Monoclonal Antibodies to the Hypervariable Region 1 of Hepatitis C Virus Capture Virus and Inhibit Virus Adsorption to Susceptible Cells in Vitro

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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In vivo and in vitro evidence that cross-reactive antibodies to C-terminus of hypervariable region 1 do not neutralize heterologous hepatitis C virus

Vaccine ◽

10.1016/s0264-410x(02)00271-2 ◽

2002 ◽

Vol 20 (25-26) ◽

pp. 3095-3103 ◽

Cited By ~ 10

Author(s):

M ESUMI

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Hypervariable Region 1 ◽

C Terminus

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In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

PLoS Pathogens ◽

10.1371/journal.ppat.1009720 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009720

Author(s):

Christina Holmboe Olesen ◽

Elias H. Augestad ◽

Fulvia Troise ◽

Jens Bukh ◽

Jannick Prentoe

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Neutralizing Antibody ◽

Virus Envelope ◽

E2 Protein ◽

Hypervariable Region 1 ◽

Increased Sensitivity ◽

Viral Attenuation

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.

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Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

Journal of Virology ◽

10.1128/jvi.02070-15 ◽

2015 ◽

Vol 89 (24) ◽

pp. 12245-12261 ◽

Cited By ~ 32

Author(s):

Yousef Alhammad ◽

Jun Gu ◽

Irene Boo ◽

David Harrison ◽

Kathleen McCaffrey ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Variable Region ◽

Antigenic Structure ◽

Variable Regions ◽

Core Domain ◽

Hypervariable Region 1 ◽

Glycoprotein E2

ABSTRACTHepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661and E2661lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion.IMPORTANCEThis study reveals conformational and antigenic differences between the Δ123 and intact E2661glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

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Chimeric monoclonal antibodies to hypervariable region 1 of hepatitis C virus

Journal of General Virology ◽

10.1099/vir.0.80912-0 ◽

2005 ◽

Vol 86 (6) ◽

pp. 1709-1716 ◽

Cited By ~ 7

Author(s):

Chengyao Li ◽

Jean-Pierre Allain

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hypervariable Region ◽

Passive Immunization ◽

Dependent Manner ◽

Variable Regions ◽

Binding Characteristics ◽

Hypervariable Region 1

Two chimeric monoclonal antibodies (cAbs), 2P24 and 15H4, to hypervariable region 1 (HVR1) of hepatitis C virus (HCV) were constructed by grafting the variable regions of murine monoclonal antibodies (mAbs) 2P24 and 15H4 to a human IgG1 kappa constant region. Two cAb-producing cell lines were adapted to serum-free media. Both cAb 2P24 and cAb 15H4 cell lines produced 3–5 μg antibodies ml−1 after 3–5 days culture. cAbs retained binding characteristics similar to those observed in the original mAbs. There was no clear difference in affinity between binding of cAbs and mAbs to seven HVR1 peptides. Mixtures of biotinylated cAbs or mAbs reacted with 32 (86 %) and 31 (84 %) of 37 HVR1 peptides, respectively, but not with non-HVR1 control peptides. HCV from 16 out of 18 (89 %) random HCV-containing plasmas was captured by the mixture of biotinylated cAbs. The capture from IgG-depleted plasmas suggested that cAbs captured mainly free rather than complexed HCV, irrespective of genotype. A mixture of the two cAbs inhibited HCV binding to Molt-4 cells in a dose-dependent manner. These cAbs may be useful for prevention of nosocomial HCV infection and passive immunization to prevent HCV reinfection after liver transplantation.

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A chimeric GB virus B encoding the hepatitis C virus hypervariable region 1 is infectious in vivo

Journal of General Virology ◽

10.1099/vir.0.82467-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 895-902 ◽

Cited By ~ 21

Author(s):

G. Haqshenas ◽

X. Dong ◽

H. Netter ◽

J. Torresi ◽

E. J. Gowans

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Immunoblot Analysis ◽

Post Inoculation ◽

Sequencing Analysis ◽

Coding Region ◽

Hypervariable Region 1

Two GB virus B (GBV-B) chimeric genomes, GBV-HVR and GBV-HVRh (with a hinge), containing the coding region of the immunodominant hypervariable region 1 (HVR1) of the E2 envelope protein of Hepatitis C virus (HCV) were constructed. Immunoblot analysis confirmed that HVR1 was anchored to the GBV-B E2 protein. To investigate the replication competence and in vivo stability of in vitro-generated chimeric RNA transcripts, two naïve marmosets were inoculated intrahepatically with the transcripts. The GBV-HVR chimeric genome was detectable for 2 weeks post-inoculation (p.i.), whereas GBV-HVRh reverted to wild type 1 week p.i. Sequencing analysis of the HVR1 and flanking regions from GBV-HVR RNA isolated from marmoset serum demonstrated that the HVR1 insert remained unaltered in the GBV-HVR chimera for 2 weeks. Inoculation of a naïve marmoset with serum collected at 1 week p.i. also resulted in viraemia and confirmed that the serum contained infectious particles. All animals cleared the infection by 3 weeks p.i. and remained negative for the remaining weeks. The chimera may prove useful for the in vivo examination of any HCV HVR1-based vaccine candidates.

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Monoclonal Antibodies with Broad Specificity for Hepatitis C Virus Hypervariable Region 1 Variants Can Recognize Viral Particles

The Journal of Immunology ◽

10.4049/jimmunol.167.7.3878 ◽

2001 ◽

Vol 167 (7) ◽

pp. 3878-3886 ◽

Cited By ~ 22

Author(s):

Antonella Cerino ◽

Annalisa Meola ◽

Laura Segagni ◽

Milena Furione ◽

Sabrina Marciano ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hypervariable Region ◽

Hypervariable Region 1 ◽

Viral Particles ◽

Broad Specificity

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Expression of Noncovalent Hepatitis C Virus Envelope E1-E2 Complexes Is Not Required for the Induction of Antibodies with Neutralizing Properties following DNA Immunization

Journal of Virology ◽

10.1128/jvi.73.9.7497-7504.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7497-7504 ◽

Cited By ~ 24

Author(s):

A. Fournillier ◽

E. Depla ◽

P. Karayiannis ◽

O. Vidalin ◽

G. Maertens ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Hypervariable Region ◽

Virus Envelope ◽

Hypervariable Region 1 ◽

Viral Particles ◽

Expression Plasmids

ABSTRACT Interactive glycoproteins present on the surface of viral particles represent the main target of neutralizing antibodies. The ability of DNA vaccination to induce antibodies directed at such structures was investigated by using eight different expression plasmids engineered either to favor or to prevent interaction between the hepatitis C virus (HCV) envelope glycoproteins E1 and E2. Independently of the injection route (intramuscular or intraepidermal), plasmids expressing antigens capable of forming heterodimers presumed to be the prebudding form of the HCV envelope protein complex failed to induce any significant, stable antibodies following injection in mice. In sharp contrast, high titers of antibodies directed at both conformational and linear determinants were induced by using plasmids expressing severely truncated antigens that have lost the ability to form native complexes. In addition, only a truncated form of E2 induced antibodies reacting against the hypervariable region 1 of E2 (specifically with the C-terminal part of it) known to contain a neutralization site. When injected intraepidermally into small primates, the truncated E2-encoding plasmid induced antibodies able to neutralize in vitro the binding of a purified E2 protein onto susceptible cells. Because such antibodies have been associated with viral clearance in both humans and chimpanzees, these findings may have important implications for the development of protective immunity against HCV.

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