Epstein-Barr virus gp42 antibodies reveal sites of vulnerability for receptor binding and fusion to B cells.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with B cell lymphomas. EBV glycoprotein 42 (gp42) binds HLA class II and activates membrane fusion with B cells. We isolated gp42-specific monoclonal antibodies (mAbs), A10 and 4C12, which use distinct mechanisms to neutralize virus infection. mAb A10 was more potent than the only known neutralizing gp42 mAb, F-2-1, in neutralizing EBV infection and blocking binding to HLA class II. mAb 4C12 was similar to mAb A10 in inhibiting glycoprotein-mediated B cell fusion but did not block receptor binding, and it was less effective in neutralizing infection. Crystallographic structures of gH/gL/gp42/A10 and gp42/4C12 complexes revealed two distinct sites of vulnerability on gp42 for receptor binding and B cell fusion. Passive transfer of mAb A10 into humanized mice conferred nearly 100% protection from viremia and EBV lymphomas after EBV challenge. These findings identify vulnerable sites on EBV that may facilitate therapeutics and vaccines.

Hepatitis C virus E1 recruits high-density lipoprotein to support infectivity and evade antibody recognition.

Hepatitis C virus (HCV) is a member of the Flaviviridae family; however, unlike other family members, the HCV virion has an unusually high lipid content. HCV has two envelope glycoproteins, E1 and E2. E2 contributes to receptor binding, cell membrane attachment, and immune evasion. In contrast, the functions of E1 are poorly characterized due, in part, to challenges in producing the protein. This manuscript describes the expression and purification of a soluble E1 ectodomain (eE1) that is recognized by conformational, human monoclonal antibodies. eE1 forms a complex with apolipoproteins AI and AII, cholesterol, and phospholipids by recruiting high-density lipoprotein (HDL) from the extracellular media. We show that HDL binding is a function specific to eE1 and HDL hinders recognition of E1 by a neutralizing monoclonal antibody. Either low-density lipoprotein or HDL increases the production and infectivity of cell culture-produced HCV, but E1 preferentially selects HDL, influencing both viral life cycle and antibody evasion.IMPORTANCEHepatitis C virus (HCV) infection is a significant burden on human health, but vaccine candidates have yet to provide broad protection against this infection. We have developed a method to produce high quantities of soluble E1 or E2, the viral proteins located on the surface of HCV. HCV has an unusually high lipid content due to the recruitment of apolipoproteins. We found that E1 (and not E2) preferentially recruits host high-density lipoprotein (HDL) extracellularly. This recruitment of HDL by E1 prevents binding of E1 by a neutralizing antibody and furthermore prevents antibody-mediated neutralization of the virus. By comparison, low-density lipoprotein does not protect the virus from antibody-mediated neutralization. Our findings provide mechanistic insight into apolipoprotein recruitment, which may be critical for vaccine development.

Concerted synergy between viral-specific IgG and CD8 + T cells is critical for clearance of an HCV-related rodent hepacivirus.

BACKGROUND AND AIMS: Evidence assessing the role of B cells and their antibodies, or lack thereof, in the spontaneous resolution of acute HCV infection is conflicting. Utilization of a strictly hepatotropic, HCV-related rodent hepacivirus (RHV) model circumvents many of the challenges facing the field in characterizing the immunological correlates of dichotomous infection outcomes. This study seeks to elucidate the importance of B cells in the clearance of acute RHV infection. APPROACH AND RESULTS: µMT mice were infected i.v. with RHV and found to develop chronic infection for over a year. Wild-type (WT) mice depleted of B cells also exhibited persistent viremia that resolved only upon B cell resurgence. The persistent infection developed by B1-8i and AID cre/cre mice revealed that antigen-specific, class-switched B cells or their antibodies were crucial for viral resolution. Virus-specific CD8 + and CD4 + T cells were characterized in these mice using newly developed major histocompatibility complex class I and II tetramers and ex vivo peptide stimulation. Immunoglobulin G (IgG) was purified from the serum of RHV- or lymphocytic choriomeningitis virus Armstrong-infected mice after viral clearance and passively transferred to AID cre/cre recipients, revealing viral clearance only in αRHV IgG recipients. Further, the transfer of αRHV IgG into B cell-depleted recipients also induced viral resolution. This ability of RHV-specific IgG to induce viral clearance was found to require the concomitant presence of CD8 + T cells. CONCLUSIONS: Our findings demonstrate a cooperative interdependence between immunoglobulins and the T cell compartment that is required for RHV resolution. Thus, HCV vaccine regimens should aim to simultaneously elicit robust HCV-specific antibody and T cell responses for optimal protective efficacy.

Learning more about hepatitis E virus.

A domain in the ORF1 polyprotein of the hepatitis E virus that was previously thought to be a protease is actually a zinc-binding domain.

Regions of hepatitis C virus E2 required for membrane association.

