Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development.
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ABSTRACT:
SUBMITTER: Toth EA
PROVIDER: S-EPMC8227786 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry.
| S-EPMC5428263 | biostudies-literature
Project description:Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.
| S-EPMC6530877 | biostudies-literature
Project description:BackgroundHepatitis C virus (HCV) causes chronic liver disease that often leads to cirrhosis and hepatocellular carcinoma. In animal studies, chimpanzees were protected against chronic infection following experimental challenge with either homologous or heterologous HCV genotype 1a strains which predominate in the USA and Canada. We describe the first in humans clinical trial of this prophylactic HCV vaccine.MethodsHCV E1E2 adjuvanted with MF59C.1 (an oil-in-water emulsion) was given at 3 different dosages on day 0 and weeks 4, 24 and 48 in a phase 1, placebo-controlled, dose escalation trial to healthy HCV-negative adults.ResultsThere was no significant difference in the proportion of subjects reporting adverse events across the groups. Following vaccination subjects developed antibodies detectable by ELISA, CD81 neutralization and VSV/HCV pseudotype neutralization. There were no significant differences between vaccine groups in the number of responders and geometric mean titers for each of the three assays. All subjects developed lymphocyte proliferation responses to E1E2 and an inverse response to increasing amounts of antigen was noted.ConclusionsThe vaccine was safe and generally well-tolerated at each of the 3 dosage levels and induced antibody and lymphoproliferative responses. A larger study to further evaluate safety and immunogenicity is warranted.
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Project description:HCV vaccine development is stymied by the high genetic diversity of the virus and the variability of the envelope glycoproteins. One strategy to overcome this is to identify conserved, functionally important regions-such as the epitopes of broadly neutralizing antibodies (bNAbs)-and use these as a basis for structure-based vaccine design. Here, we report an anti-idiotype approach that has generated an antibody that mimics a highly conserved neutralizing epitope on HCV E2. Crucially, a mutagenesis screen was used to identify the antibody, designated B2.1?A, whose binding characteristics to the bNAb AP33 closely resemble those of the original antigen. Protein crystallography confirmed that B2.1?A is a structural mimic of the AP33 epitope. When used as an immunogen B2.1?A induced antibodies that recognized the same epitope and E2 residues as AP33 and most importantly protected against HCV challenge in a mouse model.
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Project description:The E1 and E2 envelope proteins of hepatitis C virus (HCV) form a heterodimer that drives virus-host membrane fusion. Here, we analyze the role of each amino acid in E1E2 function, expressing 545 individual alanine mutants of E1E2 in human cells, incorporating them into infectious viral pseudoparticles, and testing them against 37 different monoclonal antibodies (MAbs) to ascertain full-length translation, folding, heterodimer assembly, CD81 binding, viral pseudoparticle incorporation, and infectivity. We propose a model describing the role of each critical residue in E1E2 functionality and use it to examine how MAbs neutralize infection by exploiting functionally critical sites of vulnerability on E1E2. Our results suggest that E1E2 is a surprisingly fragile protein complex where even a single alanine mutation at 92% of positions disrupts its function. The amino-acid-level targets identified are highly conserved and functionally critical and can be exploited for improved therapies and vaccines.
| S-EPMC9281441 | biostudies-literature
Project description:Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.
| S-EPMC7826332 | biostudies-literature
Project description:Hepatitis C virus (HCV) is a serious and growing public health problem despite recent developments of antiviral therapeutics. To achieve global elimination of HCV, an effective cross-genotype vaccine is needed. The failure of previous vaccination trials to elicit an effective cross-reactive immune response demands better vaccine antigens to induce a potent cross-neutralizing response to improve vaccine efficacy. HCV E1 and E2 envelope (Env) glycoproteins are the main targets for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. Therefore, a molecular-level understanding of the nAb responses against HCV is imperative for the rational design of cross-genotype vaccine antigens. Here we summarize the recent advances in structural studies of HCV Env and Env-nAb complexes and how they improve our understanding of immune recognition of HCV. We review the structural data defining HCV neutralization epitopes and conformational plasticity of the Env proteins, and the knowledge applicable to rational vaccine design.
| S-EPMC8147963 | biostudies-literature
Project description:Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.
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Project description:Although a vaccine against hepatitis B virus (HBV) has been available since 1982, it is estimated that 600,000 people die every year due to HBV. An affordable oral vaccine could help alleviate the disease burden and to this end the hepatitis B surface antigen (HBsAg) was expressed in maize. Orally delivered maize material induced the strongest immune response in mice when lipid was extracted by CO2 supercritical fluid extraction (SFE), compared to full fat and hexane-extracted material. The present study provides a biochemical and biophysical basis for these immunological differences by comparing the active ingredient in the differently treated maize material. Purified maize-derived HBsAg underwent biophysical characterization by gel filtration, transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-CD, and fluorescence. Gel filtration showed that HBsAg forms higher-order oligomers and TEM demonstrated virus-like particle (VLP) formation. The VLPs obtained from SFE were more regular in shape and size compared to hexane or full fat material. In addition, SFE-derived HBsAg showed the greatest extent of ?-helical structure by far UV-CD spectrum. Fluorescence experiments also revealed differences in protein conformation. This work establishes SFE-treated maize material as a viable oral vaccine candidate and advances the development of the first oral subunit vaccine.
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Project description:BACKGROUND:Hepatitis C virus (HCV) is one of the major causes of cirrhosis and hepatocellular carcinoma with an estimation of 185 million people with infection. The E2 is the main target for neutralizing antibody responses and the variation of this region is related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection. While both E1 and E2 are hypervariable in nature, it is difficult to design vaccines or therapeutic drugs against them. OBJECTIVES:The objective of this study was to characterize genotype 5a E1 and E2 sequences to determine possible glycosylation sites, conserved B-cell epitopes and peptides in HCV that could be useful targets in design of vaccine and entry inhibitors. PATIENTS AND METHODS:This study was conducted through PCR amplification of E1 and E2 regions, sequencing, prediction of B-cell epitopes, analysis of N-linked glycosylation and peptide design in 18 samples of HCV genotype 5a from South African. RESULTS:Differences in the probability of glycosylation in E1 and E2 regions were observed in this study. Three conserved antigenic B-cell epitopes were predicted in the E2 regions and also 11 short peptides were designed from the highly conserved residues. CONCLUSIONS:This study provided conserved B-cell epitopes and peptides that can be useful for designing entry inhibitors and vaccines able to cover a global population, especially where genotype 5a is common.
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