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

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.More than 170 million people worldwide are seropositive for hepatitis C virus (HCV) (65). Despite induction of effective immune responses, 80% of HCV-infected individuals progress from acute to chronic hepatitis, which can lead to cirrhosis and hepatocellular carcinoma (42). Escape strategies may be operating for both the innate and the adaptive immune systems, but the exact mechanisms whereby HCV establishes and maintains its persistence have not yet been determined (59). It is known that an immune response composed of both cellular (CD4 ϩ and CD8 ϩ T cells) and humoral (antibodies produced by B cells) immune responses is present during acute and chronic infections (40). Typically, HCV infection results in production of antibodies to various HCV proteins in the majority of chronically infected people. Moreover, neutralizing antibodies have been detected in sera of HCV-infected patients (2,3,19,39,41,44,69), but the role of these antibodies in host protection has been questioned since reinfection in both humans and chimpanzees has been described (18, 38). Investigations of HCV-neutralizing antibodies have long been hampered by difficulties in propagating HCV in cell culture, but the recent development of HCV pseudoparticles (HCVpp) (3, 15, 31), consisting of the native HCV envelope glycoproteins, E1 and E2, assembled onto retroviral core particles, offered new opportunities in this field (2,3,31,39,41,44,52,69).The ability of HCV to persist in its hos...

In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

“…Neutralizing antibodies are typically delayed in appearance in acute HCV infection, generally do not confer sterile immunity, and are usually present in chronically infected persons (30)(31)(32). Nonetheless, substantial data suggest that virus-specific antibodies exert a level of control over HCV infection.…”

“…This suggests that the AP33 epitope is poorly immunogenic, possibly because it is shielded by glycans. Residue N417 is a highly conserved glycosylation site, present in all HCV genotypes (32). Neutralization by the closely related rat mAb, 3/11 (22), is enhanced by mutations knocking out N-linked glycosylation at residues 417, 532, and 645, suggesting that these residues may be in close proximity to the 3/11 (and thus AP33) epitope (32).…”

“…Residue N417 is a highly conserved glycosylation site, present in all HCV genotypes (32). Neutralization by the closely related rat mAb, 3/11 (22), is enhanced by mutations knocking out N-linked glycosylation at residues 417, 532, and 645, suggesting that these residues may be in close proximity to the 3/11 (and thus AP33) epitope (32). The enhanced neutralization associated with ablation of glycosylation at N645 is particularly interesting, as it is only 10 residues distant from E655, which our data suggest contributes to AP33 recognition (Fig.…”

Structural elucidation of critical residues involved in binding of human monoclonal antibodies to hepatitis C virus E2 envelope glycoprotein

“…Previous studies showed that viral envelope proteins usually contain N-linked glycans that may play a major role in their folding, entry functions or in modulating the immune response [46,52,53]. In a recent paper, Helle et al describes the neutralizing activity of anti-HCV antibodies, and they found that this neutralization is modulated by specific glycans on the E2 protein [54]. In addition, they observed that HCVpp containing non-glycosylation mutants at positions N476 and N540 were more sensitive to neutralization by all the mAb tested, among them the CBH-5 HMAb.…”

“…This observation seems in contradiction with the mass spectrometric results which indicated that the N476 glycosylation site might be involved in the antibody binding. However, the mass spectrometric analyses were performed with intact, glycosylated wt E2 protein, whereas Helle et al used single glycosylation mutants to test their neutralizing effect against different antibodies [54]. Thus, alanine mutation of a single glycosylation site has no apparent effect on the ability of the antibody to bind E2 protein, in comparison with the intact wt glycosylated E2 protein which most probably has a conformation that is required for antibody-antigen recognition.…”