scholarly journals The Neutralizing Activity of Anti-Hepatitis C Virus Antibodies Is Modulated by Specific Glycans on the E2 Envelope Protein

2007 ◽  
Vol 81 (15) ◽  
pp. 8101-8111 ◽  
Author(s):  
François Helle ◽  
Anne Goffard ◽  
Virginie Morel ◽  
Gilles Duverlie ◽  
Jane McKeating ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Binding Site ◽  
Neutralizing Antibody ◽  
Soluble Form ◽  
Envelope Glycoproteins ◽  
Neutralizing Activity ◽  
Humoral Immune ◽  
Glycosylation Sites ◽  
High Level

ABSTRACT Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with up to 5 and 11 N-linked glycans on E1 and E2, respectively. Most of the glycosylation sites on HCV envelope glycoproteins are conserved, and some of the glycans associated with these proteins have been shown to play an essential role in protein folding and HCV entry. Such a high level of glycosylation suggests that these glycans can limit the immunogenicity of HCV envelope proteins and restrict the binding of some antibodies to their epitopes. Here, we investigated whether these glycans can modulate the neutralizing activity of anti-HCV antibodies. HCV pseudoparticles (HCVpp) bearing wild-type glycoproteins or mutants at individual glycosylation sites were evaluated for their sensitivity to neutralization by antibodies from the sera of infected patients and anti-E2 monoclonal antibodies. While we did not find any evidence that N-linked glycans of E1 contribute to the masking of neutralizing epitopes, our data demonstrate that at least three glycans on E2 (denoted E2N1, E2N6, and E2N11) reduce the sensitivity of HCVpp to antibody neutralization. Importantly, these three glycans also reduced the access of CD81 to its E2 binding site, as shown by using a soluble form of the extracellular loop of CD81 in inhibition of entry. These data suggest that glycans E2N1, E2N6, and E2N11 are close to the binding site of CD81 and modulate both CD81 and neutralizing antibody binding to E2. In conclusion, this work indicates that HCV glycans contribute to the evasion of HCV from the humoral immune response.

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles

Vaccine ◽  
2011 ◽  
Vol 30 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Alla Kachko ◽  
Galina Kochneva ◽  
Galina Sivolobova ◽  
Antonina Grazhdantseva ◽  
Tatyana Lupan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibody ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Peptide Recognition ◽  
Antibody Epitopes

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 Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Richard A. Urbanowicz ◽  
Ruixue Wang ◽  
John E. Schiel ◽  
Zhen-yong Keck ◽  
Melissa C. Kerzic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Glycosylation Sites ◽  
Complete Deletion ◽  
Global Health Challenge

ABSTRACTThe development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predictedN-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but thatN-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specificN-glycans proximal to these epitopes.IMPORTANCEThe development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

scholarly journals Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Kai Deng ◽  
Zhanxue Xu ◽  
Mingxiao Chen ◽  
Xiaoxiang Liu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Neutralizing Antibodies ◽  
Hypervariable Region ◽  
Vaccine Design ◽  
Keyhole Limpet Hemocyanin ◽  
Hcv Genotypes ◽  
Neutralizing Activity ◽  
Humoral Immune ◽  
C Terminus

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

scholarly journals Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Glycoproteins

2005 ◽  
Vol 79 (13) ◽  
pp. 8400-8409 ◽  
Author(s):  
Anne Goffard ◽  
Nathalie Callens ◽  
Birke Bartosch ◽  
Czeslaw Wychowski ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Glycosylation Site ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Wild Type ◽  
Virus Envelope ◽  
Glycosylation Sites

ABSTRACT Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.

scholarly journals Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant fab fragments

Hepatology ◽  
1998 ◽  
Vol 28 (3) ◽  
pp. 810-814 ◽  
Author(s):  
Roberto Burioni ◽  
Paola Plaisant ◽  
Aldo Manzin ◽  
Domenico Rosa ◽  
Valeria Delli Carri ◽  
...  
Keyword(s):  
Immune Response ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Humoral Immune Response ◽  
Humoral Immune ◽  
Fab Fragments ◽  
E2 Glycoprotein

scholarly journals Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

2004 ◽  
Vol 78 (6) ◽  
pp. 2994-3002 ◽  
Author(s):  
Anne Op De Beeck ◽  
Cécile Voisset ◽  
Birke Bartosch ◽  
Yann Ciczora ◽  
Laurence Cocquerel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Conformational Changes ◽  
Structural Changes ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

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