Fig 1.
The E430G Fc variant of chimeric mAb 2C7 enhances complement-dependent bactericidal activity and complement deposition on N. gonorrhoeae compared to WT Fc.
(A) Serum bactericidal activity of chimeric mAb 2C7 and its 2C7 Fc variants against N. gonorrhoeae strains FA1090 (left graph), 15253 (middle graph), MS11 (right graph) in the presence of 16.7% NHS for FA1090 and 15253, or 16.7% human complement (IgG- and IgM-depleted human complement; Pel-Freez) for MS11 as the complement sources. Human complement (IgG/IgM depleted) was used for MS11 because it displays intermediate serum resistance and is only partially resistant to NHS. X-axis, concentration of mAb (μg/mL); y-axis, percent survival (mean [range]) at 30 min relative to counts at 0 min. Two-way ANOVA was used to compare survival across WT Fc and E430G Fc at the various concentrations. *P < 0.05; ***P < 0.001; ****P < 0.0001. (B) The E430G Fc variant enhances C1q binding and C4 and C3 deposition on N. gonorrhoeae. Strain FA1090 was incubated with mAb 2C7, either with WT Fc, E430G Fc, or D27A/K322A Fc (concentrations indicated on the x-axis) followed by the addition of 16.7% human complement (IgG- and IgM-depleted NHS) for 30 min at 37°C. C1q binding and C4 and C3 deposition on N. gonorrhoeae were measured by whole-cell ELISA (mean [range] of two experiments). Differences in complement deposition between WT Fc and E430G Fc at the different concentrations were compared by two-way ANOVA. ***P < 0.001; ****P < 0.0001. Data associated with this figure can be found in the supplemental data file (S1 Data). IgG, immunoglobulin G; IgM, immunoglobulin M; mAb, monoclonal antibody; NHS, normal human serum; OD, optical density; WT, wild-type.
Fig 2.
Activity of chimeric mAb 2C7 and 2C7 Fc variant derivatives against N. gonorrhoeae FA1090 in the mouse vaginal colonization model.
(A-C) Mice (n = 10/group) were challenged with 8 × 105 CFU FA1090, and mAb 2C7 or its derivatives was administered intravaginally daily at either 10 μg/d or 1 μg/d over the 7 d course of the experiment. The vaginal cavity was swabbed daily and recovered CFU enumerated. (A) Kaplan Meier graph showing time to clearance of infection (P < 0.0001 when groups treated with 2C7-WT Fc or 2C7-E430G Fc were compared with saline [control] or D270A/K322A groups; Mantel-Cox analysis). Significance was set at 0.002 (Bonferroni correction for 7 groups). P < 0.0001 for mAb 2C7-E430G (1 μg/d) versus 1 μg/d mAb 2C7-WT Fc (1 μg/d). (B) Log10 CFU versus time (mean [SD]). (C) AUC analysis to compare bacterial burdens over time. The median and 95% confidence interval are shown for each group. A one-way ANOVA showed significant differences across treatment groups (P < 0.0001 by Kruskal-Wallis nonparametric test). Pairwise comparisons across groups was performed using Dunn’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D-F) Activity of lower doses (0.5 and 0.1 μg) given to mice (n = 10/group) intravaginally (daily) of chimeric mAb 2C7 and 2C7-E430G Fc. Mice were administered mAb 2C7 or mAb 2C7-E430G intravaginally daily at either 0.5 μg/d or 0.1 μg/d over 7 d and were challenged with 8.75 × 106 CFU FA1090. (D) Kaplan Meier graph showing time to clearance of infection. Significance was set at 0.005 (Bonferroni correction for 5 groups). P < 0.0001 for WT Fc (0.5 μg/d) and E430G Fc (0.1 and 0.5 μg/d) versus saline (control) or WT Fc at 0.1 μg/d (Mantel-Cox analysis). WT Fc (1 μg/d) versus E430G Fc (1 μg/d), P = 0.0004. (E) Log10 CFU versus time (mean [SD]). (F) AUC analysis. The median and 95% confidence interval are shown for each group. Comparison across groups by one-way ANOVA showed significance (P < 0.0001). Pairwise comparisons across groups were made using Dunn’s post hoc test. *P < 0.05; ***P < 0.001; ****P < 0.0001. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; CFU, colony-forming units; mAb, monoclonal antibody; WT, wild-type.
Fig 3.
Binding to FcγR of chimeric 2C7 Fc variant mAbs.
