Binding of complement factor H to PorB3 and NspA enhances resistance of Neisseria meningitidis to anti-factor H binding protein bactericidal activity

Abstract

Among 25 serogroup B Neisseria meningitidis clinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange of porB3 from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

FIG 1

Anti-FHbp binding in relation to complement-mediated bactericidal activity. (A and B) Binding of MAb (10 μg/ml) to live bacteria as measured by flow cytometry. Solid black line, resistant (R) isolates; dashed black line, control sensitive (S) isolates. (A) Anti-FHbp MAb JAR 5; (B) anticapsular MAb SEAM 12. Representative data are shown. The results were replicated in two experiments. (C) Complement-mediated bactericidal activity. Anti-FHbp, antisera from mice immunized with an FHbp sequence variant that matched the FHbp amino acid sequence variant of the respective isolates. Anti-capsule, anticapsular MAb SEAM 12. The data are calculated from results of three assays. Error bars represent ranges.

FIG 2

Anti-FHbp passive protective activity against bacteremia. Human FH transgenic infant rats were treated i.p. with sera from immunized mice and 2 h later challenged i.p. with 3,000 or 4,000 CFU of group B strain R1 or S1. Blood cultures were obtained 12 h after the bacterial challenge. Each symbol represents result of an individual animal. (A and D) Sera diluted 1:10 from negative-control mice immunized with aluminum hydroxide; (B and C) sera diluted 1:40 or 1:160, respectively, from mice immunized with FHbp ID 1 (antigenic variant in 4CMenB vaccine); (E and F) sera diluted 1:40 or 1:160, respectively, from mice immunized with FHbp ID 4 (matched the FHbp variant in the R1 and S1 isolates). Not shown are data from control animals treated with 10 μg of an anticapsular MAb, which resulted in sterile blood cultures. Panels with asterisks were statistically significant (panel B, P = 0.0239; panel C, P = 0.0032; panel F, P = 0.0146).

FIG 3

Effect of knocking out NspA on Anti-FHbp bactericidal activity. (A) Binding of anti-NspA MAb to live bacteria. Anti-NspA MAb 14C7 was tested at 10 μg/ml. Black solid line, resistant (R) strains; dashed black lines, susceptible (S) strains; gray-filled histogram, mutants of R and S isolates with nspA inactivated. Typical results shown; data were replicated in two experiments. (B and C) Complement-mediated bactericidal activity. Mouse anti-FHbp antisera and anticapsular MAb are described in the legend for Fig. 1B and C, respectively. Error bars represent ranges of duplicate or triplicate measurements in three experiments.

FIG 4

Effect of the addition of a nonbactericidal anti-NspA MAb on serum anti-FHbp bactericidal activity. The anti-NspA MAb (AL12) was tested at 5 μg/ml and 20% human complement. All isolates were resistant to anti-NspA bactericidal activity at MAb concentrations up to 50 μg/ml (the highest concentration tested). The anti-FHbp antisera matched the FHbp amino acid sequence of the test isolates. Ranges represent results from duplicate measurements. Representative results are shown. The data were replicated in two experiments.

FIG 5

Effect of the addition of a nonbactericidal anti-NspA MAb on complement deposition on live bacteria. (A and B) Deposition of C4b and C3b, respectively, by 1:50 dilutions of anti-FHbp antisera, anti-NspA MAb (5 μg/ml), or a combination of anti-FHbp antisera (1:50) with anti-NspA MAb (5 μg/ml). (A) S1 and R1 isolates; (B) S2 and R2 isolates. Solid black line, resistant (R) isolates; dashed black line, sensitive (S) isolates; shaded areas, negative-control without antibody. The data were replicated in three experiments testing 5% IgG-depleted human serum as a source of complement. We also obtained similar respective results for the R1 and S2 isolates testing 20% IgG-depleted human serum as a source of complement.

FIG 6

Human FH enhances survival of FHbp/NspA double-KO mutants of N. meningitidis in infant rat serum. (A) Survival in 60% infant rat serum. Bacteria were incubated in pooled sera from wild-type infant rats, to which was added different concentrations of human FH. Open squares with solid black line, double-KO mutant of R1; open circles with dashed black line, double-KO mutant of S1. The data points represent median value (ranges) of triplicate measurements. The results were replicated in an independent experiment. (B) Binding of recombinant FH domain fragments fused to mouse Fc to FHbp/NspA double-KO mutants by flow cytometry. Solid black line, resistant (R) isolates; dashed black lines, susceptible (S) isolates; gray-filled histogram, bacteria without added recombinant fragment. Left, FH domain 6,7/Fc; right, FH domain 18-20/Fc. Representative data from one assay are shown. The results were replicated in a second experiment.

FIG 7

Effect of allelic exchange of PorB3 on human FH-dependent susceptibility of FHbp and NspA double knockout mutants to killing by 60% infant rat serum. (A) Double-KO mutants of isolate R1 with its endogenous PorB3 replaced by PorB3 R1 or PorB3 S1. (B) Double-KO mutants of isolate S1 with its endogenous PorB replaced by PorB3 R1 or PorB3 S1. The data points represent median values from triplicate measurements. Error bars represent ranges. The results were replicated in an independent experiment. (C and D) Effect of PorB3 on binding of recombinant human FH domains by flow cytometry. Solid black lines, PorB3 R1; black dashed lines, PorB3 S1; gray-filled histogram, bacteria without added recombinant fragment. Panel C, double-KO isolate R1 with allelic replacement of PorB3 R1 or S1; panel D, double-KO isolate S1 with allelic replacement of PorB3 R1 or S1. The results were replicated in two independent experiments.

FIG 8

Effect of allelic replacement of PorB3 variant on anti-FHbp bactericidal activity. The sera were from mice immunized with recombinant FHbp that matched the sequence variant of the isolates (FHbp ID 4). (A) Isolate R1 expressing wild-type FHbp and NspA with either allelic exchange of PorB3 R1 (open bars) or PorB3 S1 (hatched bars); (B) isolate S1 expressing wild-type FHbp and NspA with either allelic exchange of PorB3 R1 or PorB3 S1. The data were calculated from three assays. Error bars represent ranges. Asterisks indicate that a difference in bactericidal activity between respective PorB3 variants was significant (P = 0.002, Mann-Whitney).