Characterization of antibodies induced by vaccination with hepatitis C virus envelope glycoproteins.
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ABSTRACT:
SUBMITTER: Ray R
PROVIDER: S-EPMC2931414 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly.
| S-EPMC2937754 | biostudies-literature
Project description:UnlabelledAlthough effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization.ImportanceAn effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4(+) and CD8(+) T cell responses.
| S-EPMC4249152 | biostudies-literature
Project description:Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP₁,₂ or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP₁,₂ peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Taï Forest viruses and a mucin-like domain-deleted Sudan virus GP₁,₂ (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP₁,₂ and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP₁,₂, but not particles pseudotyped with other ebolavirusGP₁,₂. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP₁,₂, as well as analysis of clinically relevant samples.
| S-EPMC3106979 | biostudies-literature
Project description:Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205-319 of E1 to aa421-716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is 285FLVGQLFTFSPRRHW299 which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.
| S-EPMC5896302 | biostudies-literature
Project description:Chronic hepatitis C virus infection often leads to liver cirrhosis and primary liver cancer. In 2015, an estimated 71 million people were living with chronic HCV. Although infection rates have decreased in many parts of the world over the last several decades, incidence of HCV infection doubled between 2010 and 2014 in the United States mainly due to increases in intravenous drug use. The approval of direct acting antiviral treatments is a necessary component in the elimination of HCV, but inherent barriers to treatment (e.g., cost, lack of access to healthcare, adherence to treatment, resistance, etc.) prevent dramatic improvements in infection rates. An effective HCV vaccine would significantly slow the spread of the disease. Difficulties in the development of an HCV culture model system and expression of properly folded- and natively modified-HCV envelope glycoproteins E1 and E2 have hindered vaccine development efforts. The recent structural and biophysical studies of these proteins have demonstrated that the binding sites for the cellular receptor CD-81 and neutralizing antibodies are highly flexible in nature, which complicate vaccine design. Furthermore, the interactions between E1 and E2 throughout HCV infection is poorly understood, and structural flexibility may play a role in shielding antigenic epitopes during infection. Here we discuss the structural complexities of HCV E1 and E2.
| S-EPMC6117417 | biostudies-literature
Project description:Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders.Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.
| S-EPMC4702674 | biostudies-literature
Project description:Background & aimsDevelopment 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.MethodsWe 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.ResultsBoth 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.ConclusionsNeutralizing 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.
| S-EPMC8792218 | biostudies-literature
Project description:Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.
| S-EPMC1143753 | biostudies-literature
Project description:Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412-425, 434-446, 502-520, and 523-535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the Leishmania tarentolae expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412-425 and 523-535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1-6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412-425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434-446, 502-520, and 523-535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412-425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.
| S-EPMC7350033 | biostudies-literature
Project description:A vaccine against Hepatitis C virus (HCV) is urgently needed to limit the spread of HCV. The large antigenic diversity of the HCV glycoprotein E1E2 makes it difficult to design a vaccine but also to fully understand the antibody response after infection or vaccination. Here we designed a panel of HCV pseudoparticles (HCVpps) that cover a wide range of genetically and antigenically diverse E1E2s. We validate our panel using neutralization and a binding antibody multiplex assay (BAMA). The panel of HCVpps includes E1E2 glycoproteins from acute and chronically infected cases in the Netherlands, as well as E1E2 glycoproteins from previously reported HCVs. Using eight monoclonal antibodies targeting multiple antigenic regions on E1E2, we could categorize four groups of neutralization sensitive viruses with viruses showing neutralization titers over a 100-fold range. One HCVpp (AMS0230) was extremely neutralization resistant and only neutralized by AR4-targeting antibodies. In addition, using binding antibody multiplex competition assay, we delineated mAb epitopes and their interactions. The binding and neutralization sensitivity of the HCVpps were confirmed using patient sera. At the end, eleven HCVpps with unique antibody binding and neutralization profiles were selected as the final panel for standardized HCV antibody assessments. In conclusion, this HCVpp panel can be used to evaluate antibody binding and neutralization breadth and potency as well as delineate the epitopes targeted in sera from patients or candidate vaccine trials. The HCVpp panel in combination with the established antibody competition assay present highly valuable tools for HCV vaccine development and evaluation.
| S-EPMC10821612 | biostudies-literature