Neutralizing Antibodies Induced by Cell Culture–Derived Hepatitis C Virus Protect Against Infection in Mice

2013 ◽  
Vol 145 (2) ◽  
pp. 447-455.e4 ◽  
Author(s):  
Daisuke Akazawa ◽  
Masaki Moriyama ◽  
Hiroshi Yokokawa ◽  
Noriaki Omi ◽  
Noriyuki Watanabe ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies

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

2008 ◽  
Vol 82 (24) ◽  
pp. 12020-12029 ◽  
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.

scholarly journals Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

2010 ◽  
Vol 84 (11) ◽  
pp. 5494-5507 ◽  
Author(s):  
Simrat Dhillon ◽  
Jeroen Witteveldt ◽  
Derek Gatherer ◽  
Ania M. Owsianka ◽  
Mirjam B. Zeisel ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies ◽  
Single Mutation ◽  
Adaptive Mutations ◽  
E2 Glycoprotein ◽  
Highly Sensitive ◽  
Infected Cells ◽  
Conserved Region

ABSTRACT 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 JFH1WT, the JFH1N415D 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 JFH1WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1T416A and JFH1I422L more efficiently than the WT virus, while neutralization of JFH1N417S and JFH1G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1N415D, highlighting the importance of the E2 412-423 region in virus entry.

scholarly journals Enhancing the antigenicity and immunogenicity of monomeric forms of hepatitis C virus E2 for use as a preventive vaccine

2020 ◽  
Vol 295 (21) ◽  
pp. 7179-7192 ◽  
Author(s):  
Rob J. Center ◽  
Irene Boo ◽  
Lilian Phu ◽  
Joey McGregor ◽  
Pantelis Poumbourios ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies ◽  
Variable Regions ◽  
Preventive Vaccine ◽  
A Cell ◽  
Homogeneous Composition ◽  
Sequential Reduction ◽  
Immunogenic Properties

The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture–derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor–binding domain lacking three variable regions, Δ123-HMW, elicits broad neutralizing activity against the seven major genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogeneous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Δ123r and Δ123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and nonneutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Δ123r and Δ123A7r recapitulated the immunogenic properties of cell culture–derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.

Investigations into the receptor requirements of Hepatitis C Virus (HCV) pseudoparticles and cell culture grown virions

2006 ◽  
Vol 44 (01) ◽  
Author(s):  
T von Hahn ◽  
M Flint ◽  
BD Lindenbach ◽  
A Boullier ◽  
O Quehenberger ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C

scholarly journals Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

2010 ◽  
Vol 84 (21) ◽  
pp. 10999-11009 ◽  
Author(s):  
Pablo Gastaminza ◽  
Kelly A. Dryden ◽  
Bryan Boyd ◽  
Malcolm R. Wood ◽  
Mansun Law ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Characteristics ◽  
Buoyant Density ◽  
Hcv Rna ◽  
Membrane Bilayer ◽  
Biophysical Characterization ◽  
Negative Stain ◽  
A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

scholarly journals Unique Features of Hepatitis C Virus Capsid Formation Revealed by De Novo Cell-Free Assembly

2004 ◽  
Vol 78 (17) ◽  
pp. 9257-9269 ◽  
Author(s):  
Kevin C. Klein ◽  
Stephen J. Polyak ◽  
Jaisri R. Lingappa
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Signal Sequence ◽  
Buoyant Density ◽  
Capsid Assembly ◽  
Culture Systems ◽  
Capsid Formation ◽  
Cell Culture Systems

ABSTRACT The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of ≈100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.

Sign in / Sign up

Export Citation Format

Share Document