Hepatitis C virus (HCV) infection is most often clinically inapparent and rarely associated with symptoms of acute hepatitis. Most patients, however, fail to resolve the acute infection and proceed to develop chronic hepatitis with the risk of liver cirrhosis and hepatocellular carcinoma later in life. Research thus far has been hampered by the fact that patients with well documented episodes of acute, self-limited hepatitis C are rare, that the intrahepatic immune responses at the site of inflammation cannot not be prospectively studied, and that sequences of the infecting virus are often unknown and rather heterogeneous. Transfection of a chimpanzee with RNA transcribed from a full length HCV cDNA clone has now provided the opportunity to prospectively study the peripheral blood as well as intrahepatic T cell response as regards to its vigor, kinetics and specificity with antigens of a single, defined sequence and to rechallenge recovered animals with virus of precisely the same sequence. We have previously shown in a cohort of patients that the HCV specific cellular immune response persists after clearance of HCV, while the humoral immune response decreases (Nat. Med. 6:578, 2000). To determine whether this immune response protects from HCV reinfection, we challenged two chimpanzees that displayed a similar cellular immune response after recovery from HCV: Chimpanzee 1 had acute hepatitis 15 months earlier after intrahepatic infection with in vitro transcribed RNA (genotype 1a) and chimpanzee 2 had acute hepatitis C six years previously after intravenous infection with (genotype 1a). Both chimpanzees were challenged with increasing concentrations of monotypic HCV obtained from a third chimpanzee four weeks after infection. Several interesting results were observed:First, significant increases in HCV specific, IFN-gamma producing CD4+ and CD8+ T cells were observed by Elispot analysis even after challenge with inoculum that did neither cause detectable viremia in the peripheral blood nor changes in antibody titers. These transient T cell responses were targeted against multiple, predominantly nonstructural proteins and disappeared from the blood by week 4 after each challenge presumably because they migrated to the liver from which HCV specific T cell clones could be isolated at that time.Second, when viremia occurred after challenge with an inoculum of higher viral titer, clinical symptoms of hepatitis were significantly milder and viremia was significantly shorter than during the previous phase of infection. Clearance was associated with a sharp increase in HCV specific, IFN-gamma secreting and proliferative T cell responses in the blood. Three to four weeks after challenge, these IFNgamma secreting, HCV specific T cells became transiently undetectable in the blood, but could still be isolated from liver biopsies. Increased antibody reactivities occurred significantly later than the T cell response, i.e. 4-6 weeks after challenge and antibodies against the envelope proteins of HCV were not observed at all.Third, during further follow up, chimpanzee 2 remained HCV-RNA negative. Chimpanzee 1 remained HCV-RNA negative during challenge with 32 CID50 and appeared to be protected from reinfection at that time, but lost its HCV specific Th1 response three months later and turned HCV RNA positive again. These results indicate, that the cellular immune response needs to be maintained to ensure complete viral clearance as previously reported for an HCV infected patient (Gastroenterology 117:933, 1999).