Hepatitis C Virus Envelope Glycoproteins Research Articles

Neutralizing antibodies evolve to exploit vulnerable sites in the HCV envelope glycoprotein E2 and mediate spontaneous clearance of infection

Individuals who clear primary hepatitis C virus (HCV) infections clear subsequent reinfections more than 80% of the time, but the mechanisms are poorly defined. Here, we used HCV variants and plasma from individuals with repeated clearance to characterize longitudinal changes in envelope glycoprotein E2 sequences, function, and neutralizing antibody (NAb) resistance. Clearance of infection was associated with early selection of viruses with NAb resistance substitutions that also reduced E2 binding to CD81, the primary HCV receptor. Later, peri-clearance plasma samples regained neutralizing capacity against these variants. We identified a subset of broadly NAbs (bNAbs) for which these loss-of-fitness substitutions conferred resistance to unmutated bNAb ancestors but increased sensitivity to mature bNAbs. These data demonstrate a mechanism by which neutralizing antibodies contribute to repeated immune-mediated HCV clearance, identifying specific bNAbs that exploit fundamental vulnerabilities in E2. The induction of bNAbs with these specificities should be a goal of HCV vaccine development.

Current Hepatitis C Vaccine Candidates Based on the Induction of Neutralizing Antibodies.

The introduction of direct-acting antivirals (DAAs) has revolutionized hepatitis C treatment. Short courses of treatment with these drugs are highly beneficial to patients, eliminating hepatitis C virus (HCV) without adverse effects. However, this outstanding success is tempered by the continuing difficulty of eradicating the virus worldwide. Thus, access to an effective vaccine against HCV is strongly needed to reduce the burden of the disease and contribute to the elimination of viral hepatitis. The recent failure of a T-cell vaccine based on the use of viral vectors expressing the HCV non-structural protein sequences to prevent chronic hepatitis C in drug users has pointed out that the induction of neutralizing antibodies (NAbs) will be essential in future vaccine candidates. To induce NAbs, vaccines must contain the main target of this type of antibody, the HCV envelope glycoproteins (E1 and E2). In this review, we summarize the structural regions in E1 and E2 proteins that are targeted by NAbs and how these proteins are presented in the vaccine candidates currently under development.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

Structural insights into hepatitis C virus receptor binding and entry.

Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.

Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice.

SummaryChronic infection with hepatitis C virus (HCV) remains a leading cause of liver‐related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus‐neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N‐glycosylation pattern. Here we report the first evidence of successful production of the full‐length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium‐mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N‐glycan analysis of HCV‐E2 produced in N. benthamiana and mammalian cells indicated host‐specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant‐derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV‐neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost‐effective production platforms for the development of HCV vaccines.

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1

Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV invitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.

Antibody Responses to Immunization With HCV Envelope Glycoproteins as a Baseline for B-Cell–Based Vaccine Development

We investigated antibody responses to hepatitis C virus (HCV) antigens E1 and E2 and the relevance of animal models for vaccine development. We compared antibody responses to vaccination with recombinant E1E2 complex in healthy volunteers, non-human primates (NHPs), and mice. We analyzed 519 serum samples from participants in a phase 1 vaccine trial(ClinicalTrials.gov identifier NCT00500747) and compared them with serum or plasma samples from C57BL/6J mice (n= 28) and rhesus macaques (n= 4) immunized with the same HCV E1E2 antigen. Blood samples were collected at different time points and analyzed for antibody binding, neutralizing activity, and epitope specificity. Monoclonal antibodies from the immunized NHPs were isolated from single plasmablasts and memory B cells, and their immunogenetic properties were characterized. Antibody responses of the volunteers, NHPs, and mice to the non-neutralizing epitopes on the E1 N-terminus and E2 hypervariable region 1 did not differ significantly. Antibodies from volunteers and NHPs that neutralized heterologous strains of HCV primarily interacted with epitopes in the antigen region 3. However, the neutralizing antibodies were not produced in sufficient levels for broad neutralization of diverse HCV isolates. Broadly neutralizing antibodies similar to the human VH1-69 class antibody specific for antigen region 3 were produced in the immunized NHPs. In an analysis of vaccinated volunteers, NHPs, and mice, we found that recombinant E1E2 vaccine antigen induces high-antibody titers that are insufficient to neutralize diverse HCV isolates. Antibodies from volunteers and NHPs bind to the same neutralizing epitopes for virus neutralization. NHPs can therefore be used as a preclinical model to develop HCV vaccines. These findings also provide useful baseline values for development of vaccines designed to induce production of neutralizing antibodies.

