Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.     

Cooperativity in Virus Neutralization by Human Monoclonal Antibodies to Two Adjacent Regions Located at the Amino Terminus of Hepatitis C Virus E2 Glycoprotein

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33 HMAbs and HMAbs to aa 434 to 446. When HMAbs to aa 434 to 446, which mediated neutralization, were combined with neutralizing HC33 HMAbs, biphasic patterns in neutralization were observed. A modest degree of antagonism was observed at lower concentrations, and a modest degree of synergism was observed at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus neutralization and that the antibodies to aa 412 to 423 and aa 434 to 446 do not hinder their respective virus-neutralizing activities.     

Determination of the Human Antibody Response to the Neutralization Epitopes Encompassing Amino Acids 313–327 and 432–443 of Hepatitis C Virus E1E2 Glycoproteins

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regionsencompassing amino acids 313–327 and 432–443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of whichwere from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes.The remaining 126 samples were isolated from patients who had spontaneously clearedHCV infection.In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313–327 and 432–443was 24.29%(51 of 210) and4.76%(10 of 210),respectively. In thespontaneousclearance group (SC group),the prevalence was 0.79%(1 of 126) and 12.70%(16 of 126), respectively.The positive serum samples that contained antibodies reactive to epitope 313–327 neutralizedHCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313–327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432–443 had cross-genotype neutralizing activities. Theneutralizing activityof SC38, SC86, SC92 and CHC75waspartiallyinhibited by peptide 432–443. However,the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVppwere notinhibited by the peptide.This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope432–443as sources for future antibody therapies.     

Structural Basis for Penetration of the Glycan Shield of Hepatitis C Virus E2 Glycoprotein by a Broadly Neutralizing Human Antibody

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412–423 (E2_412–423) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2_412–423 epitope to 2.0 Å resolution. The conformation of E2_412–423 bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2_412–423 complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2_412–423 epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.     

Longitudinal Sequence and Functional Evolution within Glycoprotein E2 in Hepatitis C Virus Genotype 3a Infection

The E2 glycoprotein of Hepatitis C virus (HCV) is a major target of the neutralizing antibody (NAb) response with the majority of epitopes located within its receptor binding domain (RBD; 384–661). Within E2 are three variable regions located at the N-terminus (HVR1; 384–411), and internally at 460–480 (HVR2) and 570–580 [intergenotypic variable region (igVR)], all of which lie outside a conserved core domain that contains the CD81 binding site, essential for attachment of virions to host cells and a major target of NAbs. In this study, we examined the evolution of the E1 and E2 region in two patients infected with genotype 3a virus. Whereas one patient was able to clear the acute infection, the other developed a chronic infection. Mutations accumulated at multiple positions within the N-terminal HVR1 as well as within the igVR in both patients over time, whereas mutations in HVR2 were observed only in the chronically infected patient. Mutations within or adjacent to the CD81 contact site were observed in both patients but were less frequent and more conservative in the patient that cleared his/her infection. The evolution of CD81 binding function and antigenicity was examined with longitudinal E2 RBD sequences. The ability of the RBD to bind CD81 was completely lost by week 108 in the patient that developed chronic HCV. In the second patient, the ability of the week 36 RBD, just prior to viral clearance, to bind CD81 was reduced ~50% relative to RBD sequences obtained earlier. The binding of a NAb specific to a conserved epitope located within E2 residues 411–428 was significantly reduced by week 108 despite complete conservation of its epitope suggesting that E2 antigenicity is allosterically modulated. The exposure of non-neutralizing antibody epitopes was similarly explored and we observed that the epitope of 3 out of 4 non-NAbs were significantly more exposed in the RBDs representing the late timepoints in the chronic patient. By contrast, the exposure of non-neutralizing epitopes was reduced in the patient that cleared his/her infection and could in part be attributed to sequence changes in the igVR. These studies reveal that during HCV infection, the exposure of the CD81 binding site on E2 becomes increasingly occluded, and the antigenicity of the E2 RBD towards both neutralizing and non-neutralizing antibodies is modulated via allosteric mechanisms.     

Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody

The E2 envelope glycoprotein of hepatitis C virus (HCV) binds to the host entry factor CD81 and is the principal target for neutralizing antibodies (NAbs). Most NAbs recognize hypervariable region 1 on E2, which undergoes frequent mutation, thereby allowing the virus to evade neutralization. Consequently, there is great interest in NAbs that target conserved epitopes. One such NAb is AP33, a mouse monoclonal antibody that recognizes a conserved, linear epitope on E2 and potently neutralizes a broad range of HCV genotypes. In this study, the X-ray structure of AP33 Fab in complex with an epitope peptide spanning residues 412 to 423 of HCV E2 was determined to 1.8 Å. In the complex, the peptide adopts a β-hairpin conformation and docks into a deep binding pocket on the antibody. The major determinants of antibody recognition are E2 residues L413, N415, G418, and W420. The structure is compared to the recently described HCV1 Fab in complex with the same epitope. Interestingly, the antigen-binding sites of HCV1 and AP33 are completely different, whereas the peptide conformation is very similar in the two structures. Mutagenesis of the peptide-binding residues on AP33 confirmed that these residues are also critical for AP33 recognition of whole E2, confirming that the peptide-bound structure truly represents AP33 interaction with the intact glycoprotein. The slightly conformation-sensitive character of the AP33-E2 interaction was explored by cross-competition analysis and alanine-scanning mutagenesis. The structural details of this neutralizing epitope provide a starting point for the design of an immunogen capable of eliciting AP33-like antibodies.     

Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ～100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.     

A Hepatitis C Virus (HCV) Vaccine Comprising Envelope Glycoproteins gpE1/gpE2 Derived from a Single Isolate Elicits Broad Cross-Genotype Neutralizing Antibodies in Humans

Although a cure for HCV is on the near horizon, emerging drug cocktails will be expensive, associated with side-effects and resistance making a global vaccine an urgent priority given the estimated high incidence of infection around the world. Due to the highly heterogeneous nature of HCV, an effective HCV vaccine which could elicit broadly cross-neutralizing antibodies has represented a major challenge. In this study, we tested for the presence of cross-neutralizing antibodies in human volunteers who were immunized with recombinant glycoproteins gpE1/gpE2 derived from a single HCV strain (HCV1 of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1–7. Vaccination induced significant neutralizing antibodies against heterologous HCV genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV genotypes. Although observed in only a minority of vaccinees, our results prove the key concept that a vaccine derived from a single strain of HCV can elicit broad cross-neutralizing antibodies against all known major genotypes of HCV and provide considerable encouragement for the further development of a human vaccine against this common, global pathogen.     

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.     

Three Different Functional Microdomains in the Hepatitis C Virus Hypervariable Region 1 (HVR1) Mediate Entry and Immune Evasion

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25–27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16–24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1–13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.     

Genotype 1 Hepatitis C Virus Envelope Features That Determine Antiviral Response Assessed through Optimal Covariance Networks

The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531–626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431–481 (EE), 500–522 (QA) in 1a, and 407–434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy.     

Identification of a Domain Containing B-Cell Epitopes in Hepatitis C Virus E2 Glycoprotein by Using Mouse Monoclonal Antibodies

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.     

Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein

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.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

Hepatitis C Virus Infection among Injection Drug Users with and without Human Immunodeficiency Virus Co-Infection

The aim of this study is to explore the prevalence of hepatitis C virus (HCV) infection among injection drug users (IDUs) with and without human immunodeficiency virus (HIV) infection in southern Taiwan. For 562 IDUs (265 anti-HIV negative, 297 anti-HIV positive), we analyzed liver function, anti-HIV antibody, anti-HCV antibody, HCV viral loads, and hepatitis B surface antigen (HBsAg). HIV RNA viral loads and CD4 cell count for anti-HIV-seropositive IDUs and the HCV genotype for HCV RNA-seropositive IDUs were measured. The seroprevalence rates of anti-HIV, anti-HCV, and HBsAg were 52.8%, 91.3%, and 15.3%, respectively. All the anti-HIV-seropositive IDUs were positive for HIV RNA. Anti-HCV seropositivity was the most important factor associated with HIV infection (odds ratio [OR], 25.06; 95% confidence intervals [CI], 8.97–74.9), followed by male gender (OR, 6.12; 95% CI, 4.05–9.39) and HBsAg seropositivity (OR, 1.90; 95% CI, 1.11–3.34). Among IDUs positive for anti-HCV, 80.7% had detectable HCV RNA. HCV viremia after HCV exposure was strongly related to HIV infection (OR, 6.262; 95% CI, 1.515–18.28), but negatively correlated to HBsAg seropositivity (OR, 0.161; 95% CI, 0.082–0.317). HCV genotype 6 was the most prevalent genotype among all IDUs (41.0%), followed by genotypes 1 (32.3%), 3 (12.8%), and 2 (5.6%). In conclusion, about half IDUs were infected with HIV and >90% with HCV infection. Male and seropositivity for HBsAg and anti-HCV were factors related to HIV infection among our IDUs. HIV was positively correlated, whereas hepatitis B co-infection was negatively correlated with HCV viremia among IDUs with HCV exposure. Different HCV molecular epidemiology was noted among IDUs.     

Amino Acid Residue-Specific Neutralization and Nonneutralization of Hepatitis C Virus by Monoclonal Antibodies to the E2 Protein

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W⁴³⁷, L⁴³⁸, L⁴⁴¹, and F⁴⁴², within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.     

Hepatitis C Virus (HCV) Infection May Elicit Neutralizing Antibodies Targeting Epitopes Conserved in All Viral Genotypes

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.     

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.     

Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions

Development of full-length hepatitis C virus (HCV) RNAs replicating efficiently and producing infectious cell-cultured virions, HCVcc, in hepatoma cells provides an opportunity to characterize immunogenic domains on viral envelope proteins involved in entry into target cells. A panel of immunoglobulin G1 human monoclonal antibodies (HMAbs) to three immunogenic conformational domains (designated A, B, and C) on HCV E2 glycoprotein showed that epitopes within two domains, B and C, mediated HCVcc neutralization, whereas HMAbs to domain A were all nonneutralizing. For the neutralizing antibodies to domain B (with some to conserved epitopes among different HCV genotypes), the inhibitory antibody concentration reducing HCVcc infection by 90%, IC90, ranged from 0.1 to 4 microg/ml. For some neutralizing HMAbs, HCVcc neutralization displayed a linear correlation with an antibody concentration between the IC50 and the IC90 while others showed a nonlinear correlation. The differences between IC50/IC90 ratios and earlier findings that neutralizing HMAbs block E2 interaction with CD81 suggest that these antibodies block different facets of virus-receptor interaction. Collectively, these findings support an immunogenic model of HCV E2 having three immunogenic domains with distinct structures and functions and provide added support for the idea that CD81 is required for virus entry.     

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.Hepatitis C virus (HCV) is a major cause of liver failure and infects more than 170 million people worldwide. HCV is a member of the Flaviviridae family and contains a 9.6-kb positive-strand RNA genome. The genome is translated into a single polypeptide that is cleaved by viral and cellular proteases into at least nine different proteins. The major HCV surface glycoproteins, E1 and E2, form a noncovalent heterodimer on the virion surface (23) and are believed to mediate viral entry via a complex set of poorly understood interactions with cellular coreceptors, including CD81 (28), claudin-1 (8), occludin (29), scavenger receptor class B type I (30), and others (38). The E2 glycoprotein has been shown to interact directly with receptors (38); currently, no function has been assigned to E1, although it is known to be required for viral infection. These viral glycoproteins provide an obvious target for neutralizing monoclonal antibodies (MAbs).Isolation of potently neutralizing HCV-specific MAbs has been complicated by the lack of an in vitro cell culture system to study the full infection cycle of the virus. Recently, systems have been developed that allow for the generation of infectious viral particles, highlighting the importance of E1 and E2 in viral binding and entry. A novel in vitro infection system employs HCV pseudotyped viral particles (HCVpp) generated from a lentivirus that are devoid of native glycoproteins and engineered to contain HCV glycoproteins E1 and E2 (4, 15). HCVpp specifically infect cell lines derived from human liver cells and can be neutralized by polyclonal and MAbs directed against the HCV envelope glycoproteins.HCVpp have allowed the identification of antibodies that can neutralize HCV infection in cell culture. E1 has proven to be a difficult target for MAb-mediated neutralization, possibly because it appears to have low immunogenicity (32), has no identified binding proteins on the cell surface, and has an undefined role in cell entry. Despite this challenge, two groups have identified HCV neutralizing MAbs directed to E1: these MAbs are H-111, which has moderate neutralizing activity (17), and the recently isolated IGH505 and IGH526, which neutralize numerous HCV genotypes (1a, 1b, 2a, 4a, 5a, and 6a but not 2b and 3a) (22). Although they are predicted to inhibit viral binding or fusion, the mechanism by which these E1-directed MAbs neutralize HCV infection is unclear.A diverse group of mouse anti-E2 antibodies, recognizing both linear and discontinuous epitopes, has been generated. Many of these MAbs showed broad neutralization of multiple HCV genotypes, but not surprisingly, several HCV isolates were refractory to neutralization. In contrast, AP33, a mouse MAb that largely recognizes a highly conserved linear epitope in the N terminus of E2 (amino acids 412 to 423), was identified as a broadly cross-reactive antibody that neutralized strains from all genotypes tested (1a, 1b, 2a, 2b, 3a, 4, 5, and 6), with the exception of one genotype 5 virus (UKN5.14.4; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY894682","term_id":"58220837","term_text":"AY894682"}}AY894682) (24). Recently, several cross-reactive neutralizing MAbs have been identified that are of human origin and have the capacity to neutralize a significant fraction of the genotypes tested (1, 5, 12, 13, 27, 31) or to neutralize all genotypes tested (16, 20, 25). As with the vast majority of previously described human MAbs (HuMAbs), these MAbs recognize conformation-dependent epitopes of E2. One broadly neutralizing human antibody, AR3B, was tested in a mouse model of infection and showed significant protection from viremia (20). Given the known function of the E2 envelope glycoprotein, the high level of immunogenicity, the surface vulnerability, and the abundance of data pertaining to E2 and HCV neutralization, E2 provides a promising target for the development of fully human neutralizing antibodies.Liver deterioration due to HCV infection is the leading reason for liver transplantation in the United States. Unfortunately, it is highly likely that the transplanted liver will also become infected with HCV, and 10 to 25% of these patients develop cirrhosis within 5 years of transplant (9, 40). Here we describe the characterization of HuMAbs directed against the HCV E2 envelope glycoprotein, generated using transgenic mice. Based on epitope conservation and broad neutralization capacity, HuMAbs HCV1 and 95-2 provide excellent candidates for prevention of graft reinfection of HCV-infected individuals undergoing liver transplantation.