We propose to undertake a detailed study of the immune responses to hepatitis C virus infection in order to develop a rational approach to vaccine and immunotherapy design. In previous work, we have constructed a full length cDNA clone of the HCV genome. Intrahepatic inoculation of RNA transcripts of that clone in chimpanzees has resulted in HCV infection and hepatitis clinically indistinguishable from infections caused by inoculation of HCV containing serum. We have followed viral sequence evolution in two, transfected chimpanzees that have developed persistent HCV infection and shown that the virus does evolve at a rate similar to other RNA viruses. However, no mutations occurred in the so called hypervariable region (HVR1) that is believed to contain the primary neutralizing epitope and to mutate under humoral immune selection pressure. We have also studied the immune responses in a cohort of patients infected 18-20 years ago in a single source outbreak of HCV. We have shown that antibodies declined to undetectable levels in 44 percent of the recovered patients while still present in most chronically infected patients 18-20 years after exposure. In contrast, recovered, but not chronically infected patients maintained vigorous T helper and CTL responses. In the proposed study, we will perform a prospective study on humoral and cellular immune responses to HCV infections and viral sequence evolution in infected chimpanzees. We will pre-select a group of chimpanzees that express an HLA-A2, -A3 or -B7 like Patr-haplotype and transfect these animals with molecularly cloned HCV to initiate infection with a uniform viral sequence. In this way we can use reagents that perfectly match the infecting virus to study humoral and cellular immune responses, the emergence of possible immune escape mutations and their effect on immune response, viral load and disease activity, and, most importantly, define how the virus-host interactions differ between animals that resolve their infections and those that become persistently infected. Recovered animals will be challenged with homologous and heterologous virus to test whether they are protected from reinfection. This may also provide clinically important information on the protection of recovered patients, since an infected chimpanzee that cleared viral infection displayed a Th response that was targeted against the same proteins and the same immunodominant epitope as the immune response of the recovered patients. We will then use this information to rationally develop strategies for both immunotherapy and prophylactic vaccination. It is believed that much of the information gained in these studies will be applicable to vaccine development for man.