single cell level from spontaneous resolvers are more activated, clonally focused and produce antibodies with greater neutralization breadth than HCV-specific B cells from persistently-infected patients (Aim 1); an early increase in Tfh cell activity during acute infection in spontaneous resolvers directly influences the rapid, clonal expansion of HCV-specific B cells and subsequent bNAb production (Aim 2). This study makes a significant contribution towards a vaccine-based approach designed to prevent primary HCV infection and/or reinfection.