Activation of the grp78 and grp94 promoters by hepatitis C virus E2 envelope protein.

The hepatitis C virus E1 and E2 envelope proteins are targeted to the endoplasmic reticulum, but instead of being secreted, they are retained in a pre-Golgi compartment, at least partly in a misfolded state. Since secretory proteins which are retained in the endoplasmic reticulum frequently can activate the transcription of intraluminal chaperone proteins, we measured the effect of the E1 and E2 proteins on the promoters of two such chaperones, GRP78 (BiP) and GRP94. We found that E2 but not E1 protein activates these two promoters, as assayed by a reporter gene system. Furthermore, E2 but not E1 protein induces the synthesis of GRP78 from the endogenous cellular gene. We also found that E2 but not E1 protein expressed in mammalian cells is bound tightly to GRP78. This association may explain the ability of E2 protein to activate transcription, since GRP78 has been postulated to be a sensor of stress in the endoplasmic reticulum. Since overexpression of GRP78 has been shown to decrease the sensitivity of cells to killing by cytotoxic T lymphocytes and to increase tumorigenicity and resistance to antitumor drugs, this activity of E2 protein may be involved in the pathogenesis of hepatitis C virus-induced diseases.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release.

Cytotoxic T lymphocytes (CTL) are important to the control of viral replication and their presence may be important to disease outcome. An understanding of the spectrum of proteins recognized by hepatitis C virus (HCV)-specific CTL and the functional properties of these cells is an important step in understanding the disease process and the mechanisms of persistent infection, which occurs in the majority of HCV-infected individuals. In this report we identify HCV-specific CTL responses restricted by the HLA class I molecules A2, A3, A11, A23, B7, B8, and B53. The epitopes recognized by these intrahepatic CTL conform to published motifs for binding to HLA class I molecules, although in some cases we have identified CTL epitopes for which no published motif exists. The use of vectors expressing two different strains of HCV, HCV-1 and HCV-H, revealed both strain-specific and cross-reactive CTL. These HCV-specific CTL were shown to produce cytokines including IFN-gamma, TNF-alpha, GM-CSF, IL-8, and IL-10 in an antigen- and HLA class I-specific manner. These studies indicate that the CTL response to HCV is broadly directed and that as many as five different epitopes may be targeted in a single individual. The identification of minimal epitopes may facilitate peptide-specific immunization strategies. In addition, the release of proinflammatory cytokines by these cells may contribute to the pathogenesis of HCV-induced liver damage.

Peptide immunogen mimicry of putative E1 glycoprotein-specific epitopes in hepatitis C virus.

Hepatitis C virus (HCV) accounts for most cases of acute and chronic non-A and non-B hepatitis with serious consequences that may lead to hepatocellular carcinoma. The putative envelope glycoproteins (E1 and E2) of HCV probably play a role in the pathophysiology of the virus. In order to map the immunodominant domains of the E1 glycoprotein, two epitopes from amino acid residues 210 to 223 (P1) and 315 to 327 (P2) were predicted from the HCV sequence. Immunization of mice with the synthetic peptides conjugated to bovine serum albumin induced an antibody response, and the antisera immunoprecipitated the E1 glycoprotein (approximately 33 kDa) of HCV expressed by recombinant vaccinia virus. A panel of HCV-infected human sera was also tested with the synthetic peptides by enzyme-linked immunosorbent assay for epitope-specific responses. Of 38 infected serum samples, 35 (92.1%) demonstrated a spectrum of reactivity to the P2 peptide. On the other hand, only 17 of 38 (44.7%) serum samples were reactive to the P1 peptide. Strains of HCV exhibit a striking genomic diversity. The predicted P1 epitope showed localization in the sequence-variable region, and the P2 epitope localized in a highly conserved domain. Results from this study suggest that the E1 glycoprotein of HCV contains at least two potential antigenic epitopes. Synthetic peptides corresponding to these epitopes and antisera to these peptides may serve as the monospecific immunological reagents to further determine the role of E1 glycoprotein in HCV infection.

Vaccination of chimpanzees against infection by the hepatitis C virus.

