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Immunoglobulin GM allotypes, genetic markers of IgG, are associated with the outcome of hepatitis C virus (HCV) infection, but the underlying mechanisms are not completely understood. HCV has evolved mechanisms for decreasing the efficacy of the host immune response. One such strategy might involve the scavenging of the Fcγ domain of the anti-HCV IgG antibodies by its Fcγ-receptor–like core protein and thus interfering with the Fcγ-mediated host defense mechanisms. We tested the hypothesis that GM allotypes modulate this viral strategy through differential binding to the core protein. Here we show that the absorbance values for binding to the core protein were higher for GM23+IgG2 than for GM23−IgG2 (p=0.027), a finding that at least in part explains the involvement of GM allotypes in the outcome of HCV infection. These findings also contribute toward our understanding of the mechanisms that maintain strong linkage disequilibrium between particular GM alleles.
Hepatitis C virus (HCV) infection is one of the most common causes of liver disease in the world. Approximately 20–40% of the acutely infected individuals spontaneously clear the virus, while the rest eventually develop chronic liver disease. Among the factors influencing the outcome of HCV infection, the host genetic factors are thought to play a predominant role (4,6). We have previously reported involvement of immunoglobulin (Ig) GM and KM allotypes—genetic markers of γ and κ chains, respectively—in the outcome of HCV infection (8). The mechanisms underlying this association are not completely understood.
In an effort to delineate these mechanisms, in a previous study involving IgG1 allotypes, we tested the hypothesis that GM allotypes act as effect modifiers of the strategies employed by the virus to evade host immunosurveillance (7). The HCV core protein has Fcγ receptor (FcγR)-like properties, which the virus probably exploits to modulate the effector functions of the host immune cells, resulting in the evasion of immunosurveillance (5). We showed that the HCV core protein had a significantly higher affinity for IgG1 with GM3 allotype than that for the allelic GM1,2,17 determinants, which explains at least in part the involvement of GM allotypes in the outcome of HCV infection (7). There is significant linkage disequilibrium between particular GM alleles expressed on different IgG subclasses (9,12), which may be a result of natural selection due to infectious agents like HCV. Therefore, for a better understanding of the mechanisms underlying the association of GM allotypes with the outcome of HCV infection, it is essential to examine the GM alleles on other subclasses for their possible role as the modulators of the core-IgG binding affinities.
In the present report we have evaluated the binding affinity of the HCV core protein to the IgG2 proteins that differ in their expression of the GM23 allotype, a valine-to-methionine substitution at position 282 of the IgG2 molecule.
The study population consisted of anti-HCV-antibody–negative blood donors—17 South American Indians and 18 Caucasians from the U.S. The study was approved by the local institutional review board for human research.
The HCV core protein was expressed and purified using a commercially available core protein recombinant DNA construct. Bacterial expression–ready full-length (191aa) recombinant HCV genotype 1 core protein clone, carrying a C terminal polyhistidine tag was purchased (Bioclone Inc., San Diego, CA, USA) and expressed in Escherichia coli BL21 (DE3) strain. The protein was purified by affinity chromatography over a Ni-NTA (nickel nitrilotriacetic acid) spin column (Qiagen, Valencia, CA, USA). Protein concentration was estimated using Bradford dye-binding reagent (Bio-Rad, Hercules, CA, USA). Purity was checked by SDS-PAGE. The amino acid sequence of this protein was the same as that used in previous studies (5,7).
IgG2 proteins were isolated from the sera by subclass-specific affinity chromatography, using a monoclonal anti-human IgG2 antibody-coupled agarose column (Sigma-Aldrich, St. Louis, MO, USA). This preparation was used for binding studies.
The binding of IgG2 proteins (GM23+or GM23−allele) to the HCV core protein was measured by an ELISA. The absorbance value for binding of each IgG2 protein to the HCV core protein is relative to its binding to an Fc-specific sheep anti-human IgG antibody (Sigma-Aldrich), which was used as a reference and had no specificity for any GM allotypes. For each affinity-purified IgG2 preparation, a full titration curve was generated on sheep anti-human-IgG–coated ELISA plates, and the dilution required to give the absorbance at the midpoint of the titration curve (mid-OD) was determined in a manner similar to that described by Shields et al. (11). This dilution was used for measuring the binding of IgG2 to the core protein. Experiments were replicated three times, and each time in duplicate. Thus, each absorbance value presented in Table 1 represents a mean of six observations.
