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A hepatitis C virus (HCV) vaccine is a critical yet unfulfilled step in addressing the global disease burden of HCV. While decades of research have led to numerous clinical and pre-clinical vaccine candidates, these efforts have been hindered by factors including HCV antigenic variability and immune evasion. Structure-based and rational vaccine design approaches have capitalized on insights regarding the immune response to HCV and the structures of antibody-bound envelope glycoproteins. Despite successes with other viruses, designing an immunogen based on HCV glycoproteins that can elicit broadly protective immunity against HCV infection is an ongoing challenge. Here, we describe HCV vaccine design approaches where immunogens were selected and optimized through analysis of available structures, identification of conserved epitopes targeted by neutralizing antibodies, or both. Several designs have elicited immune responses against HCV in vivo, revealing correlates of HCV antigen immunogenicity and breadth of induced responses. Recent studies have elucidated the functional, dynamic and immunological features of key regions of the viral envelope glycoproteins, which can inform next-generation immunogen design efforts. These insights and design strategies represent promising pathways to HCV vaccine development, which can be further informed by successful immunogen designs generated for other viruses.

Hepatitis C virus (HCV) is a major global health concern, and though therapeutic options have improved, no vaccine is available despite decades of research. As HCV can rapidly mutate to evade the immune response, an effective HCV vaccine must rely on identification and characterization of sites critical for broad immune protection and viral neutralization. This knowledge depends on structural and mechanistic insights of the E1 and E2 envelope glycoproteins, which assemble as a heterodimer on the surface of the virion, engage coreceptors during host cell entry, and are the primary targets of antibodies. Due to the challenges in determining experimental structures, structural information on E1 and E2 and their interaction is relatively limited, providing opportunities to model the structures, interactions, and dynamics of these proteins. This review highlights efforts to model the E2 glycoprotein structure, the assembly of the functional E1E2 heterodimer, the structure and binding of human coreceptors, and recognition by key neutralizing antibodies. We also discuss a comparison of recently described models of full E1E2 heterodimer structures, a simulation of the dynamics of key epitope sites, and modeling glycosylation. These modeling efforts provide useful mechanistic hypotheses for further experimental studies of HCV envelope assembly, recognition, and viral fitness, and underscore the benefit of combining experimental and computational modeling approaches to reveal new insights. Additionally, computational design approaches have produced promising candidates for epitope-based vaccine immunogens that specifically target key epitopes, providing a possible avenue to optimize HCV vaccines versus using native glycoproteins. Advancing knowledge of HCV envelope structure and immune recognition is highly applicable toward the development of an effective vaccine for HCV and can provide lessons and insights relevant to modeling and characterizing other viruses.

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. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a major global health concern, and though therapeutic options have improved, no vaccine is available despite decades of research. As HCV can rapidly mutate to evade the immune response, an effective HCV vaccine must rely on identification and characterization of sites critical for broad immune protection and viral neutralization. This knowledge depends on structural and mechanistic insights of the E1 and E2 envelope glycoproteins, which assemble as a heterodimer on the surface of the virion, engage coreceptors during host cell entry, and are the primary targets of antibodies. Due to the challenges in determining experimental structures, structural information on E1 and E2 and their interaction is relatively limited, providing opportunities to model the structures, interactions, and dynamics of these proteins. This review highlights efforts to model the E2 glycoprotein structure, the assembly of the functional E1E2 heterodimer, the structure and binding of human coreceptors, and recognition by key neutralizing antibodies. We also discuss a comparison of recently described models of full E1E2 heterodimer structures, a simulation of the dynamics of key epitope sites, and modeling glycosylation. These modeling efforts provide useful mechanistic hypotheses for further experimental studies of HCV envelope assembly, recognition, and viral fitness, and underscore the benefit of combining experimental and computational modeling approaches to reveal new insights. Additionally, computational design approaches have produced promising candidates for epitope-based vaccine immunogens that specifically target key epitopes, providing a possible avenue to optimize HCV vaccines versus using native glycoproteins. Advancing knowledge of HCV envelope structure and immune recognition is highly applicable toward the development of an effective vaccine for HCV and can provide lessons and insights relevant to modeling and characterizing other viruses. [ABSTRACT FROM AUTHOR]