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 hepatocellular 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 [E1 (gp33) and E2 (gp72)] that were copurified from HeLa cells infected with a recombinant vaccinia virus expression vector. Despite the induction of a weak humoral immune response to these viral glycoproteins in experimentally infected chimpanzees, a strong humoral immune response was obtained in all vaccines. The five highest responders showed complete protection against an i.v. challenge with homologous hepatitis C virus 1. The remaining two vaccines became infected, but both infection and disease may have been ameliorated in comparison with four similarly challenged control chimpanzees, all of which developed acute hepatitis and chronic infections. These results provide considerable encouragement for the eventual control of hepatitis C virus infection by vaccination.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes recognize epitopes in the core and envelope proteins of HCV.

Hepatitis C virus (HCV) is a major cause of posttransfusion and community-acquired hepatitis, and a majority of individuals infected with this virus will subsequently develop chronic hepatitis. Characterization of the host immune response to this infection is an important first step that should facilitate the development of immunomodulatory agents and vaccines. Cellular immune responses, especially those mediated by cytotoxic T lymphocytes (CTL), are important in the control of many viral diseases. In this study, liver-infiltrating lymphocytes from persons with chronic HCV hepatitis were examined for evidence of HCV-specific CTL by using target cells infected with recombinant vaccinia viruses expressing the HCV core, E1, E2, and part of the NS2 proteins. Bulk expansion of liver-derived CD8+ lymphocytes resulted in the detection of HCV-specific CTL activity, whereas activity could not be found in CD8+ lymphocytes expanded from peripheral blood. Epitopes recognized by these CTL were defined by using CTL clones obtained by limiting dilution and target cells sensitized with synthetic HCV peptides. Four distinct HLA class I-restricted epitopes were identified, including two epitopes in the amino-terminal portion of the core protein. These studies provide evidence that the highly conserved core protein is a target for HCV-specific CTL and identify CTL epitopes within the more highly variable E2 envelope protein. Our studies also suggest that HCV-specific CTL are localized at the site of tissue injury in infected persons with chronic hepatitis. Identification of the epitopes recognized by HCV-specific CTL will facilitate exploration of their role in disease pathogenesis and may provide information useful in development of therapeutic interventions or vaccines.

Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes.

Hepatitis C virus-specific CTL responses in the liver of chimpanzees with acute and chronic hepatitis C.

Hepatitis C virus (HCV)-specific CTL responses were evaluated in two chimpanzees (Pan troglodytes) during the acute and chronic phases of HCV infection. CD8+ T lymphocytes were isolated from liver tissue homogenates using anti-CD8 antibody-coated magnetic beads and then stimulated with anti-CD3 antibodies, IL-2, and irradiated human PBMC using limiting dilution culture conditions. HCV-specific cytotoxic activity of expanding CD8+ cell lines was assessed against autologous lymphoblastoid cell lines infected with recombinant vaccinia virus vectors encoding HCV Ag. CD8+ T cell lines specific for structural and nonstructural proteins of HCV were established from both animals. Cytolytic activity was blocked with anti-CD8 or anti-class I MHC antibodies, indicating that class I MHC molecules were involved in presentation of viral Ag to the CTL. Overlapping synthetic peptides were used to define a 12 amino acid segment of the nonstructural 3 (NS3) protein recognized by CTL lines from both chimpanzees. Studies with truncated peptides revealed that these CD8+ cell lines were directed against overlapping epitopes presented by distinct class I restriction elements of the chimpanzee MHC complex. CD8+ cell lines with identical specificities for an NS3 epitope were generated from one chronically infected animal at 16 and 28 wk postinfection. These results indicate that virus-specific CTL populations persist in the liver for months, but are unable to resolve chronic HCV infection.

Antibody response to core, envelope and nonstructural hepatitis C virus antigens: comparison of immunocompetent and immunosuppressed patients.

