Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

About two million new cases of hepatitis C virus (HCV) infections annually underscore the urgent need for a vaccine. However, this effort has proven challenging because HCV evades neutralizing antibodies (NAbs) through molecular features of viral envelope glycoprotein E2, including hypervariable region 1 (HVR1) and N-linked glycans. Here, we observe large variation in the effects of removing individual E2 glycans across HCV strains H77(genotype 1a), J6(2a), and S52(3a) in Huh7.5 cell infections. Also, glycan-mediated effects on neutralization sensitivity were completely HVR1-dependent, and neutralization data were consistent with indirect protection of epitopes, as opposed to direct steric shielding. Indeed, the effect of removing each glycan was similar both in type (protective or sensitizing) and relative strength across four nonoverlapping neutralization epitopes. Temperature-dependent neutralization (e.g., virus breathing) assays indicated that both HVR1 and protective glycans stabilized a closed, difficult to neutralize, envelope conformation. This stabilizing effect was hierarchical as removal of HVR1 fully destabilized closed conformations, irrespective of glycan status, consistent with increased instability at acidic pH and high temperatures. Finally, we observed a strong correlation between neutralization sensitivity and scavenger receptor BI dependency during viral entry. In conclusion, our study indicates that HVR1 and glycans regulate HCV neutralization by shifting the equilibrium between open and closed envelope conformations. This regulation appears tightly linked with scavenger receptor BI dependency, suggesting a role of this receptor in transitions from closed to open conformations during entry. This importance of structural dynamics of HCV envelope glycoproteins has critical implications for vaccine development and suggests that similar phenomena could contribute to immune evasion of other viruses.

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An Interplay between Hypervariable Region 1 of the Hepatitis C Virus E2 Glycoprotein, the Scavenger Receptor BI, and High-Density Lipoprotein Promotes both Enhancement of Infection and Protection against Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.79.13.8217-8229.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8217-8229 ◽

Cited By ~ 217

Author(s):

Birke Bartosch ◽

Géraldine Verney ◽

Marlène Dreux ◽

Peggy Donot ◽

Yoann Morice ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Scavenger Receptor ◽

Hypervariable Region ◽

Density Lipoprotein ◽

Cell Entry ◽

Scavenger Receptor Bi ◽

E2 Glycoprotein ◽

Hypervariable Region 1

ABSTRACT Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins. To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with naïve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV.

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A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1

Journal of Virology ◽

10.1128/jvi.00810-19 ◽

2019 ◽

Vol 93 (22) ◽

Cited By ~ 4

Author(s):

Janelle Johnson ◽

Holly Freedman ◽

Michael Logan ◽

Jason Alexander Ji-Xhin Wong ◽

Darren Hockman ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Scavenger Receptor ◽

Hypervariable Region ◽

Neutralization Sensitivity ◽

Hcv Genotype ◽

Hcv Genotypes ◽

E2 Glycoprotein ◽

Hypervariable Region 1

ABSTRACT The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine. IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

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Convergent antibody responses associated with broad neutralization of hepatitis C virus and clearance of infection

10.1101/2021.09.11.21263416 ◽

2021 ◽

Author(s):

Nicole E. Skinner ◽

Clinton O. Ogega ◽

Nicole Frumento ◽

Kaitlyn E. Clark ◽

Srinivasan Yegnasubramanian ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

B Cell ◽

Early Development ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

High Plasma ◽

Spontaneous Clearance ◽

Cell Repertoire ◽

Molecular Features

AbstractEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, much remains to be learned at a molecular level about protective E2-reactive antibodies, since HCV infection persists in some individuals despite early development of broadly neutralizing plasma. To examine B cell repertoire features associated with broad neutralization and viral clearance, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of people with cleared or persistent HCV, including subjects with high or low plasma neutralizing breadth in both clearance and persistence groups. We identified many E2-reactive public BCR clonotypes, which are antibody clones with the same V and J-genes and identical CDR3 sequences, shared among subjects grouped by either clearance or neutralization status. The majority (89) of these public clonotypes were shared by two subjects with broad plasma neutralizing activity and cleared infection, but not found in subjects with high plasma neutralizing breadth and persistent infection. We cloned a potent, cross-reactive neutralizing monoclonal antibody (mAb) by pairing the most abundant public heavy and light chains from these two subjects, providing evidence that broadly E2-reactive public clonotypes arise in a subset of individuals with broadly neutralizing plasma and spontaneous clearance of infection. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

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Identification of a Residue in Hepatitis C Virus E2 Glycoprotein That Determines Scavenger Receptor BI and CD81 Receptor Dependency and Sensitivity to Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.01569-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12020-12029 ◽

Cited By ~ 124

Author(s):

Joe Grove ◽

Søren Nielsen ◽

Jin Zhong ◽

Margaret F. Bassendine ◽

Heidi E. Drummer ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Particle Density ◽

Gradient Centrifugation ◽

Scavenger Receptor ◽

Scavenger Receptor Bi ◽

Host Immune Responses ◽

The Relationship

ABSTRACT Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

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Production and Characterization of Monoclonal Antibodies Specific for a Conserved Epitope within Hepatitis C Virus Hypervariable Region 1

Journal of Virology ◽

10.1128/jvi.75.24.12412-12420.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 12412-12420 ◽

Cited By ~ 18

Author(s):

