Hepatitis C virus (HCV) is a major etiological agent of liver disease in most regions of the world, with approximately 170 million individuals infected. Cellular and humoral immune responses are mounted but fail to clear HCV in most individuals. Although more difficult to study, the -30% of patients who do resolve infection are a valuable resource for identifying immune correlates that lead to virus control and clearance. Recent studies of re-challenge after acute resolving infection indicate that protective immunity exists, mediated largely by memory CD4+ and CD8+ T cell responses. While early and strong intrahepatic CD4+ and CD8+ T-cell responses are also believed to be important for control of primary infections, the role of neutralizing antibodies (nAbs) is less clear. This gap in our knowledge has stemmed largely from technical limitations in our ability to measure HCV neutralization. The development of retrovirus pseudotype particles (HCVpp) and cell culture infectious virus (HCVcc) now allows these studies to move forward. The aims outlined in this proposal will refine and generalize the HCVcc approach, create convenient reporter viruses for high throughput and quantitative measurements of infectivity and neutralization, and compare the celltropism, receptor usage and neutralization characteristics of HCVcc to the well-established HCVpp system. This will result in a standardized panel of virus stocks, representing all HCV genotypes, which can be used for basic and applied studies (see CRC Project 3). These tools will be used to expand longitudinal studies of nAbs in acute HCV infection in the unique cohorts described in Translational Research Core A. The underlying premise of this work, supported by preliminary data, is that nAb responses during acute infection may play a role in controlling and resolving virus infection. The nAb responses and the HCV epitopes they recognize will be defined and used to establish a database of conserved neutralization determinants that can be integrated into strategies for vaccination or passive immunotherapy. In addition, this panel of recombinant HCVcc expressing convenient reporters will facilitate screening efforts to identify pan-HCV small molecule inhibitors of virus assembly and entry. Tools and reagents developed in the course of these studies will be made available to other HCV CRCs and the broader HCV research community in an effort to speed progress on combating this important public health problem.