For binding of mAb 2C7 Fc variants to dimeric variants of ECDs of FcγRIIA allotype 131H, FcγRIIA allotype 131R, FcγRIIB, FcγRIIIA allotype 158F, and FcγRIIIA allotype 158V, increasing concentrations (x-axis) of chimeric 2C7 Fc variant mAbs were dispensed to and incubated with goat F(ab’)2-anti-human-IgG-F(ab’)2-coated microtiter wells. Subsequent binding of FcγRs is shown as absorbance at 405 nm (y-axis). For binding to monomeric ECD of FcγRIA, increasing concentrations (x-axis) of chimeric 2C7 mAbs with wild-type Fc or Fc variants were dispensed into microtiter wells coated with FcγRIA. Binding is shown as absorbance at 405 nm (y-axis). Data associated with this figure can be found in the supplemental data file (S1 Data). Ab, antibody; ECD, extracellular domain; FcγR, Fc gamma receptor; IgG, immunoglobulin G; LOS, lipooligosaccharide; mAb, monoclonal Ab.
Table 1.
Summary of binding and activity of Fc variants of chimeric mAb 2C7.
Fig 4.
Efficacy of mAb 2C7 requires complement activation by Fc.
Wild-type BALB/c mice (n = 10/group) were infected with 107 CFU N. gonorrhoeae FA1090 and treated intravaginally (daily) with 0.5 μg of each of the 2C7 mAb Fc variants. The variant 2C7-E430G Fc served as a positive control for efficacy. (A) Kaplan Meier graph showing time to clearance of infection. Significance was set at 0.008 (Bonferroni correction for 4 groups). P < 0.0001 for E430G Fc versus each of the other groups. (B) Log10 CFU versus time (mean [SD]). (C) AUC analysis. The median with 95% confidence intervals for each group is shown. Comparison across groups by one-way ANOVA showed significance (P < 0.0001 by the Kruskal-Wallis test). Pairwise comparisons across groups using Dunn’s post hoc test. **P < 0.01; ***P < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; CFU, colony-forming units; mAb, monoclonal antibody.
Fig 5.
An intact complement system is required for efficacy of mAb 2C7-E430G Fc.
(A) mAb 2C7-E430G Fc (abbreviated E430G in the figure) is not efficacious in C1q−/− mice. C1q−/− mice and WT C57BL/6 control mice (n = 9–10/group) were infected with 4 × 106 CFU N. gonorrhoeae strain FA1090 and administered saline (control) or intravaginal (daily) 0.1 μg/d of mAb 2C7-E430G Fc. Vaginas were swabbed daily to enumerate CFUs. Left graph: Kaplan Meier graph showing time to clearance of infection (P ≤ 0.0003 for E430G-treated WT mice versus all other groups; significance was set at 0.008 [Bonferroni correction for 4 groups]). Middle graph: Log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence intervals are shown for each group. Comparison across groups by one-way ANOVA were significant (P < 0.0001; Kruskal-Wallis test). Pairwise comparisons across groups were made with Dunn’s post hoc test. (B) C5 blockade function abrogates efficacy of mAb 2C7-E430G Fc. C5 function in WT BALB/c mice was blocked with mAb BB5.1 (1 mg intraperitoneally on days −1, 2, and 5). In addition, 10 μg of mAb BB5.1 was also administered intravaginally (daily for 8 d). Saline was used as a control in C5 sufficient mice. Four groups of mice (n = 10/group) infected with N. gonorrhoeae FA1090 were treated as follows: (1) WT mice, saline intravaginally; (2) C5 blockaded mice, saline intravaginally; (3) WT mice, mAb 2C7-E430G Fc, 0.1 μg intravaginally, daily; and (4) C5 blockaded mice, mAb 2C7-E430G Fc, 0.1 μg intravaginally, daily. Left graph: Kaplan Meier graph showing time to clearance of infection. Significance was set at 0.008 (Bonferroni correction for 4 groups). P < 0.0001 for E430G-treated WT mice versus all other groups. Middle graph: Log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence intervals are shown for each group. Comparison across groups by one-way ANOVA by Kruskal-Wallis test showed significance (P < 0.0001). Pairwise comparisons across groups were made with Dunn’s post hoc test. (C) mAb 2C7-E430G Fc loses efficacy in C9−/− mice. Four groups of mice (n = 5–6/group) were infected with N. gonorrhoeae FA1090 (3.6 × 107) and were treated as follows: (1) WT mice, saline intravenously; (2) WT mice, mAb 2C7-E430G intravenously (5 μg intravenously, single dose on day +1); (3) C9−/− mice, saline intravenously; and (4) C9−/− mice, mAb 2C7 E430G intravenously (5 μg intravenously, single dose on day +1). Left graph: Kaplan Meier graph showing time to clearance of infection. Significance was set at 0.008 (Bonferroni correction for 4 groups). P = 0.0009 for E430G-treated WT mice versus all other groups. Middle graph: Log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence intervals are shown for each group. Comparison across groups by one-way ANOVA by Kruskal-Wallis test showed significance (P = 0.0028). Pairwise comparisons across groups were made with Dunn’s post hoc test. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; CFU, colony-forming units; mAb, monoclonal antibody; NS, not significant; WT, wild type.
