The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

To investigate the type of immunity responsible for resolution of hepatitis C virus (HCV) infection, we monitored antibody and intrahepatic cytotoxic T lymphocyte (CTL) responses during acute (<20 weeks) infection in chimpanzees. Two animals who terminated infection made strong CTL but poor antibody responses. In both resolvers, CTL targeted at least six viral regions. In contrast, animals developing chronic hepatitis generated weaker acute CTL responses. Extensive analysis of the fine specificity of the CTL in one resolver revealed nine peptide epitopes and restriction by all six MHC class I allotypes. Every specificity shown during acute hepatitis persisted in normal liver tissue more than 1 yr after resolution. These results suggest that CD8+CTL are better correlated with protection against HCV infection than antibodies.

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3–4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C.

A high incidence of community-acquired hepatitis C virus infection that can lead to the progressive development of chronic active hepatitis, liver cirrhosis, and primary hepatoceflular carcinoma occurs throughout the world. A vaccine to control the spread of this agent that represents a major cause of chronic liver disease is therefore needed. Seven chimpanzees (Pan troglodytes) have been immunized with both putative envelope glycoproteins [El (gp33) (4) and leads to the development of chronic hepatitis and liver cirrhosis in "50%o and 10% of cases, respectively (5). A significant proportion of patients with liver cirrhosis will also develop primary hepatocellular carcinoma (6). The prevalence of HCV infection around the world is generally between 0.4 and 2% (7-10), although a much higher level has been reported in Egypt (14%; see ref. 11). Therefore, HCV constitutes a major cause of chronic liver disease throughout the world. With the recent development of recombinant-based diagnostic assays for the detection of circulating HCV antibodies (2, 3), the risk of being infected with HCV after transfusion of blood or cellular components has been substantially reduced (12,13). However, community-acquired infection is much more common and occurs at various frequencies in high-risk groups such as i.v. drug users, health-care workers, and sexual and household contacts ofhepatitis patients, although -40%o of cases in the United States appear to have no known risk factor for acquisition of infection (14). Thus, the development of an HCV vaccine to prevent transmission within the community is highly desirable.HCV is distantly related genetically to both the pestiviruses and flaviviruses and, like these relatives, appears to process virion structural proteins from the N-terminal region of the polyprotein precursor encoded by the positivestranded RNA genome (15). The host signal peptidase mediates the cleavage of a basic, presumed nucleocapsid protein (C; -20 kDa) from the N terminus of the polyprotein precursor followed by two glycoproteins (El, glycoproteins (gp33 and gp72) under nondenaturing conditions from the endoplasmic reticulum. A fraction of the purified material was shown to exist in the form of a large E1/E2 oligomeric complex (22). We now report on the efficacy of this purified preparation in vaccinating chimpanzees against experimental infection with HCV-1. MATERIALS AND METHODSVaccine Preparation. A Stu I-Bgl II cDNA restriction fragment ofthe HCV-1 genome (nt 63 to +2901; aa 1-967; ref. 15) encoding the complete C (20 kDa), El (gp33 kDa), and E2 (gp72 kDa) proteins along with a C-terminally truncated NS2 product was cloned into the Sma I site of plasmid SC59 downstream of a hybrid early/late vaccinia promoter (S. Chakrabarti and B.M., unpublished work). BSC40 cells preinfected with wild-type WR vaccinia were transfected with the SC59 recombinant and thymidine kinase-negative recombinants, selected, and purified through three rounds ofplaque purification (23). Spinner cultures of HeLa cells (109 c...

Hepatitis