Effect of Different Adjuvants on the Longevity and Strength of Humoral and Cellular Immune Responses to the HCV Envelope Glycoproteins.

Infection by Hepatitis C virus (HCV) can lead to liver cirrhosis/hepatocellular carcinoma and remains a major cause of serious disease morbidity and mortality worldwide. However, current treatment regimens remain inaccessible to most patients, particularly in developing countries, and, therefore, the development of a novel vaccine capable of protecting subjects from chronic infection by HCV could greatly reduce the rates of HCV infection, subsequent liver pathogenesis, and in some cases death. Herein, we evaluated two different semi-synthetic archaeosome formulations as an adjuvant to the E1/E2 HCV envelope protein in a murine model and compared antigen-specific humoral (levels of anti-E1/E2 IgG and HCV pseudoparticle neutralization) and cellular responses (numbers of antigen-specific cytokine-producing T cells) to those generated with adjuvant formulations composed of mimetics of commercial adjuvants including a squalene oil-in-water emulsion, aluminum hydroxide/monophosphoryl lipid A (MPLA) and liposome/MPLA/QS-21. In addition, we measured the longevity of these responses, tracking humoral, and cellular responses up to 6 months following vaccination. Overall, we show that the strength and longevity of anti-HCV responses can be influenced by adjuvant selection. In particular, a simple admixed sulfated S-lactosylarchaeol (SLA) archaeosome formulation generated strong levels of HCV neutralizing antibodies and polyfunctional antigen-specific CD4 T cells producing multiple cytokines such as IFN-γ, TNF-α, and IL-2. While liposome/MPLA/QS-21 as adjuvant generated superior cellular responses, the SLA E1/E2 admixed formulation was superior or equivalent to the other tested formulations in all immune parameters tested.

Hepatitis C Virus Structure: Defined by What It Is Not.

Hepatitis C virus (HCV) represents an important and growing public health problem, chronically infecting an estimated 70 million people worldwide. This blood-borne pathogen is generating a new wave of infections in the United States, associated with increasing intravenous drug use over the last decade. In most cases, HCV establishes a chronic infection, sometimes causing cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Although a curative therapy exists, it is extremely expensive and provides no barrier to reinfection; therefore, a vaccine is urgently needed. The virion is asymmetric and heterogeneous with the buoyancy and protein content similar to low-density lipoparticles. Core protein is unstructured, and of the two envelope glycoproteins, E1 and E2, the function of E1 remains enigmatic. E2 is responsible for specifically binding host receptors CD81 and scavenger receptor class B type I (SR-BI). This review will focus on structural progress on HCV virion, core protein, envelope glycoproteins, and specific host receptors.

Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.

Abstract B109: Broadly neutralizing antibodies against HCV use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design