Fig 6.
mAb 2C7 activity in the mouse vaginal colonization model does not require PMNs or C5aR1.
(A) Efficacy of murine mAb 2C7 (mouse IgG3) in the absence of PMNs. Five groups of BALB/c mice (n = 10/group) infected with 106 CFU N. gonorrhoeae FA1090 were treated as follows: (1) saline (control), (2) RB6 mAb (depletes PMNs), administered IP, (3) RB6 IP plus mAb 2C7 intravaginally, (4) rat IgG2b (control for RB6) IP, and (5) rat IgG2b IP plus mAb 2C7 intravaginally. Left graph: Kaplan Meier curves showing time to clearance of infection. Significance was set at 0.005 (Bonferroni correction for 5 groups). P < 0.0001 (Mantel-Cox log-rank test) for each of the groups that received mAb 2C7 versus each of the groups that did not get mAb 2C7. Middle graph: log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence interval are indicated for each group. Comparison across groups by one-way ANOVA showed significance (P < 0.0001). Groups that received mAb 2C7 showed significantly lower AUCs than each group that did not get mAb 2C7 when compared in a pairwise manner (Dunn’s post hoc test). *P < 0.05; ***P < 0.001; ****P < 0.0001. (B) Chimeric mAb 2C7-E430G maintains efficacy in the absence of PMNs. Four groups of BALB/c mice (n = 10/group) infected with N. gonorrhoeae FA1090 (3.5 × 107 CFU) were treated as follows: (1) saline (control), (2) RB6 mAb, IP, (3) 2C7-E430G (5 μg intravenously on day 1), and (4) RB6 IP and 2C7-E430G intravenously. Left graph: Kaplan Meier curves. Significance was set at 0.008 (Bonferroni correction for 4 groups). P = 0.0012 for the two groups that received 2C7-E430G versus each of the groups that did not get 2C7-E430G. Middle graph: log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence intervals for each group are shown. Comparison across groups by one-way ANOVA (Kruskal-Wallis test) showed significance (P < 0.0001). Pairwise comparisons were made by Dunn’s post hoc test. **P < 0.01; ***P < 0.001. (C) Chimeric mAb 2C7-E430G is effective when C5aR1 is blocked. Mice that had been administered PMX205 (C5aR1 inhibitor) subcutaneously and intravaginally were used in the following experiment. Four groups of BALB/c mice (n = 10/group) infected with N. gonorrhoeae FA1090 (3.2 × 107 CFU) were treated as follows: (1) saline (control), (2) PMX205 (C5aR1 inhibitor), (3) 2C7-E430G (5 μg intravenously on day 1), and (4) PMX205 (C5aR1 inhibitor) and 2C7-E430G. Left graph: Kaplan Meier curves. Significance was set at 0.008 (Bonferroni correction for 4 groups). P < 0.0001 for each of the groups that received 2C7-E430G versus each of the groups that did not get 2C7-E430G (Mantel-Cox log-rank test). Middle graph: log10 CFU versus time (mean [SEM]). Right graph: AUC analysis. The median and 95% confidence interval are shown for each group. Comparison across groups by one-way ANOVA showed significance (P < 0.0001). Pairwise comparisons were made by Dunns’s post hoc test. **P < 0.01; ***P < 0.001. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; C5aR1, C5a receptor; CFU, colony-forming units; IgG, immunoglobulin G; IP, intraperitoneally; mAb, monoclonal antibody; PMN, polymorphonuclear leukocyte.
Fig 7.
Efficacy of chimeric mAb 2C7 and its Fc variants against N. gonorrhoeae in human FH/C4BP dual Tg mice.
