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Background & Aims: In order to design an effective vaccine against hepatitis C virus (HCV) infection, it is necessary to understand immune protection. A number of broadly reactive neutralizing antibodies have been isolated from B cells of HCV-infected patients. However, it remains unclear whether B cells producing such antibodies contribute to HCV clearance and long-term immune protection against HCV., Methods: We analysed the B cell repertoire of 13 injecting drug users from the Amsterdam Cohort Study, who were followed up for a median of 17.5 years after primary infection. Individuals were classified into 2 groups based on the outcome of HCV infection: 5 who became chronically infected either after primary infection or after reinfection, and 8 who were HCV RNA negative following spontaneous clearance of ≥1 HCV infection(s). From each individual, 10,000 CD27+IgG+B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire., Results: Using a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of 1 or multiple infections (p = 0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in 4 individuals who were HCV RNA negative following spontaneous clearance of 1 or multiple infections. Interestingly, the cross-genotype antibodies were mainly antigenic region 3 (AR3)-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, 3 individuals developed antibodies recognizing antigenic region 4, of which 1 monoclonal antibody showed cross-neutralizing capacity., Conclusions: Together, these data suggest that a strong B cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contributes to HCV clearance and long-term immune protection against HCV., Lay Summary: Although effective treatments against hepatitis C virus (HCV) are available, 500,000 people die from liver disease caused by HCV each year and approximately 1.75 million people are newly infected. This could be prevented by a vaccine. To design a vaccine against HCV, more insight into the role of antibodies in the protection against HCV infection is needed. In a cohort of injecting drug users, we found that antibodies interfering with virus cell entry, and recognizing multiple HCV genotypes, conferred long-term protection against chronic HCV infection., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) pseudoparticles (HCVpp) are generated by cotransfection of HCV envelope (E1 and E2) genes along with a retroviral packaging/reporter construct into HEK293T cells. Enveloped particles bearing HCV E1E2 proteins on their surface are released through a retroviral budding process into the supernatant. Viral E1E2 glycoproteins facilitate a single round of receptor-mediated entry of HCVpp into hepatoma cells, which can be quantified by reporter gene expression. These HCVpp have been employed to study mechanisms of HCV entry into hepatoma cells, as well as HCV neutralization by immune sera or HCV-specific monoclonal antibodies.

Global eradication of hepatitis C virus (HCV) infection will require an efficacious vaccine capable of eliciting protective immunity against genetically diverse HCV strains. Natural spontaneous resolution of HCV infection is associated with production of broadly-neutralizing antibodies targeting the HCV glycoproteins E1 and E2. As such, production of cross-neutralizing antibodies is an important endpoint for experimental vaccine trials. Varying success generating cross-neutralizing antibodies has been achieved with immunogens derived from naturally-occurring HCV strains. In this study the challenge of minimising the genetic diversity between the vaccine strain and circulating HCV isolates was addressed. Two novel synthetic E2 glycoprotein immunogens (NotC1 and NotC2) were derived from consensus nucleotide sequences deduced from samples of circulating genotype 1 HCV strains. These two synthetic sequences differed in their relative positions in the overall genotype 1a/1b phylogeny. Expression of these constructs in Drosophila melanogaster S2 cells resulted in high yields of correctly-folded, monomeric E2 protein, which were recognised by broadly neutralizing monoclonal antibodies. Immunization of guinea pigs with either of these consensus immunogens, or a comparable protein representing a circulating genotype 1a strain resulted in high titres of cross-reactive anti-E2 antibodies. All immunogens generated antibodies capable of neutralizing the H77 strain, but NotC1 elicited antibodies that more potently neutralized virus entry. These vaccine-induced antibodies neutralized some viruses representing genotype 1, but not strains representing genotype 2 or genotype 3. Thus, while this approach to vaccine design resulted in correctly folded, immunogenic protein, cross-neutralizing epitopes were not preferentially targeted by the host immune response generated by this immunogen. Greater immunofocussing of vaccines to common epitopes is necessary to successfully elicit broadly neutralizing antibodies., (Copyright © 2018. Published by Elsevier B.V.)

Unlabelled: Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies., Importance: Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design., (Copyright © 2016 Urbanowicz et al.)

Unlabelled: Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses., Importance: Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Hepatitis C virus (HCV) is a major cause of liver disease and hepatocellular carcinoma worldwide. HCV establishes a chronic infection in the majority of cases. However, some individuals clear the virus, demonstrating a protective role for the host immune response. Although new all-oral drug combinations may soon replace traditional ribavirin-interferon therapy, the emerging drug cocktails will be expensive and associated with side-effects and resistance, making a global vaccine an urgent priority. T cells are widely accepted to play an essential role in clearing acute HCV infection, whereas the role antibodies play in resolution and disease pathogenesis is less well understood. Recent studies have provided an insight into viral neutralizing determinants and the protective role of antibodies during infection. This review provides a historical perspective of the role neutralizing antibodies play in HCV infection and discusses the therapeutic benefits of antibody-based therapies. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication.", (Copyright © 2014. Published by Elsevier B.V.)

