A broadly cross-reactive monoclonal antibody against an epitope on the n-terminus of meningococcal fHbp

Abstract

Meningococcal factor H binding protein (fHbp) is an important vaccine antigen for prevention of disease caused by capsular group B strains. The protein has been sub-classified into three variant groups. Most anti-fHbp antibodies are variant group-specific and recognize epitopes on the C-terminal domain. We report a murine IgG1 mAb, JAR 41, which broadly cross-reacted with fHbp sequence variants from all variant groups. The mAb bound to the surface of live meningococci with fHbp from each of the three variant groups. In combination with second non-bactericidal anti-fHbp mAbs, JAR 41 elicited complement-mediated bactericidal activity in vitro, and augmented passive protection against meningococcal bacteremia in human fH transgenic rats. The epitope was located on a conserved region of the N-terminal portion of the fHbp molecule opposite that of fH contact residues. The data underscore the importance of broadly cross-reactive, surface-exposed epitopes on the N-terminal domain in the design of protective fHbp vaccines.

Figure 1. Binding of anti-fHbp mAb JAR 41 to recombinant fHbp in variant groups 1, 2 or 3.

Grey circles, JAR 41. Open triangles, JAR 5. Xs, JAR 11. Open squares, JAR 35. Panel a, fHbp ID 1 from variant group 1. Panel b, fHbp ID 77 from variant group 2. Panel c, fHbp ID 28 from variant group 3.

Figure 2. Relatedness of unique fHbp amino acid sequences.

Network analysis illustrating the relatedness of unique fHbp amino acid sequences, as generated using the program SplitsTree. Panel a, the relatedness of 531 unique fHbp amino acid sequences available from the public database www.pubmlst.org. Each “twig” (node) represents a unique sequence. Sub-families, as described by Murphy et al; variant groups as described by Masignani et al, and modular groups (denoted using roman numbers as described by Beernink and Granoff. Modular Group X is a new group reported in this work. Panel b, the relatedness of the 21 fHbp sequence variants, which were selected for production of recombinant proteins (See Table 1). Each filled circle represents a unique sequence variant designated by fHbp ID number from the public database http://pubmlst.org/neisseria/fHbp.

Figure 3. Binding of JAR 41 to 21 fHbp sequence variants as measured by ELISA.

Panel a, recombinant fHbps from variant group 1. Panel b, recombinant fHbps from variant group 2. Panel c, recombinant fHbps from variant group 3. Panel d, natural hybrids between variant groups 1 and 2, or 1 and 3. Panel e, solid lines, Data from JAR 41 binding to all 21 recombinant sequence variants (solid lines); or with with negative control recombinant proteins NadA and GNA1030 (dashed lines).

Figure 4. Binding of mAbs to live N. meningitidis strains as measured by flow cytometry.

Panel a, Comparison of JAR 41 binding (25 µg/ml) to wild-type strain H44/76 (H44/76-WT, solid black line) with fHbp ID 1 in variant group 1, or an isogenic mutant with lower expression of fHbp ID 1 (H44/76-LE, blue dashed line). Solid green line, H44/76-WT incubated with 25 µg/ml of a negative control IgG mAb (JAR 11, specific for fHbp in variant groups 2 and 3); green-shaded area, H44/76-LE incubated with JAR 11. Panel b, binding of a control anti-PorA mAb. Orange line, H44/76-WT; orange-shaded area, H44/76-LE. Panels c-f, Concentration-dependent binding of JAR 41 with strain H44/76-WT (panel c), mutant strain H44/76-LE (panel d), strain 8047 with fHbp in variant group 2 (panel e), or M1239 with fHbp in variant group 3 (panel f). Panels c, d and e, thick black line, 25 µg/ml of JAR 41; dark grey-shaded area, 5 µg/ml; dashed black line, 1 µg/ml; thin black line, 0.2 µg/ml; light grey-shaded area, 25 µg/ml JAR 41 with a negative control H44/76 fHbp knock-out mutant; solid blue line, 25 µg/ml of JAR 5, which is specific for fHbp in variant group 1 and a negative control mAb for variants 2 and 3. Panel f, thick black line, 50 µg/ml of JAR 41 with wild-type strain M1239 with fHbp in variant group 3; dark shaded area, 5 µg/ml of JAR 41 (lower concentrations of JAR 41 were not tested); blue line, 50 µg/ml of JAR 5 (specific for fHbp variant group 1); Light gray shade area, 50 µg/ml of JAR 41 incubated with M1239 fHbp knock-out mutant.

Figure 5. Cooperative bactericidal activity elicited by JAR 41, individually or in combination with second anti-fHbp mAbs.

Panel a, strain H44/76 (fHbp ID 1, variant group 1). Panel b, mutant of strain H44/76 engineered to have lower expression of fHbp ID 1 as that of the wild-type strain (See Figure 4a). Panel c, strain 8047 (fHbp ID 77, variant group 2). BC_50%, concentration of mAb (µg/ml) resulting in 50% decrease in CFU/ml after 1 hr incubation with human complement, compared to time 0. The combinations contained 1∶1 mixtures (i.e, BC_50% of 1 µg/ml contained 0.5 µg/ml of each of the mAbs in the combination). The anti-capsular mAb (SEAM 12) or anti-PorA mAbs (P1.7, panels a and b; or P1.2, panel c) served as positive controls. Error bars represent range of results from two to three independent assays.

Figure 6. JAR 41 augments passive protective activity of anti-fHbp mAb JAR 5 against bacteremia caused by group B strain H44/76 in human fH transgenic infant rats.

Panel a, Experiment 1, 8- to 9-day old rats challenged IP with 4900 CFU/rat; Panel b, Experiment 2, 6- to 7-day old rats challenged IP with 760 CFU/rat. In both experiments, rats were given a total dose of 25 µg of mAb or phosphate buffered saline alone (PBS, negative control) 1 hr before the bacterial challenges. The combination contained 12.5 µg of each mAb. Blood cultures were obtained 6 hrs after challenge. Compared to PBS, rats given JAR 5 alone had a lower geometric mean CFU/ml (Experiment 1, p = 0.04 and Experiment 2, p<0.03)) but there was no protection by JAR 41 alone (p>0.05). In Experiment 2, the combination of JAR 41 and JAR 5 (0/8 with bacteremia) had greater protective activity than JAR 5 alone (6 out of 9 with bacteremia, p = 0.009, Fisher's exact test). ND, not done.

Figure 7. Effect of amino acid substitutions on binding of mAbs to recombinant fHbp.

(Xs with solid line), wild-type fHbp ID 1; (Open squares with dotted line), H26A; (Open triangles with dashed line), K27A; (Open circles with solid line), D25A. Panel a, JAR 41. Panel b, JAR 4. Panel c, JAR 1. Panel d, JAR 5.