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ABSTRACT: The E2 envelope glycoprotein of 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 has been determined to 1.8Å. 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.
Journal of Virology 09/2012; · 5.40 Impact Factor
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Isabel Fofana,
Samira Fafi-Kremer,
Patric Carolla,
Catherine Fauvelle,
Muhammad Nauman Zahid,
Marine Turek,
Laura Heydmann,
Karine Cury,
Juliette Hayer,
Christophe Combet, [......],
Thomas Pietschmann,
Marie-Sophie Hiet,
Ralf Bartenschlager,
François Habersetzer,
Michel Doffoël, Zhen-Yong Keck,
Steven K H Foung,
Mirjam B Zeisel,
Françoise Stoll-Keller,
Thomas F Baumert
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ABSTRACT: The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion.
We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors.
By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general.
We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines.
Gastroenterology 04/2012; 143(1):223-233.e9. · 11.68 Impact Factor
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Zhen-yong Keck,
Jinming Xia,
Yong Wang,
Wenyan Wang,
Thomas Krey,
Jannick Prentoe,
Thomas Carlsen,
Angela Ying-Jian Li,
Arvind H Patel,
Stanley M Lemon,
Jens Bukh,
Felix A Rey,
Steven K H Foung
[show abstract]
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ABSTRACT: 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.
PLoS Pathogens 04/2012; 8(4):e1002653. · 9.13 Impact Factor
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ABSTRACT: A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development.
Journal of Biological Chemistry 12/2011; 286(51):44218-33. · 4.77 Impact Factor
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ABSTRACT: A critical first step in a "rational vaccine design" approach for hepatitis C virus (HCV) is to identify the most relevant mechanisms of immune protection. Emerging evidence provides support for a protective role of virus neutralizing antibodies, and the ability of the B cell response to modify the course of acute HCV infection. This has been made possible by the development of in vitro cell culture models, based on HCV retroviral pseudotype particles expressing E1E2 and infectious cell culture-derived HCV virions, and small animal models that are robust tools in studies of antibody-mediated virus neutralization. This review is focused on the immunogenic determinants on the E2 glycoprotein mediating virus neutralization and the pathways in which the virus is able to escape from immune containment. Encouraging findings from recent studies provide support for the existence of broadly neutralization antibodies that are not associated with virus escape. The identification of conserved epitopes mediating virus neutralization that are not associated with virus escape will facilitate the design of a vaccine immunogen capable of eliciting broadly neutralizing antibodies against this highly diverse virus.
Viruses 11/2011; 3(11):2127-45. · 1.50 Impact Factor
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[show abstract]
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ABSTRACT: A potent neutralizing antibody to a conserved HCV epitope might overcome its extreme variability, allowing immunotherapy.
The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed
that HC-1 neutralized most HCV genotypes but having modest potency. To improve neutralization, we affinity-matured HC-1 by
constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants and using for selection an E2 antigen from one
of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and kappa
chain variable (Vk) genes separately and then combining the optimized VH and Vk mutants. This resulted in the generation
of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After
conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon
resonance and solution kinetic exclusion analysis showed the increase in affinity was largely due to a lowering of the dissociation
rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the
affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized
by wildtype (wt) HC-1. Moreover, propagating 2a HCVcc under the selective pressure of wt HC-1 or affinity-matured HC-1 antibodies
yielded no viral escape mutants, and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus
elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic
development.
Journal of Biological Chemistry 10/2011; · 4.77 Impact Factor
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ABSTRACT: Understanding the interaction between broadly neutralizing antibodies and their epitopes provides a basis for the rational design of a preventive hepatitis C virus (HCV) vaccine. CBH-2, HC-11, and HC-1 are representatives of antibodies to overlapping epitopes on E2 that mediate neutralization by blocking virus binding to CD81. To obtain insights into escape mechanisms, infectious cell culture virus, 2a HCVcc, was propagated under increasing concentrations of a neutralizing antibody to isolate escape mutants. Three escape patterns were observed with these antibodies. First, CBH-2 escape mutants that contained mutations at D431G or A439E, which did not compromise viral fitness, were isolated. Second, under the selective pressure of HC-11, escape mutations progressed from a single L438F substitution at a low antibody concentration to double substitutions, L438F and N434D or L438F and T435A, at higher antibody concentrations. Escape from HC-11 was associated with a loss of viral fitness. An HCV pseudoparticle (HCVpp) containing the L438F mutation bound to CD81 half as efficiently as did wild-type (wt) HCVpp. Third, for HC-1, the antibody at a critical concentration completely suppressed viral replication and generated no escape mutants. Epitope mapping revealed contact residues for CBH-2 and HC-11 in two regions of the E2 glycoprotein, amino acids (aa) 425 to 443 and aa 529 to 535. Interestingly, contact residues for HC-1 were identified only in the region encompassing aa 529 to 535 and not in aa 425 to 443. Taken together, these findings point to a region of variability, aa 425 to 443, that is responsible primarily for viral escape from neutralization, with or without compromising viral fitness. Moreover, the region aa 529 to 535 is a core CD81 binding region that does not tolerate neutralization escape mutations.
