Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships

Rychłowska, Małgorzata; Owsianka, Ania M.; Foung, Steven K. H.; Dubuisson, Jean; Bieńkowska-Szewczyk, Krystyna; Patel, Arvind H.

doi:10.1099/vir.0.034314-0

Cited by 14 publications

(17 citation statements)

References 57 publications

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“…D698 and D639 of E2 were critical for the binding of AR4A and AR5A, respectively. Interestingly, D698 is within the highly conserved membrane-proximal external region (MPER), which is important for E1E2 heterodimerization and membrane fusion (57). There is still much to be discovered about how the epitopes of these MAbs are formed by E1E2 heterodimerization.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

Wong

Bhat

Hockman

et al. 2014

J Virol

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Although effective hepatitis C virus (HCV) antivirals are on the horizon, a global prophylactic vaccine for HCV remains elusive. The diversity of the virus is a major concern for vaccine development; there are 7 major genotypes of HCV found globally. Therefore, a successful vaccine will need to protect against HCV infection by all genotypes. Despite the diversity, many monoclonal antibodies (MAbs) with broadly cross-neutralizing activity have been described, suggesting the presence of conserved epitopes that can be targeted to prevent infection. Similarly, a vaccine comprising recombinant envelope glycoproteins (rE1E2) derived from the genotype 1a HCV-1 strain has been shown to be capable of eliciting cross-neutralizing antibodies in guinea pigs, chimpanzees, and healthy human volunteers. In order to investigate the basis for this cross-neutralization, epitope mapping of anti-E1E2 antibodies present within antisera from goats and humans immunized with HCV-1 rE1E2 was conducted through peptide mapping and competition studies with a panel of cross-neutralizing MAbs targeting various epitopes within E1E2. The immunized-goat antiserum was shown to compete with the binding of all MAbs tested (AP33, HC33.4, HC84.26, 1:7, AR3B, AR4A, AR5A, IGH526, and A4). Antisera showed the best competition against HC84.26 and AR3B and the weakest competition against AR4A. Furthermore, antisera from five immunized human vaccinees were shown to compete with five preselected MAbs (AP33, AR3B, AR4A, AR5A, and IGH526). These data show that immunization with HCV-1 rE1E2 elicits antibodies targeting multiple cross-neutralizing epitopes. Our results further support the use of such a vaccine antigen to induce cross-genotype neutralization. IMPORTANCEAn effective prophylactic vaccine for HCV is needed for optimal control of the disease burden. The high diversity of HCV has posed a challenge for developing vaccines that elicit neutralizing antibodies for protection against infection. Despite this, we have previously shown that a vaccine comprising recombinant envelope glycoproteins derived from a single genotype 1a strain was capable of eliciting a cross-neutralizing antibody response in human volunteers. Here, we have used competition binding assays and peptide binding assays to show that antibodies present in the antisera from vaccinated goats and humans bind epitopes overlapping with those of a variety of well-characterized cross-neutralizing monoclonal antibodies. This provides a mechanism for the cross-neutralizing human antisera: antibodies present in the antisera bind to conserved regions associated with cross-neutralization. Importantly, this work provides further support for a vaccine comprising recombinant envelope glycoproteins, perhaps in a formulation with a vaccine component eliciting strong anti-HCV CD4 ؉ and CD8 ؉ T cell responses.

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Section: Discussionmentioning

confidence: 99%

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

Wong

Bhat

Hockman

et al. 2014

J Virol

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“…HCVpp were generated in HEK-293T cells (49). Virus particles were filtered through 0.45-µm-pore-size membrane and concentrated 10-fold using Amicon Ultra-15 centrifugal units with a 100,000 molecular weight cutoff membrane.…”

Section: Methodsmentioning

confidence: 99%

Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2

Vasiliauskaite

Owsianka

England

et al. 2017

mBio

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The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like ␤-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a ␤-strand of this Ig-like domain forms an ␣-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design.IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development.

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“…( A ) Genomic organization and mutations of envelope glycoproteins of escape variant VL and nonselected variants VC and VA. HVR1 and HVR2 are depicted in green ; E2 domains are depicted in red (DI), yellow (DII), and blue (DIII); and CD81 binding domains are depicted in dark blue . 29,33,39 Positions 447, 458, and 478 are highlighted in black vertical lines . Differences between VL, VC, and VA in region E1E2 384–483 are displayed.…”

Section: Figurementioning

confidence: 99%

Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

et al. 2012

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BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

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Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure–function relationships

Cited by 14 publications

References 57 publications

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

Recombinant Hepatitis C Virus Envelope Glycoprotein Vaccine Elicits Antibodies Targeting Multiple Epitopes on the Envelope Glycoproteins Associated with Broad Cross-Neutralization

Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2

Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

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