Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein

Abstract

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Fig 1

Binding of anti-fHbp MAb MAbs to live bacteria of group B strain H44/76 and a mutant (H44/76-OE) with increased fHbp expression. (A) Binding of control murine anticapsular MAb (5 μg/ml). Gray line, wild-type strain; black line, H44/76-OE mutant; filled gray histogram, mutant incubated with 100 μg of an irrelevant MAb/ml. (B) Binding of murine anti-fHbp MAbs (50 μg of JAR 3 + JAR 4/ml). Symbols are as defined in panel A.

Fig 2

Binding and fH inhibition by chimeric human IgG subclass mouse anti-fHbp MAbs. (A) Binding to fHbp by ELISA. (B) Binding to live N. meningitidis bacterial cells of strain H44/76 by flow cytometry. The MAbs were tested at 4 μg/ml. IgG1, black solid lines; IgG2, gray solid lines; IgG3, black dashed lines (the respective binding lines are superimposed). An irrelevant human IgG1 MAb (100 μg/ml) was used as a negative control (dark gray filled histograms). (C) Inhibition of fH binding by flow cytometry using 20% IgG-depleted human serum as a source of fH (∼90 μg/ml) and 2 μg (JAR 3 or JAR 5) or 50 μg (MAb502) of chimeric antibody/ml. Light gray filled area, fH without MAb; dark gray filled area, bacteria without fH as a negative control. Symbols for IgG1, IgG2, and IgG3 MAbs are as defined for panel B.

Fig 3

Deposition of human C4b and C3b on live bacterial cells of group B strain H44/76 as measured by flow cytometry. The bacteria were incubated with human complement and 4 μg of chimeric anti-fHbp MAbs/ml. IgG1, black solid lines; IgG2, gray solid lines; and IgG3, black dashed lines. An irrelevant human MAb (4 μg/ml) was used as a negative control (gray filled histograms). (A) Activation of C4b deposition using 10% commercial C6-depleted human serum, which also had been depleted of IgG as described in Materials and Methods. (B) Activation of C3b deposition. The MAb concentrations and complement were the same as those for panel A. Similar respective results were obtained in two independent experiments with C6-depleted human serum as the complement source and in a third experiment with a different human complement source that had not been C6-depleted (data not shown).

Fig 4

Complement-mediated bactericidal activity of chimeric anti-fHbp MAbs. (A to C) Survival of wild-type N. meningitidis group B strain H44/76 after 60 min of incubation with anti-fHbp chimeric MAbs and 20% human complement. Crosses with solid line, IgG1; open triangles with solid line, IgG2; open circles with dashed line, IgG3. (A) JAR 3; (B) JAR 5; (C) MAb502. (D) Geometric mean MAb concentrations (μg/ml) for 50% human complement-mediated bactericidal activity in three to four independent assays. Error bars represent two standard errors. For all three paratopes, bacteriolysis occurred at lower concentrations of IgG3 (hatched bars) than the respective IgG2 (gray bars) or IgG1 (white bars; P < 0.0001) subclasses. For JAR 3 and JAR 5, bacteriolysis occurred at lower concentrations of IgG1 than with the respective IgG2 subclass MAbs (P < 0.0001 and P = 0.01, respectively).

Fig 5

Effect of increased fHbp expression on IgG subclass anti-fHbp complement deposition and bactericidal activity. (A to C) Flow cytometry with live bacterial cells of a mutant strain with overexpressed fHbp (H44/76-OE). (A) Binding of 4 μg of chimeric anti-fHbp MAbs/ml (IgG1, solid black lines; IgG2, gray lines; IgG3, dashed lines; the respective lines are superimposed). An irrelevant MAb (100 μg/ml) was used as a negative control (gray filled histograms). (B and C) Deposition of C4b and C3b, respectively, elicited by 4 μg of anti-fHbp MAb/ml and 10% C6-depleted human serum as a complement source, which also had been depleted of IgG. (D) Anti-fHbp MAb complement-mediated bactericidal activity against mutant strain H44/76-OE. For JAR 3 and JAR 5, the geometric IgG1 concentrations required for 50% bactericidal activity (BC₅₀) (white bars) were lower than the respective IgG3 concentrations (black diagonal hatched bars P ≤ 0.014). For MAb502, the geometric mean BC₅₀ of IgG1 was higher than for IgG3 (P = 0.005). For all three paratopes, the IgG2 BC₅₀ values were higher than the respective IgG1 or IgG3 values (P < 0.0001). The data are from three independent experiments. Error bars represent two standard errors.

Fig 6

Effect of blocking properdin on anti-fHbp MAb bactericidal activity. Survival of N. meningitidis bacteria incubated for 60 min with 20% IgG-depleted human serum and anti-fHbp MAbs in the presence or absence of 50 μg of an anti-properdin MAb inhibitor/ml. Results for the H44/76 wild-type strain without anti-properdin inhibitor (open blue squares with dashed line), the H44/76 wild-type strain with anti-properdin inhibitor (blue filled squares with black solid line), the H44/76-OE mutant with overexpressed fHbp without inhibitor (open orange circles with dashed line), and the H44/76-OE mutant with anti-properdin inhibitor (orange filled circles with solid black line) are indicated. The data points and error bars represent respective means and ranges from two to three independent experiments.

Fig 7

Effect of the addition of an anti-Bb MAb on anti-fHbp MAb bactericidal activity. Survival of N. meningitidis bacteria incubated for 60 min with 20% IgG-depleted human serum and anti-fHbp MAbs in the presence or absence of 100 μg of an anti-Bb MAb inhibitor/ml. The results for the H44/76 wild-type strain without anti-Bb MAb inhibitor (open blue squares with dashed line), the H44/76 wild-type strain with anti-Bb MAb inhibitor (blue filled squares with solid line), the H44/76-OE mutant with overexpressed fHbp without anti-Bb MAb inhibitor (open orange circles with dashed line), and the H44/76-OE mutant with anti-Bb MAb inhibitor (orange filled circles with solid line) are given. The data points and error bars represent respective means and ranges from two to three independent experiments.