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Find video protocols related to scientific articles indexed in Pubmed.
Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.
PLoS Pathog.
PUBLISHED: 08-14-2014
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A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which can co-exist with lower potency or levels of neutralizing activities.
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Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a.
Hepatology
PUBLISHED: 01-16-2014
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Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc-expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50 ) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 ?g/mL. Interestingly, IC50 values against the different HCVcc's exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50 values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc's when combined individually with AR4A.
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Structural basis of HCV neutralization by human monoclonal antibodies resistant to viral neutralization escape.
PLoS Pathog.
PUBLISHED: 05-01-2013
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The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434-446 and aa610-619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434-446. The structures show that the peptide adopts an ?-helical conformation with the main contact residues F??² and Y??³ forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development.
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Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies.
Hepatology
PUBLISHED: 03-14-2013
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Immunotherapy and vaccine development for hepatitis C virus (HCV) will depend on broadly reactive neutralizing antibodies (NAbs). However, studies in infectious strain JFH1-based culture systems expressing patient-derived Core-NS2 proteins have suggested neutralization resistance for specific HCV strains, in particular, of genotype 2. To further examine this phenomenon, we developed a panel of HCV genotype 2 recombinants for testing of sensitivity to neutralization by chronic-phase patient sera and lead human monoclonal antibodies (HMAbs). The novel Core-NS2 recombinants, with patient-derived genotype 2a (strain T9), 2b (strains DH8 and DH10), and 2c (strain S83) consensus sequences, were viable in Huh7.5 hepatoma cells without requirement for adaptive mutations, reaching HCV infectivity titers of 3.9-4.5 log10 focus-forming units per milliliter. In in vitro neutralization assays, we demonstrated that the novel genotype 2 viruses as well as prototype strains J6/JFH1(2a) and J8/JFH1(2b), all with authentic envelope proteins, were resistant to neutralization by genotype 2a, 2b, 2c, 2j, 2i, and 2q patient sera. However, these patient sera had high titers of HCV-specific NAbs, because they efficiently reduced the infectivity of J6(2a) and J8(2b) with deleted hypervariable region 1. The genotype 2a, 2b, and 2c viruses, found resistant to polyclonal patient sera neutralization, were efficiently neutralized by two lead HMAbs (AR4A and HC84.26). Conclusion: Using novel 2a, 2b, and 2c cell-culture systems, expressing authentic envelope proteins, we demonstrated resistance of HCV to patient-derived polyclonal high-titer NAbs. However, the same genotype 2 culture viruses were all sensitive to HMAbs recognizing conformational epitopes, indicating that neutralization resistance of HCV can be overcome by applying recombinant antibodies. These findings have important implications for HCV immunotherapy and vaccine development. (Hepatology 2013).
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Contrasting roles of mitogen-activated protein kinases in cellular entry and replication of hepatitis C virus: MKNK1 facilitates cell entry.
J. Virol.
PUBLISHED: 01-30-2013
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The human kinome comprises over 800 individual kinases. These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability. We propose a model by which MKNK1 acts to facilitate viral entry downstream of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), both of which have been implicated in the entry process.
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Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus.
J. Biol. Chem.
PUBLISHED: 10-14-2011
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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 ?-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.
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Neutralizing antibody response to hepatitis C virus.
Viruses
PUBLISHED: 09-14-2011
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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.
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Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations.
J. Virol.
PUBLISHED: 08-03-2011
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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.
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Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure-function relationships.
J. Gen. Virol.
PUBLISHED: 06-22-2011
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Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal stem of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system.
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The hepatitis C virus E1 glycoprotein undergoes productive folding but accelerated degradation when expressed as an individual subunit in CHO cells.
PLoS ONE
PUBLISHED: 01-21-2011
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Hepatitis C Virus E1E2 heterodimers are components of the viral spike. Although there is a general agreement on the necessity of the co-expression of both E1 and E2 on a single coding unit for their productive folding and assembly, in a previous study using an in vitro system we obtained strong indications that E1 can achieve folding in absence of E2. Here, we have studied the folding pathway of unescorted E1 from stably expressing CHO cells, compared to the folding observed in presence of the E2 protein. A DTT-resistant conformation is achieved by E1 in both situations, consistent with the presence of an E2-independent oxidative pathway. However, while the E1E2 heterodimer is stable inside cells, E1 expressed alone is degraded within a few hours. On the other hand, the oxidation and stability of individually expressed E2 subunits is dependent on E1 co-expression. These data are consistent with E1 and E2 assisting each other for correct folding via different mechanisms: E2 assists E1 by stabilizing a semi-native conformation meanwhile E1 drives E2 towards a productive folding pathway.
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The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae.
