An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Detection of Antibodies to HCV E1E2 by Lectin-Capture ELISA.

Enzyme-linked immunosorbent assays (ELISAs) enable rapid detection and quantitation of antibodies in samples. Such assays can be highly sensitive and can be performed in most laboratories with basic equipment. Although detecting binding antibodies to the surface proteins of most pathogens by ELISA is not always indicative of antibody function, i.e., neutralizing activity of antibodies, the results can be used as a first step toward more in-depth analysis of antibody responses. Here we describe a method that can be used to standardize ELISAs for the detection of HCV envelope antibodies across laboratories and provide adaptations of the method to further characterize antibody responses in serum samples.

Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry.

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81.IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

Qualitative differences in cellular immunogenicity elicited by hepatitis C virus T-Cell vaccines employing prime-boost regimens.

T-cell based vaccines have been considered as attractive candidates for prevention of hepatitis C virus (HCV) infections. In this study we compared the magnitude and phenotypic characteristics of CD8+ T-cells induced by three commonly used viral vectors, Adenovirus-5 (Ad5), Vaccinia virus (VV) and Modified Vaccinia Ankara (MVA) expressing the HCV NS3/4A protein. C57/BL6 mice were primed with DNA expressing NS3/4A and boosted with each of the viral vectors in individual groups of mice. We then tracked the vaccine-induced CD8+ T-cell responses using pentamer binding and cytokine production analysis. Overall, our data indicate that the memory cells induced by Ad5 were inferior to those induced by VV or MVA. We found that Ad5 boosting resulted in rapid expansion and significantly higher frequencies of NS3-specific T-cells compared to VV and MVA boosting. However, the functional profiles, assessed through analysis of the memory cell marker CD127 and the anti-apoptotic molecule Bcl-2 in the blood, spleen, and liver; and measurements of interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 production indicated significantly lower frequencies of long-lived memory T-cells following Ad5 boosting compared to VV and MVA. This same set of analyses suggested that the memory cells induced following boosting with MVA were superior to those induced by both Ad5 and VV. This superiority of the MVA-induced CD8+ T-cells was confirmed following surrogate challenge of mice with a recombinant mouse herpes virus expressing the HCV NS3 protein. Higher levels of NS3-specific CD8+ T-cells displaying the functional markers CD69, Ki67 and Granzyme B were found in the spleens of mice boosted with MVA compared to VV and Ad5, both alone and in combination. These data suggest that MVA may be a more successful viral vector for induction of effective CD8+ T-cell responses against hepatitis C virus.

Hepatitis C virus clearance correlates with HLA-DR expression on proliferating CD8+ T cells in immune-primed chimpanzees.

UNLABELLED: Vaccination of chimpanzees against hepatitis C virus (HCV) using T-cell-based vaccines targeting nonstructural proteins has not resulted in the same levels of control and clearance as those seen in animals reexposed after HCV clearance. We hypothesized that the outcome of infection depends on the different subtypes of activated T cells. We used multicolor flow cytometry to evaluate activation (CD38+/HLA-DR+) and proliferation (Ki67+/Bcl-2-low) profiles of CD4+ and CD8+ T cells in peripheral blood before and after challenge in chimpanzees vaccinated using DNA/adenovirus, mock-vaccinated, and chimpanzees that had spontaneously cleared infection (rechallenged). The frequencies of activated or proliferating CD8+ T cells peaked at 2 weeks postchallenge in the vaccinated and rechallenged animals, coinciding with reductions in viral titers. However, the magnitude of the responses did not correlate with outcome or sustained control of viral replication. In contrast, proliferation of the CD8+ T cells coexpressing HLA-DR either with or without CD38 expression was significantly higher at challenge in animals that rapidly cleared HCV and remained so throughout the follow-up period. CONCLUSION: Our data suggest that the appearance of proliferating HLA-DR+/CD8+ T cells can be used as a predictor of a successfully primed memory immune response against HCV and as a marker of effective vaccination in clinical trials.