Injection drug users uninfected by hepatitis C virus ( HCV) despite likely repeated exposure through high-risk behaviour are well documented. Factors preventing infection in these individuals are incompletely understood. Here, we looked for anti- HCV-envelope antibody responses in a cohort of repeatedly exposed but uninfected subjects. Forty-two hepatitis C diagnostic antibody- and RNA-negative injection drug users at high risk of exposure were studied and findings compared to healthy controls and cases with chronic HCV infection. Purified IgGs from sera were tested by ELISA for binding to genotype 1a and 3a envelope glycoproteins E1E2 with further testing for IgG and IgM reactivity against soluble E2. Virus-neutralizing activity was assessed using an HCV pseudoparticle system. Uninfected subjects demonstrated significantly greater IgG and IgM reactivities to envelope glycoproteins than healthy controls with IgG from 6 individuals additionally showing significant neutralization. This study is the first to describe humoral immunological responses targeting the HCV envelope, important for viral neutralization, in exposed uninfected individuals. A subset of these cases also had evidence of viral neutralization via anti-envelope antibodies. In addition to confirming viral exposure, the presence of specific anti-envelope antibodies may be a factor that helps these individuals resist HCV infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] A scFv from the anti-HCV E1E2 D32.10 mAb was successfully expressed. [•] This scFv consisted of functional monomers as shown by MALLS methodology. [•] The scFv D32.10 mimicked the antibody in binding to patient-derived HCV particles. [•] The scFv D32.10 competed with the parental IgG for binding to antigen. [•] This scFv is the basis of a drug discovery approach for tackling HCV reinfection. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hepatitis 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. Methods: HCV 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. Results: There 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

Abstract: Neutralizing antibody responses elicited during infection generally confer protection from infection. Hepatitis C virus (HCV) encodes two glycoproteins E1 and E2 that are essential for virus entry and are the major target for neutralizing antibodies. To assess whether both glycoproteins are required for the generation of a neutralizing antibody response, rodents were immunized with a series of glycoproteins comprising full length and truncated versions. Guinea pigs immunized with HCV-1 genotype 1a E1E2p7, E1E2 or E2 generated high titer anti-glycoprotein antibody responses that neutralized the infectivity of HCVpp and HCVcc expressing gps of the same genotype as the immunizing antigen. Less potent neutralization of viruses bearing the genotype 2 strain J6 gps was observed. In contrast, immunized mice demonstrated reduced anti-gp antibody responses, consistent with their minimal neutralizing activity. Immunization with E2 alone was sufficient to induce a high titer response that neutralized HCV pseudoparticles (HCVpp) bearing diverse glycoproteins and cell culture grown HCV (HCVcc). The neutralization titer was reduced 3-fold by the presence of lipoproteins in human sera. Cross-competition of the guinea pig anti-E1E2 immune sera with a panel of epitope mapped anti-E2 monoclonal antibodies for binding E2 identified a series of epitopes within the N-terminal domain that may be immunogenic in the immunized rodents. These data demonstrate that recombinant E2 and E1E2 can induce polyclonal antibody responses with cross-reactive neutralizing activity, supporting the future development of prophylactic and therapeutic vaccines. [Copyright &y& Elsevier]

A hepatitis C virus (HCV) vaccine is a critical yet unfulfilled step in addressing the global disease burden of HCV. While decades of research have led to numerous clinical and pre-clinical vaccine candidates, these efforts have been hindered by factors including HCV antigenic variability and immune evasion. Structure-based and rational vaccine design approaches have capitalized on insights regarding the immune response to HCV and the structures of antibody-bound envelope glycoproteins. Despite successes with other viruses, designing an immunogen based on HCV glycoproteins that can elicit broadly protective immunity against HCV infection is an ongoing challenge. Here, we describe HCV vaccine design approaches where immunogens were selected and optimized through analysis of available structures, identification of conserved epitopes targeted by neutralizing antibodies, or both. Several designs have elicited immune responses against HCV in vivo, revealing correlates of HCV antigen immunogenicity and breadth of induced responses. Recent studies have elucidated the functional, dynamic and immunological features of key regions of the viral envelope glycoproteins, which can inform next-generation immunogen design efforts. These insights and design strategies represent promising pathways to HCV vaccine development, which can be further informed by successful immunogen designs generated for other viruses.

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., Competing Interests: Declaration of interests The authors declare no conflict of interest. M.H., M.E.F, A.G, and B.J.D. are current or former employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.