Chengyao Li ◽

Daniel Candotti ◽

Jean-Pierre Allain

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Hypervariable Region ◽

Passive Immunization ◽

Immune Recognition ◽

Hypervariable Region 1 ◽

Neutralizing Capacity ◽

Conserved Epitope

ABSTRACT Frequent mutations in hypervariable region 1 (HVR1) of the main envelope protein of hepatitis C virus (HCV) is a major mechanism of persistence by escaping the host immune recognition. HVR1 contains an epitope eliciting neutralizing antibodies. This study was aimed to prepare broadly cross-reacting, high-affinity, monoclonal antibodies (MAb) to the HVR1 C terminus of HCV with potential therapeutic neutralizing capacity. A conserved amino residue group of glycine (G) at position 23 and glutamic acid (Q) at position 26 in HVR1 was confirmed as a key epitope against which two MAbs were selected and characterized. MAbs 2P24 and 15H4 were immunoglobulin G1 kappa chain [IgG1(κ)], cross-reacted with 32 and 30 of 39 random C-terminal HVR1 peptides, respectively, and did not react with other HCV peptides. The VH of 2P24 and 15H4 heavy chains originated from Igh germ line v gene family 1 and 8, respectively. In contrast, the VL κ sequences were highly homologous. The affinity (K d ) of 2P24 and 15H4 (10−9 or 10−8 M with two immunizing peptides and 10−8 M with two nonimmunizing HVR1 peptides) paralleled the reactivity obtained with peptide enzyme immunoassay. MAbs 2P24 and 15H4 captured 25 of 31 (81%) HCV in unselected patients' plasmas. These antibodies also blocked HCV binding to Molt-4 cells in a dose-dependent fashion. The data presented suggest that broadly cross-reactive MAbs to a conserved epitope within HCV HVR1 can be produced. Clinical application for passive immunization in HCV-related chronic liver disease and after liver transplantation is considered.

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Hypervariable region 1 shielding of hepatitis C virus is a main contributor to genotypic differences in neutralization sensitivity

Hepatology ◽

10.1002/hep.28705 ◽

2016 ◽

Vol 64 (6) ◽

pp. 1881-1892 ◽

Cited By ~ 41

Author(s):

Jannick Prentoe ◽

Rodrigo Velázquez-Moctezuma ◽

Steven K.H. Foung ◽

Mansun Law ◽

Jens Bukh

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Genotypic Differences ◽

Neutralization Sensitivity ◽

Main Contributor ◽

Hypervariable Region 1

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Humoral immune response to hypervariable region 1 of a hepatitis C viral envelope glycoprotein (gp70) obtained from an asymptomatic carrier

International Hepatology Communications ◽

10.1016/0928-4346(94)00157-z ◽

1995 ◽

Vol 3 (2) ◽

pp. 77-81

Author(s):

T NAKAZAWA ◽

N KATO ◽

T FUJIOKA ◽

A SHIBUYA ◽

K SHIMOTOHNO

Keyword(s):

Immune Response ◽

Hepatitis C ◽

Humoral Immune Response ◽

Envelope Glycoprotein ◽

Asymptomatic Carrier ◽

Hypervariable Region ◽

Viral Envelope ◽

Humoral Immune ◽

Viral Envelope Glycoprotein ◽

Hypervariable Region 1

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Antibody Response to Hypervariable Region 1 Interferes with Broadly Neutralizing Antibodies to Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.02458-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3112-3122 ◽

Cited By ~ 35

Author(s):

Zhen-yong Keck ◽

Christine Girard-Blanc ◽

Wenyan Wang ◽

Patrick Lau ◽

Adam Zuiani ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Persistent Infection ◽

Neutralizing Antibodies ◽

Hypervariable Region ◽

Humoral Response ◽

Broadly Neutralizing Antibodies ◽

Alanine Scanning Mutagenesis ◽

Effect Analysis ◽

Hypervariable Region 1

ABSTRACTHypervariable region 1 (HVR1) (amino acids [aa] 384 to 410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412 to 423 revealed two subsets, designated HC33 HMAbs. While both subsets have contact residues within aa 412 to 423, alanine-scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412 to 423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412 to 423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside aa 412 to 423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection.IMPORTANCEHVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from their epitopes. This study provides insight into a new immune antagonism mechanism by which the binding of antibodies to HVR1 blocks the binding and activity of broadly neutralizing antibodies to HCV. Immunization strategies that avoid the induction of HVR1 antibodies should increase the inhibitory activity of broadly neutralizing anti-HCV antibodies elicited by candidate vaccines.

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Global and local envelope protein dynamics of hepatitis C virus determine broad antibody sensitivity

Science Advances ◽

10.1126/sciadv.abb5938 ◽

2020 ◽

Vol 6 (35) ◽

pp. eabb5938 ◽

Cited By ~ 1

Author(s):

Elias H. Augestad ◽

Matteo Castelli ◽

Nicola Clementi ◽

Luisa J. Ströh ◽

Thomas Krey ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Envelope Protein ◽

Neutralizing Antibodies ◽

Scavenger Receptor ◽

Hypervariable Region ◽

Antigenic Site ◽

Conformational Space ◽

Type I ◽

Class B

Broad antibody sensitivity differences of hepatitis C virus (HCV) isolates and their ability to persist in the presence of neutralizing antibodies (NAbs) remain poorly understood. Here, we show that polymorphisms within glycoprotein E2, including hypervariable region 1 (HVR1) and antigenic site 412 (AS412), broadly affect NAb sensitivity by shifting global envelope protein conformation dynamics between theoretical “closed,” neutralization-resistant and “open,” neutralization-sensitive states. The conformational space of AS412 was skewed toward β-hairpin–like conformations in closed states, which also depended on HVR1, assigning function to these enigmatic E2 regions. Scavenger receptor class B, type I entry dependency of HCV was associated with NAb resistance and correlated perfectly with decreased virus propensity to interact with HCV co-receptor CD81, indicating that decreased NAb sensitivity resulted in a more complex entry pathway. This link between global E1/E2 states and functionally distinct AS412 conformations has important implications for targeting AS412 in rational HCV vaccine designs.

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