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.

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 Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system

Hepatology ◽  
2006 ◽  
Vol 44 (6) ◽  
pp. 1626-1634 ◽  
Author(s):  
Muriel Lavie ◽  
Cécile Voisset ◽  
Ngoc Vu-Dac ◽  
Virginie Zurawski ◽  
Gilles Duverlie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Culture System ◽  
Serum Amyloid A ◽  
Virus Entry ◽  
Cell Culture System ◽  
Amyloid A ◽  
A Cell

scholarly journals Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins

2007 ◽  
Vol 88 (9) ◽  
pp. 2495-2503 ◽  
Author(s):  
David Delgrange ◽  
André Pillez ◽  
Sandrine Castelain ◽  
Laurence Cocquerel ◽  
Yves Rouillé ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Potential Effect ◽  
Structural Proteins ◽  
Wild Type ◽  
Genotype 1A ◽  
Viral Particles ◽  
A Cell

Recently, the characterization of a cell culture system allowing the amplification of an authentic virus, named hepatitis C virus cell culture (HCVcc), has been reported by several groups. To obtain higher HCV particle productions, we investigated the potential effect of some amino acid changes on the infectivity of the JFH-1 isolate. As a first approach, successive infections of naïve Huh-7 cells were performed until high viral titres were obtained, and mutations that appeared during this selection were identified by sequencing. Only one major modification, N534K, located in the E2 glycoprotein sequence was found. Interestingly, this mutation prevented core glycosylation of E2 site 6. In addition, JFH-1 generated with this modification facilitated the infection of Huh-7 cells. In a second approach to identify mutations favouring HCVcc infectivity, we exploited the observation that a chimeric virus containing the genotype 1a core protein in the context of JFH-1 background was more infectious than wild-type JFH-1 isolate. Sequence alignment between JFH-1 and our chimera, led us to identify two major positions, 172 and 173, which were not occupied by similar amino acids in these two viruses. Importantly, higher viral titres were obtained by introducing these residues in the context of wild-type JFH-1. Altogether, our data indicate that a more robust production of HCVcc particles can be obtained by introducing a few specific mutations in JFH-1 structural proteins.

scholarly journals Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

2010 ◽  
Vol 84 (22) ◽  
pp. 11905-11915 ◽  
Author(s):  
François Helle ◽  
Gabrielle Vieyres ◽  
Laure Elkrief ◽  
Costin-Ioan Popescu ◽  
Czeslaw Wychowski ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies ◽  
Point Mutations ◽  
Envelope Proteins ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Humoral Immune ◽  
A Cell

ABSTRACT Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a “glycan shield” at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.

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.

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