Inhibition of hepatitis C virus by the cyanobacterial protein Microcystis viridis lectin: mechanistic differences between the high-mannose specific lectins MVL, CV-N, and GNA.

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein.

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W(437), L(438), L(441), and F(442), within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles.

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.

Delayed induction, not impaired recruitment, of specific CD8⁺ T cells causes the late onset of acute hepatitis C.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is characterized by lack of immune-mediated liver injury despite a high level of HCV replication during the incubation phase, which lasts about 8 weeks. We investigated whether this results from delayed recruitment of HCV-specific T cells and whether it facilitates HCV persistence. METHODS: Six chimpanzees were infected with HCV; blood and liver samples were collected for 28 weeks and analyzed for immune cells and chemokines. RESULTS: Two chimpanzees developed self-limited infections, whereas the remaining 4 developed chronic infections. Levels of the chemokines CXCL10, CXCL11, CCL4, and CCL5 increased in blood and liver samples from all chimpanzees within 1 month of HCV infection. Chemokine induction correlated with intrahepatic type I interferon (IFN) responses in vivo and was blocked by neutralizing antibodies against IFN-ß in vitro. Despite the early-stage induction of chemokines, the intrahepatic lymphocytic infiltrate started to increase no earlier than 8 weeks after HCV infection, when HCV-specific, tetramer-positive CD8(+) T cells appeared in the circulation. The HCV-specific CD8(+) T cells expressed chemokine receptors when they were initially detected in blood samples, so they could be recruited to the liver as soon as they entered the circulation. CONCLUSIONS: Chemokines are induced during early stages of HCV infection, which requires a type I IFN-mediated response. The delayed onset of acute hepatitis does not result from delayed recruitment of HCV-specific T cells, but could instead be related to a primary delay in the induction of HCV-specific T cells. Divergent outcomes occur without evident differences in chemokine induction and T-cell recruitment.

Meta-analysis of hepatitis C virus vaccine efficacy in chimpanzees indicates an importance for structural proteins.

BACKGROUND & AIMS: Studies in patients and chimpanzees that spontaneously cleared hepatitis C virus (HCV) infections demonstrated that natural immunity to the virus is induced during primary infections and that this immunity can be cross protective. These discoveries led to optimism about prophylactic HCV vaccines, and several studies were performed in chimpanzees, although most included fewer than 6 animals. To draw meaningful conclusions about the efficacy of HCV vaccines in chimpanzees, we performed statistical analyses of data from previously published studies from different groups. METHODS: We performed a meta-analysis that compared parameters among naïve (n = 63), vaccinated (n = 53), and rechallenged (n = 36) animals, including peak RNA titer postchallenge, time points of peak RNA titer, duration of viremia, and proportion of persistent infections. RESULTS: Each vaccination study induced immune responses that were effective in rapidly controlling HCV replication. Levels of induced T-cell responses did not indicate vaccine success. There was no reduction in the rate of HCV persistence in vaccinated animals, compared with naïve animals, when nonstructural proteins were included in the vaccine. Vaccines that contained only structural proteins had clearance rates that were significantly higher than vaccines that contained nonstructural components (P = .015). CONCLUSIONS: The inclusion of nonstructural proteins in HCV vaccines might be detrimental to protective immune responses, and/or structural proteins might activate T-cell responses that mediate viral clearance.

Hepatitis C virus with a naturally occurring single amino-acid substitution in the E2 envelope protein escapes neutralization by naturally-induced and vaccine-induced antibodies.

Mutations arising in neutralizing epitopes of hepatitis C virus may play a role in the ability of the virus to escape control by neutralizing antibodies and in the establishment of chronic infections. An amino-acid substitution, Q412H, within a major conserved neutralization epitope EP I (aa 412-426) in the E2 glycoprotein is observed in chronic HCV carriers. We found that naturally acquired polyclonal EP I-specific antibodies have an equivalent binding capacity toward either the wild type or the Q412H mutant peptide encompassing the EP I epitope. While EP I-specific antibodies neutralized J6/JFH1 virus in vitro, they did not neutralize J6/JFH1 virus containing the Q412H mutation. Furthermore, we found that plasma obtained from a chimpanzee that had anti-E1/E2 antibodies following experimental immunization, neutralized the wild type J6/JFH1 virus but failed to neutralize the mutant virus. Thus, mutation Q412H found in naturally occurring variants could represent an antibody escape mutation. These data may have important implications for vaccine design.

Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity.

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412-426 (epitope I) and 434-446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.

Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6/JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.

Long-term persistence of infection in chimpanzees inoculated with an infectious hepatitis C virus clone is associated with a decrease in the viral amino acid substitution rate and low levels of heterogeneity.

Two chimpanzees, 1535 and 1536, became persistently infected following inoculation with RNA transcripts from cDNA clones of hepatitis C virus (HCV). Analysis of the HCV genomes from both animals showed an accumulation of amino acid substitutions over time. The appearance of substitutions in the envelope genes was associated with increased antienvelope antibody titers. However, extensive mutations were not incorporated into hypervariable region 1 (HVR1). A comparison of the nonsynonymous substitution rate/synonymous substitution rate was made at various time points to analyze selective pressure. The highest level of selective pressure occurred during the acute phase and decreased as the infection continued. The nonsynonymous substitution rate was initially higher than the synonymous substitution rate but decreased over time from 3.3 x 10(-3) (chimpanzee 1535) and 3.2 x 10(-3) (chimpanzee 1536) substitutions/site/year at week 26 to 1.4 x 10(-3) (chimpanzee 1535) and 1.7 x 10(-3) (chimpanzee 1536) at week 216, while the synonymous substitution rate remained steady at approximately 1 x 10(-3) substitutions/site/year. Analysis of PCR products using single-stranded conformational polymorphism indicated a low level of heterogeneity in the viral genome. The results of these studies confirm that the persistence of infection is not solely due to changes in HVR1 or heterogeneity and that the majority of variants observed in natural infections could not arise simply through mutation during the time period most humans and chimpanzees are observed. These data also indicate that immune pressure and selection continue throughout the chronic phase.

Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection.

Two chimpanzees, one naïve (Ch1601) and one recovered from hepatitis C virus (HCV) acute infection (Ch1587), were vaccinated with recombinant envelope glycoproteins (E1E2) and then challenged with 100 CID50 of HCV. Results of the challenge were compared to infection in a non-vaccinated control animal. Immunization generated high antibody titers to E1E2 including antibody specifically directed to the hypervariable region 1 (HVR1) in addition to strong and specific HVR1 T-cell proliferative responses. Upon challenge with HCV, viremia was delayed 3 weeks in both vaccinated animals compared to the non-immunized (control) animal. Ch1601 HCV RNA titers were maintained below 5 x 10(4) copies/ml, and alanine aminotransferase levels were only minimally elevated. An increase in intrahepatic cytokine mRNA levels coincided with a fall in HCV RNA to non-quantifiable levels. Despite this apparent control of virus replication the animal became persistently infected. Ch1587 had a significantly shorter and milder viremia, compared to the re-infection of the non-vaccinated control animal. This data indicates that a strategy inducing a T-cell immune response combined with antibody responses to E1E2 would make a viable candidate for an HCV vaccine.

Transcriptional upregulation of interferon-induced protein kinase, PKR, in breast cancer.

PKR (double-stranded RNA activated protein kinase) is overexpressed and overactive in human breast carcinoma (BC) cells. Here, we report that BC cells also have higher PKR mRNA levels and exhibit increased transcription from the PKR promoter. Mutational analysis of the PKR promoter indicated that the interferon stimulation response element (ISRE) is responsible for the increased transcription in BC cells. By gel retardation assay, ISRE-protein complexes formed by BC and non-transformed nuclear extracts were compared. A BC-specific ISRE-protein complex resembles the multimeric factor, ISGF3.

Previously infected and recovered chimpanzees exhibit rapid responses that control hepatitis C virus replication upon rechallenge.

Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 10(6) copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 10(3) copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-gamma) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-gamma in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.