Chronic hepatitis C virus (HCV) infection can persist even in the presence of a broadly neutralizing antibody response. Various mechanisms that underpin viral persistence have been proposed, and one of the most recently proposed mechanisms is the presence of interfering antibodies that negate neutralizing responses. Specifically, it has been proposed that antibodies targeting broadly neutralizing epitopes located within a region of E2 encompassing residues 412 to 423 can be inhibited by nonneutralizing antibodies binding to a less conserved region encompassing residues 434 to 446. To investigate this phenomenon, we characterized the neutralizing and inhibitory effects of human-derived affinity-purified immunoglobulin fractions and murine monoclonal antibodies and show that antibodies to both regions neutralize HCV pseudoparticle (HCVpp) and cell culture-infectious virus (HCVcc) infection albeit with different breadths and potencies. Epitope mapping revealed the presence of overlapping but distinct epitopes in both regions, which may explain the observed differences in neutralizing phenotypes. Crucially, we failed to demonstrate any inhibition between these two groups of antibodies, suggesting that interference by nonneutralizing antibodies, at least for the region encompassing residues 434 to 446, does not provide a mechanism for HCV persistence in chronically infected individuals.

Hepatitis C virus (HCV) is a blood-borne virus estimated to infect around 170 million people worldwide and is, therefore, a major disease burden. In some individuals the virus is spontaneously cleared during the acute phase of infection, whilst in others a persistent infection ensues. Of those persistently infected, severe liver diseases such as cirrhosis and primary liver cancer may develop, although many individuals remain asymptomatic. A range of factors shape the course of HCV infection, not least host genetic polymorphisms and host immunity. A number of studies have shown that neutralizing antibodies (nAb) arise during HCV infection, but that these antibodies differ in their breadth and mechanism of neutralization. Recent studies, using both mAbs and polyclonal sera, have provided an insight into neutralizing determinants and the likely protective role of antibodies during infection. This understanding has helped to shape our knowledge of the overall structure of the HCV envelope glycoproteins--the natural target for nAb. Most nAb identified to date target receptor-binding sites within the envelope glycoprotein E2. However, there is some evidence that other viral epitopes may be targets for antibody neutralization, suggesting the need to broaden the search for neutralization epitopes beyond E2. This review provides a comprehensive overview of our current understanding of the role played by nAb in HCV infection and disease outcome and explores the limitations in the study systems currently used. In addition, we briefly discuss the potential therapeutic benefits of nAb and efforts to develop nAb-based therapies.

Infection with Hepatitis C Virus (HCV) causes chronic disease in approximately 80% of cases, resulting in chronic inflammation and cirrhosis. Current treatments are not completely effective, and a vaccine has yet to be developed. Spontaneous resolution of infection is associated with effective host adaptive immunity to HCV, including production of both HCV-specific T cells and neutralizing antibodies. However, the supporting role of soluble innate factors in protection against HCV is less well understood. The innate immune system provides an immediate line of defense against infections, triggering inflammation and playing a critical role in activating adaptive immunity. Innate immunity comprises both cellular and humoral components, the humoral arm consisting of pattern recognition molecules such as complement C1q, collectins and ficolins. These molecules activate the complement cascade, neutralize pathogens, and recruit antigen presenting cells. Here we review the current understanding of anti-viral components of the humoral innate immune system that play a similar role to antibodies, describing their role in immunity to HCV and their potential contribution to HCV pathogenesis.

Neutralizing antibodies have a role in controlling hepatitis C virus (HCV) infection. A successful vaccine will need to elicit potently neutralizing antibodies that are capable of preventing the infection of genetically diverse viral isolates. However, the specificity of the neutralizing antibody response in natural HCV infection still is poorly understood. To address this, we examined the reactivity of polyclonal antibodies isolated from chronic HCV infection to the diverse patient-isolated HCV envelope glycoproteins E1 and E2 (E1E2), and we also examined the potential to neutralize the entry of pseudoparticles bearing these diverse E1E2 proteins. The genetic type of the infection was found to determine the pattern of the antibody recognition of these E1E2 proteins, with the greatest reactivity to homologous E1E2 proteins. This relationship was strongest when the component of the antibody response directed only to linear epitopes was analyzed. In contrast, the neutralization serotype did not correlate with genotype. Instead, serum-derived antibodies displayed a range of neutralization breadth and potency, while different E1E2 glycoproteins displayed different sensitivities to neutralization, such that these could be divided broadly into neutralization-sensitive and -resistant phenotypes. An important additional observation was that entry mediated by some E1E2 proteins was enhanced in the presence of some of the polyclonal antibody fractions isolated during chronic infection. These data highlight the need to use diverse E1E2 isolates, which represent extremes of neutralization sensitivity, when screening antibodies for therapeutic potential and for testing antibodies generated following immunization as part of vaccine development.