FH/C4BP Tg mice were infected with 8.75 × 105 CFU N. gonorrhoeae FA1090, which were administered (daily, for 10 d) intravaginally: saline; chimeric WT mAb 2C7 (abbreviated WT Fc) or mAb 2C7-E430G Fc (abbreviated E430G Fc), each of these mAbs at dosages of 1, 0.5, or 0.1 μg/d; or the “complement inactive” 2C7 mAb D270A/K322A Fc (abbreviated D270A/K322A Fc) at 10 μg/d. Vaginal bacterial CFU burdens were enumerated daily. (A) Kaplan Meier graph showing time to clearance of infection. Significance was set at 0.0018 (Bonferroni correction for 8 groups). Groups that received either 1 or 0.5 μg of (2C7) WT Fc cleared infection significantly faster than saline controls, WT Fc and E430G Fc given at the lowest dose (0.1 μg/d), or D270A/K322A Fc. Groups that received 1 or 0.5 μg/d of E430G Fc cleared infection faster than groups that received the corresponding doses of WT Fc (P < 0.0001 in all instances). (B) Bacterial burdens (expressed as log10 CFU) over time (mean [SEM]). (C) AUC analysis. The median and 95% confidence interval are shown for each group. Comparison across groups by one-way ANOVA were significant (P < 0.0001; Kruskal-Wallis test). Pairwise comparisons across groups made using Dunn’s post hoc test. **P < 0.01; ***P < 0.001; ****P < 0.0001. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; C4BP, C4b-binding protein; CFU, colony-forming units; FH, factor H; Tg, transgenic; WT, wild-type.
Fig 8.
Systemically administered mAb 2C7-E430G Fc (abbreviated E430G) administered at either high (10 μg or 50 μg) or low (1 μg, 5 μg, and 10 μg [the latter as overlapping]) doses clears vaginal gonococcal infection in human FH/C4BP transgenic mice.
(A-C) Human FH/C4BP female transgenic mice (n = 10/group) were infected intravaginally with 6 × 107 CFU N. gonorrhoeae FA1090 and treated 24 h later with a single dose of 2C7-E430G (either 10 μg or 50 μg) or with saline (vehicle control). Vaginas were swabbed daily to enumerate gonococcal CFU. (A) Kaplan Meier curves showing time to clearance. Significance was set at 0.017 (Bonferroni correction for 3 groups). P < 0.0001 for each treatment group versus the saline control group. (B) Log10 CFU versus time (mean [SEM]). (C) AUC analysis. The median and 95% confidence interval are shown for each group. Comparison across groups by one-way ANOVA were significant (P < 0.0001 by the Kruskal-Wallis test). Pairwise comparisons across groups were made using Dunn’s nonparametric test. **P < 0.01; ****P < 0.0001. (D-F) Efficacy of a single 1 μg, 5 μg, and 10 μg IV dose of 2C7-E430G Fc against N. gonorrhoeae in dual FH/C4BP transgenic mice. Four groups of FH/C4BP transgenic mice (n = 6/group) were infected intravaginally with 6.6 × 107 CFU N. gonorrhoeae FA1090 and treated 24 h later with a single IV injection of 2C7-E430G Fc at the indicated doses. The control group received saline. (D) Kaplan Meier curves showing time to clearance. Significance was set at 0.008 (Bonferroni correction for 4 groups). P < 0.0001 for each treatment group versus the saline control group. (E) Log10 CFU versus time (mean [SEM]). (F) AUC analysis. Comparisons across groups made using one-way ANOVA were significant (P = 0.0012; Kruskal-Wallis test). Pairwise comparisons across groups were made using Dunn’s nonparametric test. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; C4BP, C4b-binding protein; CFU, colony-forming units; FH, factor H; IV, intravenous; WT, wild-type.
Fig 9.
mAb 2C7-E430G Fc (abbreviated 2C7-E430G) is efficacious against N. gonorrhoeae in FH/C4BP transgenic mice coinfected with C. muridarum.
Human FH/C4BP transgenic mice were infected intravaginally with 2.5 × 106 IFU C. muridarum on days −4, −3, and −2, followed by infection with 7 x 107 CFU N. gonorrhoeae FA1090 on day 0 (“Cm+Ng”; n = 9/group). Mice infected with N. gonorrhoeae only (“Ng”; n = 8/group) were used as controls. Mice were treated with 2C7-E430G Fc (1 μg intravaginally, daily for 9 d) or with saline (vehicle controls). (A) Kaplan Meier curves show time to clearance. Significance was set at 0.008 (Bonferroni correction for 4 groups). P < 0.0001 for each treatment group versus the corresponding saline control group (P < 0.0001). (B) Log10 CFU versus time (mean [SEM]). (C) AUC analysis. The median and 95% confidence interval are shown for each group. Comparison across groups made by one-way ANOVA was significant (P < 0.0001; Kruskal-Wallis test). Pairwise comparisons across groups were made with Dunn’s nonparametric post hoc test. Data associated with this figure can be found in the supplemental data file (S1 Data). AUC, area under curve; C4BP, C4b-binding protein; CFU, colony-forming units; FH, factor H; IFU, infectious units.