Approximately 170 million people worldwide are infected with the hepatitis C virus (HCV) with another 3 to 4 million new infections each year. Chronically infected persons are at risk of developing severe and potentially life-threatening liver disease, including cirrhosis and hepatocellular carcinoma. There is currently no vaccine against HCV, and the available therapies have limited efficacy and significant toxicities. Consequently, there is a great need to develop novel therapeutic agents that specifically target different stages of HCV replication, including viral entry. HCV entry is mediated by the viral envelope glycoproteins E1 and E2. However, the cellular receptors and/or factors that combine to mediate this entry process remain to be elucidated. A major advance in the field of HCV entry was the recent discovery that unmodified HCV envelope glycoproteins E1 and E2 (E1E2) can pseudotype retroviral core particles, which in turn can enter primary hepatocytes and a subset of human hepatoma cell lines. Importantly, the HCV pseudovirus particles (HCVpp) accurately recapitulate the essential biology of HCV entry, and monoclonal antibodies (MAbs) to CD81 or to HCV E1E2 block entry. For the first time, suitable tools are now available to explore the specific cellular proteins involved in HCV entry. In this project, we seek to leverage this technology in order to identify novel MAbs that broadly and potently inhibit HCV by binding requisite cellular receptors for the virus. In this Phase I project, we will utilize cells that express the primary receptor for HCV (as defined by permissiveness for HCVpp entry) as immunogens in order to elicit inhibitory MAbs in mice. Hybridomas will be generated using established methods, and high throughput screening (HTS) technologies will be used to screen over 20,000 hybridoma supernatants to identify novel MAbs that specifically block entry of HCVpp into liver cells. The most promising MAbs will be screened on diverse cell types and characterized for specificity, potency and spectrum of inhibitory activity against diverse HCVpp representing the major viral genotypes. MAbs also will be tested against authentic HCV in the recently reported HCV cell culture system. In addition, MAbs to novel, hepatocyte-specific receptors will be used to determine the molecular nature of the putative primary cellular receptor for HCV entry. Success in the Phase I project will permit testing of these murine MAbs in established in vivo models for prophylaxis and therapy of HCV infection in Phase II, and the most promising inhibitory MAb will be humanized using established procedures to support human testing for the management of HCV infection. Our goal in this project is to translate recent discoveries in HCV entry into novel therapeutic drug candidates and to ultimately identify the primary cellular receptor for HCV. [unreadable] [unreadable] [unreadable]