Some immunosuppressed patients with hepatitis C virus infection do not have detectable levels of antibody to hepatitis C virus on second-generation enzyme immunoassay. Antibodies to the envelope and nonstructural region 5 proteins have not been examined. Four groups of patients with hepatitis C virus infection were studied: (a) 20 immunocompetent patients, (b) 15 hemodialysis patients, (c) 17 kidney transplant recipients and (d) 3 acute leukemia patients who underwent bone marrow transplantation. Serum samples were tested for antibody to hepatitis C virus with a second-generation enzyme immunoassay and multi-antigen enzyme immunoassays and for hepatitis C virus RNA with a nested polymerase chain reaction assay. All the immunocompetent patients reacted to C25, C22 and C33C; 90% reacted to nonstructural region 5 antigen and 80% reacted to C100-3. Only 55% reacted against yeast-derived e1 and e2 antigens, but all reacted against vaccinia virus--expressed N e1 and e2 antigens, indicating that the envelope epitopes are conformational and glycosylated. Sixty-five percent to 90% of dialysis and kidney transplant patients reacted to C25, C22 and N e1 and e2, but only 12% to 60% reacted to C100-3, C33C and nonstructural region 5 antigen. Diminution or loss of reactivity to hepatitis C virus antigens was observed after kidney and bone marrow transplantation, with C25 and N e1 and e2 less affected. Our data suggest that incorporation of C25 and N e1 and e2 antigens in the assay for antibody to hepatitis C virus would improve the detection of hepatitis C virus infection in immunosuppressed patients.

Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome.

We have expressed the full-length coding region and selected domains of the hepatitis C virus (HCV) cDNA in mammalian cells by transfection. Using HCV antibody-positive human sera and monospecific antibodies the proteins encoded by the putative structural and non-structural regions of the open reading frame of HCV were identified as core (p22), E1 (gp32-35), E2 (gp68-72), NS2 (p23), NS3 (p72), NS4a and b (p10 and p27) and NS5a and b (p56 and p70). We have also defined the subcellular localizations of the HCV proteins using indirect immunofluorescence assays.

The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) domain has been predicted from sequence comparisons to represent a trypsin-like serine protease. By expressing wild-type and mutant HCV-1 cDNAs in transfected mammalian cells, we have identified putative nonstructural proteins 3 (72 kDa), 4 (10 kDa and 27 kDa) and 5 (58 kDa) and have shown that their processing from the viral polyprotein precursor is dependent on Ser1165 located in the proposed protease catalytic site. Data obtained from in vitro RNA translations indicate that unlike the processing of the NS2/NS3 junction, NS3/NS4 processing is dependent on Ser1165. In contrast to the situation for the related flaviviral NS3 proteases, the HCV NS3-mediated cleavage of the NS3/NS4 junction does not require the upstream NS2 domain and may not occur at dibasic sites.

T-lymphocyte response to hepatitis C virus in different clinical courses of infection.

BACKGROUND: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. METHODS: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. RESULTS: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. CONCLUSIONS: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease.

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification.

Hepatitis C virus (HCV) antigen was detected immunohistochemically using fluorescein isothiocyanate-labeled immunoglobulin G fractions from chimpanzee and human sera strongly reactive with recombinant hepatitis C virus structural and non-structural proteins. The antigen was localized in the cytoplasm of hepatocytes in all 9 chimpanzees with acute hepatitis C, in 5 of 10 chimpanzees with chronic HCV infection, and in 11 of 12 patients with chronic hepatitis C. The specificity of the hepatocellular HCV and FITC-labeled probes for HCV was ascertained by blocking studies with paired serum samples obtained from 8 infected and uninfected chimpanzees or from 14 patients during the acute and chronic phases of HCV infection. Absorption experiments on FITC-labeled probes with selected host proteins (normal liver homogenate, plasma proteins, red blood cells) did not indicate cross reactivity of the probes with these antigens. Direct immunomorphologic evidence for the HCV specificity of hepatocellular HCV antigen deposits and the FITC-labeled polyclonal anti-HCVAg probe was established in absorption experiments using recombinant HCV nonstructural proteins. The putative HCV NS3 protein was the most prominent component of hepatocellular HCV antigen.

Characterization of the hepatitis C virus E2/NS1 gene product expressed in mammalian cells.

Truncated and full-length versions of the hepatitis C virus protein domain encoding a presumptive envelope glycoprotein designated E2/NS1 were stably expressed in CHO cell lines. Characterization of the processing events involved in the maturation of E2/NS1 revealed that a high-mannose form resident in the endoplasmic reticulum was the most abundant form detected intracellularly. The ionophore carboxyl cyanide m-chlorophenyl-hydrazone was used to show that the E2/NS1 glycoprotein resided in the endoplasmic reticulum. The full-length form of E2/NS1 appeared to be cell-associated and could not be detected as a secreted product. C-terminal truncated molecules could be detected in the extracellular media as fully processed glycoproteins containing terminal sialic acid additions. These truncated glycoproteins are predicted to be biologically relevant targets of the host immune response and are therefore potential subunit vaccine candidates.