The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.Hepatitis C virus (HCV) is an important cause of morbidity and mortality worldwide (1-3). Infection with HCV becomes chronic in Ͼ80% of the cases and is a major cause of liver cirrhosis (4) and hepatocellular carcinoma (5). Although the development of a broadly reactive vaccine would be the most effective method for its control, concerns have been raised because of the high degree of genetic heterogeneity of HCV (6) and the lack of protective immunity against reinfection (7,8) or superinfection (9, 10) documented both in humans and in chimpanzees. Viral isolate-restricted neutralizing antibodies against HCV have been demonstrated recently in infected individuals (11, 12), but their molecular target is presently unknown.Several observations have suggested that the hypervariable region 1 (HVR1) could be involved in the neutralization of HCV. This assumption is based on the fact that the HVR1, which is located at the N terminus of the envelope glycoprotein 2 (E2) gene and consists of 34 amino acids spanning map position 384-414 (13), is the most variable region of the HCV genome (14, 15), contains linear epitopes that are recognized by patients' antibodies (16-22) and mutates rapidly in vivo (23)(24)(25)(26), suggesting that it is under the selective pressure of the host immune system. This hypothesis is further substantiated by the lack of variability in the HVR1 observed in an agammaglobulinemic patient over a period of 2.5 years (27). Recent data obtained in vitro suggest that antibodies, present in human sera and directed against the HVR1 as well as against the E2 protein of HCV, can prevent the binding of HCV to cells (28,29). The potential importance of the HVR1 for HCV neutralization is also underscored by the analogy with the V3 loop of human immunodeficiency virus, w...

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis -C virus (HCV) infection. The cell cultures were inoculated with a serum containin HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigeus showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of <1.09 g/ml (principally-1.06 g/ml), while that of strain F was found in fractions with densities of-1.06 and-1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.

We report here an unusual case of long-term HEV infection in a patient with T-cell lymphoma. Persistent infection with HEV was probably due to the absence of anti-HEV antibodies, which was caused by lymphoma and chemotherapy.

To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.

Nine of 15 specimens of human origin thought to contain non-A, non-B hepatitis agents caused hepatitis in recipient chimpanzees. Two have been further characterized. One inoculum, designated strain F, has been reported to produce unique cytoplasmic changes detected by electron microscopy in liver biopsy specimens; the other, strain H, produced distinctive nuclear changes. It is not yet clear whether these two changes result from infection by different agents; they have been useful markers of non-A, non-B hepatitis in chimpanzees. Strain F was serially passaged six times in chimpanzees, and the infectivity titer of the strain F plasma was estimated to be less than 10(2)/ml. Strain H had an infectivity titer in chimpanzees of at least 10(6)/ml. Both the strain F and strain H agents have been successfully transmitted and serially passaged in marmosets. Although hepatitis was detected in a lower percentage of marmosets than chimpanzees given either the strain F or H inoculum, the infectivity titer of the strain H agent appeared to be greater than or equal to 10(8) marmoset infectious doses/ml.

Two different ultrastructural alterations were observed in liver cells of chimpanzees inoculated with plasma derived from two different patients with non-A, non-B hepatitis. During the acute phase of illness in one group of four chimpanzees, peculiar tubular structures, composed of two unit membranes with electron-opaque material in between, were observed in the cytoplasm of hepatocytes. In contrast, these structures were never detected in the liver cells of the second group of five chimpanzees that received the second inoculum, However, nuclear changes, usually associated with aggregates of 20- to 27-nanometer particles, were found in hepatocytes of the latter animals. Although these particles resembled viruses, they were not as uniform as small virus particles often appear. In five other chimpanzees inoculated with non-A, non-B hepatitis material not known to be related to the first two inocula, cytoplasmic structures were found in four, and nuclear structures were found in the remaining one. Thus, all 14 chimpanzees inoculated with transmissible non-A, non-B hepatitis agents could be classified as having either nuclear or cytoplasmic changes. These observations add support to epidemiologic data suggesting that there may be more than one agent of non-A, non-B hepatitis.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.