Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Abstract

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.

FIG. 1.

Neutralization of the HSV-1 WT and gC/gE mutant viruses by antibody. The WT virus or the gC/gE mutant virus was incubated for 1 h at 37°C with 1% serum treated with EDTA to inactivate complement. Comparing antibody-mediated neutralization of the gC/gE mutant with that of the WT virus: HIV negative, P = 0.18; CD4 <200/μl, P < 0.01; CD4 200 to 500/μl, P < 0.06; and CD4 >500/μl, P < 0.04; all four groups combined, P < 0.0001.

FIG. 2.

Neutralization of the WT and gC/gE mutant viruses by pooled human IgG. The WT and gC/gE mutant viruses were incubated with various concentrations of pooled human IgG and neutralization determined. Results shown here represent the means and standard errors of three determinations. Comparing the WT and gC/gE mutant viruses at 25, 50, and 100 μg/ml, P < 0.01.

FIG. 3.

The gC/gE mutant and WT viruses express comparable concentrations of HSV-1 glycoproteins on the virion. Purified gC/gE mutant virus and WT virus were evaluated for VP5, gB, gC, gD, gE, gH, gL, and gI expression by Western blotting and densitometry analysis to compare relative glycoprotein concentrations. Each lane contains 1 × 10⁶ PFU of the WT or gC/gE mutant virus. The number associated with each gel represents the ratio of the density of the glycoprotein band above the number to the density of the VP5 band below the number. The results are representative of two separate experiments that yielded similar findings.

FIG. 4.

The role of the HSV-1 FcγR in antibody neutralization. (A) Possible model to explain the greater susceptibility of the gC/gE mutant virus to neutralization by antibody alone. On the left side of the WT virus model, gE binds the Fc domain of IgG, preventing the F(ab′)₂ from binding antigen (shown here as gD). On the right side of the WT virus model, antibody bipolar bridging is shown in which the Fab domain binds to gD and the Fc domain of the same IgG molecule binds to gE (10). If antibody binding occurs as shown on the left side but not the right side of the WT virus model, the HSV-1 FcγR (comprised by gE/gI) may prevent some F(ab′)₂ domains from interacting with their target antigens. In the model of the gC/gE mutant virus, ΔgE fails to bind the IgG Fc domain, allowing the F(ab′)₂ domain to bind antigen (shown as gD) and to neutralize the virus. (B) A nonfunctional viral FcγR does not explain the increased susceptibility of the gC/gE mutant virus to antibody neutralization. Viruses were incubated with 100 μg/ml of pooled human IgG, and the amount of neutralization was determined. Results shown represent mean titers ± standard errors (n = 8 to 17 for each virus). No significant differences were detected between the antibody neutralization of the FcγR-defective virus (NS-gE339 [gE mutant FcR−]) and that of the FcγR-intact virus (NS-gCΔC3 [gC mutant FcR+]); P = 0.47. The gC/gE mutant virus was neutralized significantly more than either the FcγR− virus or the FcγR+ virus; P < 0.05 and P < 0.02, respectively.

FIG. 5.

Neutralization by IgG antibodies to HSV-1 glycoproteins. (A to E) The WT and gC/gE mutant viruses were incubated with 1, 10, 30, or 100 μg/ml of anti-gB, gC, gD, gH/gL, or gI IgG. Each experiment was performed once.

FIG. 6.

Neutralization by combinations of antibodies to HSV-1 glycoproteins involved in entry. The WT and gC/gE mutant viruses were incubated with a total of 10 μg/ml of IgG consisting of 5 μg/ml each of anti-gB and anti-gD IgG, anti-gB and anti-gH/gL IgG, anti-gD and anti-gH/gL IgG, or anti-gD and anti-gI IgG. Results represent the means ± standard errors of three determinations for each combination (P < 0.005, for a comparison of the WT and gC/gE mutant viruses for each combination, except anti-gD and anti-gI, for which P = 0.2).

FIG. 7.

Model of gC and gE on the virion envelope, blocking antibody access and enabling the interaction of glycoproteins that are required for fusion and entry. At the left, gC and gE on the WT virus block access of the antibodies, which enables the interaction between gB and gD that is required for fusion and entry. On the right, gC and gE on the gC/gE mutant virus fail to block antibody access. Our results suggest that antibodies must bind to at least two of the glycoproteins gB, gD, or gH/gL, to prevent glycoprotein interactions and inhibit entry.