Abstract Hepatocellular carcinoma is the most common type of liver cancer and is responsible for approximately one million deaths each year. In the United States, persistent infection with hepatitis C virus (HCV) is among the leading causes of hepatocellular carcinoma. HCV vaccine efforts are hampered by genetic diversity of HCV envelope glycoproteins E1 and E2. Structural studies of broadly neutralizing antibodies (bNAbs) (e.g., HEPC3 and HEPC74) isolated from individuals who spontaneously cleared HCV infection will facilitate immunogen design to elicit antibodies against multiple HCV variants. However, challenges in expressing HCV glycoproteins previously limited bNAb-HCV structures to complexes with truncated E2 cores.New expression and purification techniques allowed us to produce bNAb complexes with full-length E2 ectodomains for structural studies. We solved crystal structures of HEPC3 and HEPC74 alone and complexed with E2 ectodomains. We measured the binding and neutralization potency of HEPC3/HEPC74-like germline precursors. Despite derivation from different individuals, HEPC3 and HEPC74 use the same germline heavy chain gene segments and possess a limited number of somatic mutations. We describe a shared CDRH3 disulfide motif that exhibits common interactions with a conserved epitope in the front layer of E2. The germline-encoded CDRH3 motif plays a critical role in initial recognition of HCV by naïve B cells. The structures of E2 ectodomain reveal regions (including a target of immunogen design) that were truncated or disordered in E2 core. We also identify candidate immunogens: E2 variants that bind to HEPC3/HEPC74-like germline precursors. These data reveal unusual structural features relevant to the fundamental understanding of antibody-antigen interactions. The identification of common genetic signatures of HCV bNAbs will facilitate lineage-targeted immunogen design to raise bNAbs against the conserved epitope in the E2 front layer. Citation Format: Andrew I. Flyak, Stormy Ruiz, Michelle Colbert, Tiffany Luong, James E. Crowe, Jr., Justin R. Bailey, Pamela J. Bjorkman. Broadly neutralizing antibodies against HCV use a CDRH3 disulfide motif to recognize an E2 glycoprotein site that can be targeted for vaccine design [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B109.

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

BackgroundDespite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.MethodsWe have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.ResultsWe found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10−7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.ConclusionsOur findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale.

Interrogation of Antigen Display on Individual Vaccine Nanoparticles for Achieving Neutralizing Antibody Responses against Hepatitis C Virus.

Elicitation of neutralizing antibody responses against hepatitis C virus (HCV) has been a challenging goal. While the E2 subunit of the HCV envelope glycoprotein complex is a promising target for generating cross-genotype neutralizing antibodies, vaccinations with soluble E2 immunogens generally induce weak neutralizing antibody responses. Here, E2 immunogens (i.e., E2.661 and E2c.661) were loaded into lipid-based nanovaccines and examined for induction of neutralizing antibody responses. Compared with soluble E2 immunogens, E2 nanoparticles elicited 6- to 20-fold higher E2-specific serum IgG titers in mice. Importantly, E2 vaccine nanoparticles analyzed at a single particle level with a flow cytometry-based method revealed interesting dynamics between epitope display on the surfaces of nanoparticles in vitro and induction of neutralizing antibody responses in vivo. E2c.661 nanoparticles that are preferentially bound by a broadly neutralizing antibody, HCV1, in vitro elicit neutralizing antibody responses against both autologous and heterologous HCV virions in vivo. In stark contrast, E2.661 nanoparticles with reduced HCV1-antibody binding in vitro mainly induce autologous neutralizing antibody responses in vivo. These results show that rationale antigen design coupled with interrogation of epitope display on vaccine nanoparticles at a single particle level may aid in vaccine development toward achieving neutralizing antibody responses in vivo.

Role of Hepatitis C Virus Envelope Glycoprotein E1 in Virus Entry and Assembly.

Hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer to constitute viral envelope proteins, which play an essential role in virus entry. E1 does not directly interact with host receptors, and its functions in viral entry are exerted mostly through its interaction with E2 that directly binds the receptors. HCV enters the host cell via receptor-mediated endocytosis during which the fusion of viral and host endosomal membranes occurs to release viral genome to cytoplasm. A putative fusion peptide in E1 has been proposed to participate in membrane fusion, but its exact role and underlying molecular mechanisms remain to be deciphered. Recently solved crystal structures of the E2 ectodomains and N-terminal of E1 fail to reveal a classical fusion-like structure in HCV envelope glycoproteins. In addition, accumulating evidence suggests that E1 also plays an important role in virus assembly. In this mini-review, we summarize current knowledge on HCV E1 including its structure and biological functions in virus entry, fusion, and assembly, which may provide clues for developing HCV vaccines and more effective antivirals.

