Article Figures & Data
Figures
- FIG. 1.
(A) Western blot analysis examining gC2 expression in HSV-2 wild-type- and gC2-null-infected cell extracts. The blot was probed with rabbit anti-gC2 antibody R81 or rabbit anti-VP5 as a loading control. (B to E) Neutralization of HSV-1 (NS) and HSV-2 (G, 333, and 2.12) wild-type and gC-null strains by NHS. Viruses were incubated with PBS as a control or NHS as the source of complement at the concentrations indicated for 1 h at 37°C. Results are expressed as PFU/ml (% of control) and were calculated as follows: (PFU with NHS/PFU with PBS) × 100. Results represent the mean titers ± standard deviations of four separate experiments for strain G and of three experiments for strains 333, 2.12, and NS. AUC comparing wild-type and gC-null viruses: P < 0.0001 for strain G, P < 0.002 for strain 333, P < 0.001 for strain 2.12, and P < 0.05 for strain NS.
- FIG. 2.
(A) Complement-mediated neutralization of HSV-1 and HSV-2 gC-null viruses does not require activation of the alternative complement pathway. The gC1-null and gC2-null viruses were incubated with 50% NHS or NHS treated with EDTA (NHS+EDTA) or EGTA (NHS+EGTA). Virus treated with PBS served as the control. Results shown represent mean titers ± standard deviations of three independent experiments. P < 0.02, NHS with PBS for NS-gC1null or G-gC2null. P values were not significant for EDTA- or EGTA-treated NHS versus PBS for NS-gC1null or G-gC2null. (B) Complement-mediated neutralization of the gC1-null and gC2-null viruses is not dependent upon activation of the mannan-binding lectin complement pathway. Neutralization assays were performed on gC1-null and gC2-null viruses incubated with PBS, PBS containing d-mannose, 50% NHS, or NHS treated with either d-mannose or sucrose. Results shown represent mean titers ± standard deviations of three independent experiments. P < 0.02, comparing virus incubated with PBS or PBS containing d-mannose with virus incubated with NHS or NHS treated with d-mannose or sucrose.
- FIG. 3.
(A) Total hemolytic complement activity of NHS, NHS depleted of C1q (C1q depleted), and C1q-depleted serum reconstituted with C1q (C1q depleted+C1q). EA were incubated with serum and the percentage of EA lysed was determined. (B) gC1-null and gC2-null viruses are neutralized by the classical complement pathway. Neutralization experiments were performed with 20% NHS that was C1q depleted or reconstituted. Results shown represent mean titers ± standard deviations of three independent experiments. P < 0.02, comparing heat-inactivated NHS and either NHS or C1q-restored serum for NS-gC1null and G-gC2null. In contrast, values are not significant, P = 0.23 and 0.86, comparing heat-inactivated NHS with C1q-depleted serum for NS-gC1null and G-gC2null, respectively.
- FIG. 4.
Neutralization of HSV-1 and HSV-2 gC-null viruses by NHS from four donors occurs in the absence of specific antibodies against HSV. Neutralization experiments were performed on virus incubated with PBS, heat-inactivated NHS (inactive NHS), or 20% NHS. The four serum samples are labeled 1 to 4. Results shown represent the mean titers ± standard deviations of three separate experiments. P was <0.001 for all four sera, comparing PBS with NHS for NS-gC1null, and P ranged from 0.006 to <0.001 for G-gC2null viruses. In contrast, values are not significant for PBS versus heat-inactivated NHS.
- FIG. 5.
Neutralization of HSV-1 and HSV-2 gC-null viruses is dependent upon C3 activation. Neutralization experiments were performed on virus treated with PBS, 20% NHS, or NHS treated with either inactive compstatin (NHS+Linear) or active compstatin (NHS+4W9A). Results represent mean titers ± standard deviations of four independent experiments for NS-gC1null and three independent experiments for G-gC2null. For both viruses, differences were significant between PBS and NHS (P < 0.01) and PBS and NHS treated with inactive compstatin (P < 0.05). However, no significant differences were detected between PBS and NHS treated with active compstatin.
- FIG. 6.
(A) Total hemolytic complement activity of NHS, NHS depleted of C5 (C5 depleted), and C5-restored serum (C5 depleted+C5). (B) Neutralization of gC1-null and gC2-null viruses requires the presence of C5. Virus was incubated with PBS, 20% NHS, 20% NHS depleted of C5 (C5 depleted), or C5-restored serum (C5 depleted+C5). Results are expressed as the mean titers ± standard deviations of four independent experiments for NS-gC1null and two for G-gC2null. P < 0.005 for both viruses, comparing NHS and C5-restored NHS with PBS. No significant differences were detected between PBS and C5-depleted NHS. (C) Total hemolytic complement activity of NHS, NHS depleted of C6 (C6 depleted), and C6-restored serum (C6 depleted+C6). (D) Neutralization of gC1-null or gC2-null virus is not dependent on the presence of C6. Neutralization assays were performed on virus treated with PBS, 20% NHS, C6-depleted NHS (C6 depleted), and C6-restored serum (C6 depleted+C6). The results represent the mean titers ± standard deviations of three independent experiments for NS-gC1null and seven experiments for G-gC2null. P < 0.01 for both viruses, comparing PBS with NHS, C6-depleted, and C6-reconstituted serum.
- FIG. 7.
(A) Total hemolytic complement activity using NHS and NHS depleted of IgM (IgM depleted). Hemolytic assays were performed using antibody-coated sheep erythrocytes to demonstrate an intact classical complement pathway in IgM-depleted serum. (B) Natural IgM antibody is required for neutralization of HSV-1 and HSV-2 gC-null viruses. The gC-null viruses were incubated with 20% NHS, heat-inactivated NHS (inactive NHS), 20% NHS depleted of IgM (IgM depleted), and IgM-restored serum (IgM depleted+IgM). The results shown represent the mean titers ± standard deviations of four independent experiments. P < 0.05, comparing IgM-depleted with reconstituted sera for NS-gC1null and G-gC2null. In contrast, values are not significant when comparing PBS with IgM-depleted serum for NS-gC1null and G-gC2null, P = 0.83 and 0.31, respectively. (C to F) ELISA detects natural IgM antibody binding to gC2-null virus. Heat-inactivated 20% NHS from four donors was serially diluted and added to microtiter wells coated with G-gC2null or control wells. The experiment was performed twice with similar results. The results of one experiment are shown.