For statistical analyses, absorbance values were log10 transformed to obtain residual homoscedasticity. For comparison of the absorbance values for binding of GM23+and GM23−proteins to the core protein, a mixed linear regression model (SAS v9.1 Proc Mixed, Cary, NC) was used. This model included a random subject effect with a compound symmetry covariance structure to account for the intraclass correlation among individual subjects' six repeated measurements. All tests were two-tailed, and the statistical significance was defined as p<0.05.
As presented in Table 1, the results of the mixed model show that the mean absorbance values were significantly higher for the IgG2 proteins expressing the GM 23+allotype than those expressing the allelic GM23−allotype (0.54 versus 0.32; p=0.027).
As mentioned before, the IgG2 proteins in these experiments were isolated from sera that were negative for anti-HCV antibodies. Since previous studies have established that the recombinant HCV core protein does not bind to the Fab fragments of “nonimmune” IgG (5), the binding between IgG2 and the HCV core protein reported here must involve the Fc region of the IgG2 molecules. These results, together with those reported for the IgG1 allotypes (7), could, at least in part, explain the involvement of GM allotypes in the outcome of HCV infection. The differential binding of the HCV core protein to the GM23-disparate IgG2 proteins shown here could influence the strategies employed by this virus to evade host immunosurveillance. FcγR-like HCV core protein could bind anti-core antibodies by “bipolar bridging.” In this model, the Fab part of the antibody molecule (paratope) binds to its antigenic target (epitope), whereas the Fcγ part of the antibody binds to the FcγR-like binding site on the viral protein (2,5). This binding may offer survival advantage to the virus by sterically hindering the access of FcγR-expressing effector cells to HCV-infected cells. The HCV core protein may scavenge the Fcγ domains of anti-core antibodies after the binding of their paratopes to their antigenic target, thereby interfering with the effector functions—such as antibody-dependent cellular cytotoxicity (ADCC)—mediated by the Fcγ domain of the bound antibody. Although antibody responses to HCV proteins are predominantly of IgG1 and IgG3 subclasses, antibodies of the IgG2 isotype have also been reported (1,14).
Since binding of IgG2 of GM23+allotype to HCV core protein was significantly higher than that of GM23−allotype, IgG2 antibodies directed to the core protein in subjects with this determinant are more likely to have their Fc domains scavenged, thereby reducing their immunological competence to eliminate the virus or circulating nucleocapsids through ADCC and other Fc-mediated effector mechanisms. This would suggest a higher prevalence of GM23 in subjects with persistent HCV infection compared to those who have cleared the virus. This appears to be the case in a study population we reported on earlier (8). Among subjects with persistent HCV infection (n=196), there were 51 subjects (26%) carrying the GM23 allele, while among patients that cleared the virus (n=99), there were 22 subjects (22%) carrying the GM23 allele. This trend in prevalence of GM23 in subjects with persistence/clearance of HCV infection is similar, but not as pronounced, to that reported for the GM3 allele, which is in linkage disequilibrium with the GM23 allele, and also has higher affinity for the core protein than its allelic counterpart (7). Thus it is tempting to speculate that one mechanism underlying the maintenance of linkage disequilibrium between the two determinants may be their similar contribution to the differential immunity to infectious agents such as HCV, which probably have played and continue to play a significant role in our evolutionary history.
The host determinants of the outcome of HCV infection are clearly polygenic. Simultaneous examination of other candidate genes and how they modulate other viral immune evasion strategies—molecular mimicry between certain Ig sequences and the HCV envelope protein E2, for instance (3)—would be necessary to better understand the mechanisms responsible for the inter-individual differences in the outcome of HCV infection.
This work was supported in part by grant R01 DK070877 from the National Institutes of Health.