Recombinant Flag-tagged E1E2 glycoproteins from three hepatitis C virus genotypes are biologically functional and elicit cross-reactive neutralizing antibodies in mice

Hepatitis C virus (HCV) is a globally disseminated human pathogen for which no vaccine is currently available. HCV is highly diverse genetically and can be classified into 7 genotypes and multiple sub-types. Due to this antigenic variation, the induction of cross-reactive and at the same time neutralizing antibodies is a challenge in vaccine production. Here we report the analysis of immunogenicity of recombinant HCV envelope glycoproteins from genotypes 1a, 1b and 2a, with a Flag tag inserted in the hypervariable region 1 of E2. This modification did not affect protein expression or conformation or its capacity to bind the crucial virus entry factor, CD81. Importantly, in immunogenicity studies on mice, the purified E2-Flag mutants elicited high-titer, cross-reactive antibodies that were able to neutralize HCV infectious particles from two genotypes tested (1a and 2a). These findings indicate that E1E2-Flag envelope glycoproteins could be important immunogen candidates for vaccine aiming to induce broad HCV-neutralizing responses.

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP, a STING activator or archaeosomes.

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice. Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4+ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.

An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry.

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies in vivo and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design. A high-resolution structural definition of the intact HCV envelope glycoprotein complex containing E1 and E2 remains to be elucidated, while crystallographic structures of a recombinant E2 ectodomain failed to resolve HVR1, HVR2, and a major neutralization determinant adjacent to HVR1. To obtain further information on E2, we characterized the role of all three variable regions in E2 ectodomain folding and function in the context of a recombinant ectodomain fragment (rE2). We report that removal of the variable regions accelerates binding to the major host cell receptor CD81 and that simultaneous deletion of HVR2 and the igVR is required to maintain wild-type CD81-binding characteristics. The removal of the variable regions also rescued the ability of rE2 to form a functional homodimer. We propose that the rE2 core provides novel insights into the role of the variable motifs in the higher-order assembly of the E2 ectodomain and may have implications for E1E2 structure on the virion surface. IMPORTANCE Hepatitis C virus (HCV) infection affects ∼2% of the population globally, and no vaccine is available. HCV is a highly variable virus, and understanding the presentation of key antigenic sites at the virion surface is important for the design of a universal vaccine. This study investigates the role of three surface-exposed variable regions in E2 glycoprotein folding and function in the context of a recombinant soluble ectodomain. Our data demonstrate the variable motifs modulate binding of the E2 ectodomain to the major host cell receptor CD81 and have an impact on the formation of an E2 homodimer with high-affinity binding to CD81.

Hepatitis C Virus E2 Protein Induces Upregulation of IL-8 Pathways and Production of Heat Shock Proteins in Human Thyroid Cells.

Thyroiditis is one of the most common extrahepatic manifestations of hepatitis C virus (HCV) infection. By binding to surface cell receptor CD81, HCV envelope glycoprotein E2 mediates entry of HCV into cells. Studies have shown that different viral proteins may individually induce host responses to infection. We hypothesized that HCV E2 protein binding to CD81 expressed on thyroid cells activates a cascade of inflammatory responses that can trigger autoimmune thyroiditis in susceptible individuals. Human thyroid cell lines ML-1 and human thyrocytes in primary cell culture were treated with HCV recombinant E2 protein. The expression of major proinflammatory cytokines was measured at the messenger RNA and protein levels. Next-generation transcriptome analysis was used to identify early changes in gene expression in thyroid cells induced by E2. HCV envelope protein E2 induced strong inflammatory responses in human thyrocytes, resulting in production of interleukin (IL)-8, IL-6, and tumor necrosis factor-α. Furthermore, the E2 protein induced production of several heat shock proteins including HSP60, HSP70p12A, and HSP10, in human primary thyrocytes. In thyroid cell line ML-1, RNA sequencing identified upregulation of molecules involved in innate immune pathways with high levels of proinflammatory cytokines and chemokines and increased expression of costimulatory molecules, specifically CD40, known to be a major thyroid autoimmunity gene. Our data support a key role for HCV envelope protein E2 in triggering thyroid autoimmunity through activation of cytokine pathways by bystander mechanisms.

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.