Journal of Virology 08/2011; 85(20):10451-63. · 5.40 Impact Factor
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Dorothea Bankwitz,
Eike Steinmann,
Julia Bitzegeio,
Sandra Ciesek,
Martina Friesland,
Eva Herrmann,
Mirjam B Zeisel,
Thomas F Baumert, Zhen-yong Keck,
Steven K H Foung,
Eve-Isabelle Pécheur,
Thomas Pietschmann
[show abstract]
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ABSTRACT: The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (Delta HVR1) are less infectious, and separation by density gradients revealed that the population of Delta HVR1 virions comprises fewer particles with low density. Strikingly, Delta HVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, Delta HVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, Delta HVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.
Journal of Virology 03/2010; 84(11):5751-63. · 5.40 Impact Factor
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Zhen-yong Keck,
Sophia H Li,
Jinming Xia,
Thomas von Hahn,
Peter Balfe,
Jane A McKeating,
Jeroen Witteveldt,
Arvind H Patel,
Harvey Alter,
Charles M Rice,
Steven K H Foung
[show abstract]
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ABSTRACT: Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.
Journal of Virology 04/2009; 83(12):6149-60. · 5.40 Impact Factor
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Jeroen Witteveldt,
Matthew J Evans,
Julia Bitzegeio,
George Koutsoudakis,
Ania M Owsianka,
Allan G N Angus, Zhen-Yong Keck,
Steven K H Foung,
Thomas Pietschmann,
Charles M Rice,
Arvind H Patel
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ABSTRACT: Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1(DeltaE1E2)-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1(W529A) was unaffected by the presence of neutralizing antibodies that inhibit E2-CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.
Journal of General Virology 02/2009; 90(Pt 1):48-58. · 3.36 Impact Factor
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ABSTRACT: Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.
Proceedings of the National Academy of Sciences 01/2009; 105(49):19450-5. · 9.68 Impact Factor
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ABSTRACT: 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.
Journal of Virology 07/2008; 82(12):6061-6. · 5.40 Impact Factor
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ABSTRACT: A challenge in hepatitis C virus (HCV) vaccine development is defining conserved protective epitopes. A cluster of these epitopes comprises an immunodominant domain on the E2 glycoprotein, designated domain B. CBH-2 is a neutralizing human monoclonal antibody to a domain B epitope that is highly conserved. Alanine scanning demonstrated that the epitope involves residues G523, G530, and D535 that are also contact residues for E2 binding to CD81, a coreceptor required for virus entry into cells. However, another residue, located at position 431 and thus at a considerable distance in the linear sequence of E2, also contributes to the CBH-2 epitope. A single amino acid substitution at this residue results in escape from CBH-2-mediated neutralization in a genotype 1a virus. These results highlight the challenges inherent in developing HCV vaccines and show that an effective vaccine must induce antibodies to both conserved and more invariant epitopes to minimize virus escape.
Journal of Virology 07/2008; 82(12):6067-72. · 5.40 Impact Factor
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ABSTRACT: 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.
Journal of General Virology 04/2008; 89(Pt 3):653-9. · 3.36 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) often causes a persistent infection associated with hypergammaglobulinemia, high levels of antiviral antibody and circulating immune complexes, and immune complex disease. We previously reported that only a limited neutralizing activity to vesicular stomatitis virus or HCV pseudotype is generated in animals immunized with recombinant HCV envelope proteins and chronically infected HCV patient sera. Interestingly, when some of these neutralizing sera were diluted into a range of concentrations below those that reduced virus plaque number, an increase in pseudotype plaque formation was observed. Purified HCV E2-specific human monoclonal antibodies were used to further verify the specificity of this enhancement, and one- to twofold increases were apparent on permissive Huh-7 cells. The enhancement of HCV pseudotype titer could be inhibited by the addition of a Fc-specific anti-human immunoglobulin G Fab fragment to the virus-antibody mixture prior to infection. Treatment of cells with antibody to Fc receptor I (FcRI) or FcRII, but not FcRIII, also led to an inhibition of pseudotype titer enhancement in an additive manner. Human lymphoblastoid cell line (Raji), a poor host for HCV pseudotype infection, exhibited a four- to sixfold enhancement of pseudotype-mediated cell death upon incubation with antibody at nonneutralizing concentrations. A similar enhancement of cell culture-grown HCV infectivity by a human monoclonal antibody was also observed. Taken together, antibodies to viral epitopes enhancing HCV infection need to be taken into consideration for pathogenesis and in the development of an effective vaccine.