Development of a successful hepatitis C virus (HCV) vaccine requires the definition of neutralization epitopes that are conserved among different HCV genotypes. Five human monoclonal antibodies (HMAbs) are described that cross-compete with other antibodies to a cluster of overlapping epitopes, previously designated domain B. Each HMAb broadly neutralizes retroviral pseudotype particles expressing HCV E1 and E2 glycoproteins, as well as the infectious chimeric genotype 1a and genotype 2a viruses. Alanine substitutions of residues within a region of E2 involved in binding to CD81 showed that critical E2 contact residues involved in the binding of representative antibodies are identical to those involved in the binding of E2 to CD81.

The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1-6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.

Liver failure associated with hepatitis C virus (HCV) accounts for a substantial portion of liver transplantation. Although current therapy helps some patients with chronic HCV infection, adverse side effects and a high relapse rate are major problems. These problems are compounded in liver transplant recipients as reinfection occurs shortly after transplantation. One approach to control reinfection is the combined use of specific antivirals together with HCV-specific antibodies. Indeed, a number of human and mouse monoclonal antibodies to conformational and linear epitopes on HCV envelope proteins are potential candidates, since they have high virus neutralization potency and are directed to epitopes conserved across diverse HCV genotypes. However, a greater understanding of the factors contributing to virus escape and the role of lipoproteins in masking virion surface domains involved in virus entry will be required to help define those protective determinants most likely to give broad protection. An approach to immune escape is potentially caused by viral infection of immune cells leading to the induction hypermutation of the immunoglobulin gene in B cells. These effects may contribute to HCV persistence and B cell lymphoproliferative diseases.

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and there is a pressing need for the development of a preventative vaccine as well as new treatments. It was recently demonstrated that the mouse monoclonal antibody (mAb) AP33 potently neutralizes infectivity of HCV pseudoparticles (HCVpp) carrying E1E2 envelopes representative of all of the major genotypes of HCV. This study determined the prevalence of human serum antibodies reactive to the region of HCV E2 recognized by AP33. Antibodies recognizing this region were present in less than 2.5 % of sera obtained from individuals with chronic HCV infection. A similar prevalence was found in a smaller cohort of individuals who had experienced an acute infection, suggesting that AP33-like antibodies do not play a major role in natural clearance of HCV infection. Sera exhibited different patterns of reactivity to a panel of peptides representing circulating variants, highlighting the presence of distinct epitopes in this region. Only two sera contained antibodies that could recognize a specific AP33-reactive peptide mimotope. AP33-like antibodies made a measurable contribution to the ability of these sera to inhibit E2-CD81 interaction, but not to the overall neutralization of cell entry. Together, these data show that antibodies to the AP33 epitope are not commonly generated during natural infection and that generation of such antibodies via vaccination may require modified immunogens to focus the generation of specific antibodies. Importantly, individuals harbouring AP33-like antibodies are an important potential source of human mAbs for future therapeutic development.

One way to dissect the antibody response to an invading microorganism is to clone the antibody repertoire from immune donors and subsequently characterize the specific antibodies. Recently, methodological advances have allowed investigations of neutralizing antibodies against hepatitis C virus (HCV) in vitro. We have investigated three human mAbs, previously isolated from an individual infected with HCV of genotype 2b, that are known to cross-react in a binding assay to the envelope E2 protein of genotypes 1a and 1b. We now report that two of them have a neutralizing activity with a breadth not previously observed. Indeed, mAbs 1:7 and A8 recognized E2 from all of the six major genotypes, and they neutralized retroviral pseudoparticles [HCV pseudoparticles (HCVpp)] carrying genetically equally diverse HCV envelope glycoproteins. Importantly, these antibodies were also able to neutralize the cell culture infectious HCV clone JFH-1 in vitro, with IC(50) values of 60 ng/ml and 560 ng/ml, respectively. The conformational epitopes of these two broadly reactive antibodies were overlapping yet distinct and involved amino acid residues in the 523-535 region of E2, known to be important for the E2-CD81 interaction. The third antibody clone, representing a dominant population in the initial screen for these antibodies, was less broadly reactive and was unable to neutralize the genotype 2a infectious clone JFH-1. Our results confirm at the clonal level that broadly neutralizing human anti-HCV antibodies can be elicited and that the region amino acids 523-535 of the HCV envelope glycoprotein E2 carries neutralizing epitopes conserved across all genotypes.

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 mug/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.