Hepatitis C virus (HCV) uses a hybrid entry mechanism. Current structural data suggest that upon exposure to low pH and Cluster of Differentiation 81 (CD81), the amino terminus of envelope glycoprotein E2 becomes ordered and releases an internal loop with two invariant aromatic residues into the host membrane. Here, we present the structure of an amino-terminally truncated E2 with the membrane binding loop in a bent conformation and the aromatic side chains sequestered. Comparison with three previously reported E2 structures with the same Fab indicates that this internal loop is flexible, and that local context influences the exposure of hydrophobic residues. Biochemical assays show that the amino-terminally truncated E2 lacks the baseline membrane-binding capacity of the E2 ectodomain. Thus, the amino terminal region is a critical determinant for both CD81 and membrane interaction. These results provide new insights into the HCV entry mechanism.

HCV neutralization goes elite.

A vaccine remains the most promising option to eradicate HCV. In this issue of Immunity, Weber et al. identified HCV elite neutralizers, isolated exceptionally potent and broad VH1-69 CD81-binding site neutralizing antibodies that used a shared mode of antigen recognition, and developed a computational approach that predicted mutations relevant to gain-of-function for this bnAb class, which can inform immunogen designs.

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.

Early T follicular helper cell activity accelerates hepatitis C virus-specific B cell expansion.

Early appearance of neutralizing antibodies during acute hepatitis C virus (HCV) infection is associated with spontaneous viral clearance. However, the longitudinal changes in antigen-specific memory B cell (MBCs) associated with divergent HCV infection outcomes remain undefined. We characterized longitudinal changes in E2 glycoprotein-specific MBCs from subjects who either spontaneously resolved acute HCV infection or progressed to chronic infection, using single-cell RNA-seq and functional assays. HCV-specific antibodies in plasma from chronically infected subjects recognized multiple E2 genotypes, while those from spontaneous resolvers exhibited variable cross-reactivity to heterotypic E2. E2-specific MBCs from spontaneous resolvers peaked early after infection (4-6 months), following expansion of activated circulating T follicular helper cells (cTfh) expressing interleukin 21. In contrast, E2-specific MBCs from chronically infected subjects, enriched in VH1-69, expanded during persistent infection (> 1 year), in the absence of significantly activated cTfh expansion. Early E2-specific MBCs from spontaneous resolvers produced monoclonal antibodies (mAbs) with fewer somatic hypermutations and lower E2 binding but similar neutralization as mAbs from late E2-specific MBCs of chronically infected subjects. These findings indicate that early cTfh activity accelerates expansion of E2-specific MBCs during acute infection, which might contribute to spontaneous clearance of HCV.

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.

Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue.

Vatreptacog alfa (VA), a recombinant activated human factor VII (rFVIIa) variant with 3 amino acid substitutions, was developed to provide increased procoagulant activity in hemophilia patients with inhibitors to factor VIII or factor IX. In phase 3 clinical trials, changes introduced during the bioengineering of VA resulted in the development of undesired anti-drug antibodies in some patients, leading to the termination of a potentially promising therapeutic protein product. Here, we use preclinical biomarkers associated with clinical immunogenicity to validate our deimmunization strategy applied to this bioengineered rFVIIa analog. The reengineered rFVIIa analog variants retained increased intrinsic thrombin generation activity but did not elicit T-cell responses in peripheral blood mononuclear cells isolated from 50 HLA typed subjects representing the human population. Our algorithm, rational immunogenicity determination, offers a broadly applicable deimmunizing strategy for bioengineered proteins.

Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants.

BACKGROUND & AIMS: Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. METHODS: We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. RESULTS: Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. CONCLUSIONS: Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies. LAY SUMMARY: Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.

Overcoming Challenges of Hepatitis C Virus Envelope Glycoprotein Production in Mammalian Cells.

Posttranslational modifications (PTMs) are often required for proper folding and physiological function of proteins, including the envelope glycoproteins 1 and 2 (E1 and E2) of hepatitis C virus (HCV). Commonly used expression systems such as bacteria, yeast, and baculovirus produce soluble HCV E1 and E2 at very low yields, as the cellular environment and molecular machinery necessary for PTMs may be suboptimal or missing. Here, we describe an expression system for HCV E2 ectodomain (eE2) with 11 N-linked glycans and eight disulfide bonds, which combines lentivirus transduction of mammalian cells and a continuous growth, adherent cell bioreactor. It is environmentally friendly, as well as cost- and time-efficient compared to other methods of recombinant protein expression in mammalian systems with final yields of eE2 approaching 60 mg/L of cell culture supernatant. eE2 produced by this system is amenable to a variety of biophysical studies, including structural determination by X-ray crystallography. Considering the ease of use and flexibility, this method can be applied to express an array of difficult target proteins in a variety of mammalian cell lines.

HDX-MS reveals dysregulated checkpoints that compromise discrimination against self RNA during RIG-I mediated autoimmunity.

Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.

RIG-I Uses an ATPase-Powered Translocation-Throttling Mechanism for Kinetic Proofreading of RNAs and Oligomerization.

RIG-I has a remarkable ability to specifically select viral 5'ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven "kinetic proofreading" mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off rates. ATP binding facilitates dsRNA engagement but, interestingly, makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merten syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5'ppp dsRNA but, more importantly, drives RIG-I oligomerization through translocation, which we show to be regulated by helicase motif IVa. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5'ppp end "throttles" translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.

Hepatitis C Virus Envelope Glycoproteins: A Balancing Act of Order and Disorder.

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.

CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells.

Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer.

Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.

Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2.

The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like ß-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a ß-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design.IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development.

Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening.

Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.