Journal of Virology 04/2008; 82(5):2140-9. · 5.40 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.
Journal of Virology 09/2007; 81(15):8101-11. · 5.40 Impact Factor
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ABSTRACT: 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.
Journal of Virology 02/2007; 81(2):1043-7. · 5.40 Impact Factor
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Rachel Eren,
Dorit Landstein,
Dov Terkieltaub,
Ofer Nussbaum,
Arie Zauberman,
Judith Ben-Porath,
Judith Gopher,
Rachel Buchnick,
Riva Kovjazin,
Ziva Rosenthal-Galili, [......],
Ofer Ben-Moshe,
Alberto Kischitsky,
Steven K H Foung, Zhen-Yong Keck,
Orit Pappo,
Ahmed Eid,
Oded Jurim,
Gidi Zamir,
Eithan Galun,
Shlomo Dagan
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ABSTRACT: Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.
Journal of Virology 04/2006; 80(6):2654-64. · 5.40 Impact Factor
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ABSTRACT: Hepatitis C (HCV) E2 glycoprotein is involved in virus attachment and entry, and its structural organization is largely unknown. Characterization of a panel of human monoclonal antibodies (HMAbs) to HCV by competition studies has led to an immunogenic organization model of E2 with three domains designated A, B, and C and epitopes in each domain having similar structural and functional properties. Domain A contains nonneutralizing epitopes, and domains B and C contain neutralizing epitopes. The isolation and characterization of three new HMAbs within domain A for a total of six provide support for this model. All six domain A HMAbs do not neutralize HCV retroviral pseudotype particle (HCVpp) infection on Huh-7 cells, and all six HMAbs have similar binding affinity and maximum binding, B(max), a relative indicator of epitope density, as other neutralizing HMAbs, suggesting that neutralization is epitope specific and not by binding to any surface epitope. The dose-dependent neutralizing activity of CBH-7, an HMAb to a domain C epitope in spatial proximity to domain A, and of CBH-5, a domain B HMAb to a more distant epitope, were tested in the presence and absence of each domain A HMAb. No enhancement or reduction in CBH-7 or CBH-5 neutralizing activity was observed, indicating that the potential induction of nonneutralizing antibodies should not be a central issue for HCV vaccine design. To assess whether domain A is involved in the structural changes as part of a pH-dependent virus envelope fusion process, changes in antibody binding patterns to normal pH and acid pH-treated HCVpp were measured. Antibody binding affinity of HMAbs to HCVpp was not affected by low pH. However, the B(max) values for low-pH-treated HCVpp with antibodies to domain A increased 46%, for domain C (CBH-7) they increased 23%, and for domain B (CBH-5) there was a decrease of 12%. Collectively, the organization and function of HCV E2 antigenic domains are roughly analogous to the large envelope glycoprotein E organizational structure for other flaviviruses with three distinct structural and functional domains.
Journal of Virology 12/2005; 79(21):13199-208. · 5.40 Impact Factor
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ABSTRACT: Mechanisms of virion attachment, interaction with its receptor, and cell entry are poorly understood for hepatitis C virus (HCV) because of a lack of an efficient and reliable in vitro system for virus propagation. Infectious HCV retroviral pseudotype particles (HCVpp) were recently shown to express native E1E2 glycoproteins, as defined in part by HCV human monoclonal antibodies (HMAbs) to conformational epitopes on E2, and some of these antibodies block HCVpp infection (A. Op De Beeck, C. Voisset, B. Bartosch, Y. Ciczora, L. Cocquerel, Z. Y. Keck, S. Foung, F. L. Cosset, and J. Dubuisson, J. Virol. 78:2994-3002, 2004). Why some HMAbs are neutralizing and others are nonneutralizing is looked at in this report by a series of studies to determine the expression of their epitopes on E2 associated with HCVpp and the role of antibody binding affinity. Antibody cross-competition defined three E2 immunogenic domains with neutralizing HMAbs restricted to two domains that were also able to block E2 interaction with CD81, a putative receptor for HCV. HCVpp immunoprecipitation showed that neutralizing and nonneutralizing domains are expressed on E2 associated with HCVpp, and affinity studies found moderate-to-high-affinity antibodies in all domains. These findings support the perspective that HCV-specific epitopes are responsible for functional steps in virus infection, with specific antibodies blocking distinct steps of virus attachment and entry, rather than the perspective that virus neutralization correlates with increased antibody binding to any virion surface site, independent of the epitope recognized by the antibody. Segregation of virus neutralization and sensitivity to low pH to specific regions supports a model of HCV E2 immunogenic domains similar to the antigenic structural and functional domains of other flavivirus envelope E glycoproteins.
Journal of Virology 10/2004; 78(17):9224-32. · 5.40 Impact Factor