J. Gen. Virol.
PUBLISHED: 11-17-2010
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In 1967, it was reported that experimental inoculation of serum from a surgeon (G.B.) with acute hepatitis into tamarins resulted in hepatitis. In 1995, two new members of the family Flaviviridae, named GBV-A and GBV-B, were identified in tamarins that developed hepatitis following inoculation with the 11th GB passage. Neither virus infects humans, and a number of GBV-A variants were identified in wild New World monkeys that were captured. Subsequently, a related human virus was identified [named GBV-C or hepatitis G virus (HGV)], and recently a more distantly related virus (named GBV-D) was discovered in bats. Only GBV-B, a second species within the genus Hepacivirus (type species hepatitis C virus), has been shown to cause hepatitis; it causes acute hepatitis in experimentally infected tamarins. The other GB viruses have however not been assigned to a genus within the family Flaviviridae. Based on phylogenetic relationships, genome organization and pathogenic features of the GB viruses, we propose to classify GBV-A-like viruses, GBV-C and GBV-D as members of a fourth genus in the family Flaviviridae, named Pegivirus (pe, persistent; g, GB or G). We also propose renaming GB viruses within the tentative genus Pegivirus to reflect their host origin.
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Hepatitis C virus e2 protein ectodomain is essential for assembly of infectious virions.
Int J Hepatol
PUBLISHED: 07-14-2010
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The Hepatitis C virus E1 and E2 envelope proteins are the major players in all events required for virus entry into target cells. In addition, the recently developed HCV cell culture system has indicated that E1E2 heterodimer formation is a prerequisite for viral particle production. In this paper, we explored a new genetic approach to construct intergenotypic 2a/1b chimeras, maintaining the structural region of the infectious strain JFH1 and substituting the soluble portion of E1 and/or E2 proteins. This strategy provides useful information on the role of the surface-exposed domain of the envelope proteins in virus morphogenesis and allows comparative analysis of different HCV genotypes. We found that substituting the E2 protein ectodomain region abolishes the production of chimeric infectious particles. Our data indicate that the soluble part of the E2 protein is involved in a genotype-specific interplay with remaining viral proteins that affect the HCV assembly process.
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Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes.
J. Virol.
PUBLISHED: 03-31-2010
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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.
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Mutations within a conserved region of the hepatitis C virus E2 glycoprotein that influence virus-receptor interactions and sensitivity to neutralizing antibodies.
J. Virol.
PUBLISHED: 03-17-2010
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Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1(WT), the JFH1(N415D) was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1(WT) under MAb AP33 selective pressure. MAb AP33 neutralized JFH1(T416A) and JFH1(I422L) more efficiently than the WT virus, while neutralization of JFH1(N417S) and JFH1(G418D) was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1(N415D), highlighting the importance of the E2 412-423 region in virus entry.
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Antigen-specific proteolysis by hybrid antibodies containing promiscuous proteolytic light chains paired with an antigen-binding heavy chain.
J. Biol. Chem.
PUBLISHED: 06-19-2009
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The antigen recognition site of antibodies consists of the heavy and light chain variable domains (V(L) and V(H) domains). V(L) domains catalyze peptide bond hydrolysis independent of V(H) domains (Mei, S., Mody, B., Eklund, S. H., and Paul, S. (1991) J. Biol. Chem. 266, 15571-15574). V(H) domains bind antigens noncovalently independent of V(L) domains (Ward, E. S., Güssow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989) Nature 341, 544-546). We describe specific hydrolysis of fusion proteins of the hepatitis C virus E2 protein with glutathione S-transferase (GST-E2) or FLAG peptide (FLAG-E2) by antibodies containing the V(H) domain of an anti-E2 IgG paired with promiscuously catalytic V(L) domains. The hybrid IgG hydrolyzed the E2 fusion proteins more rapidly than the unpaired light chain. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. The hybrid IgG displayed noncovalent E2 binding in enzyme-linked immunosorbent assay tests. Immunoblotting studies suggested hydrolysis of FLAG-E2 at a bond within E2 located approximately 11 kDa from the N terminus. GST-E2 was hydrolyzed by the hybrid IgG at bonds in the GST tag. The differing cleavage pattern of FLAG-E2 and GST-E2 can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. These studies provide proof-of-principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit.
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Mutations in hepatitis C virus E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity.
J. Virol.
PUBLISHED: 03-25-2009
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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.
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CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.
J. Gen. Virol.
PUBLISHED: 02-13-2009
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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.
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Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry.
J. Virol.
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The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.
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Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein.
J. Virol.
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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.
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Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry.
J. Biol. Chem.
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The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.
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Toward a hepatitis C virus vaccine: the structural basis of hepatitis C virus neutralization by AP33, a broadly neutralizing antibody.
J. Virol.
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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 ?. In the complex, the peptide adopts a ?-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.
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Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.
PLoS Pathog.
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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.
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Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
Gastroenterology
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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.
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