Cooperative serum bactericidal activity between human antibodies to meningococcal factor H binding protein and neisserial heparin binding antigen

Abstract

A meningococcal group B vaccine containing multiple protein antigens including factor H binding protein (fHbp) and Neisserial heparin binding antigen (NHba) is in clinical development. The ability of antibodies against individual antigens to interact and augment protective immunity is unknown. We assayed human complement-mediated bactericidal activity (SBA) in stored sera from six immunized adults before and after depletion of antibodies to fHbp and/or NHba. All six subjects developed ≥ 4-fold increases in SBA titer against a test strain with fHbp in the variant 1 group with an amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:139 after 3 doses of vaccine). By adsorption 88 to >95% of the SBA was directed against fHbp. Four subjects developed ≥ 4-fold increases in SBA titer against a test strain with a heterologous fHbp variant 2 antigen and a homologous NHba amino acid sequence that matched the vaccine antigen (GMT <1:4 baseline, to 1:45). SBA was directed primarily against NHba in one subject, against fHbp in a second, while depletion of either anti-NHba or anti-fHbp antibody removed the majority of SBA in sera from two subjects. In all four subjects, depletion of both anti-fHbp and anti-NHba antibodies removed more SBA than depletion of either antibody individually. Mixing a mouse non-bactericidal anti-fHbp variant 1 antiserum with a mouse anti-NHba antiserum also augmented the anti-NHba SBA titer against this test strain. For meningococcal vaccines that target relatively sparsely exposed antigens such fHbp or NHba, non-bactericidal antibodies against individual antigens can cooperate and elicit SBA.

Figure 1

Adequacy of adsorption of post-immunization serum anti-NHba (Panel A) and anti-fHbp antibodies (Panel B) as measured by ELISA. The Y-axis shows the mean antibody concentrations and ranges of duplicate independent dilutions in arbitrary units (U) per ml (See methods). On some bars, the ranges are not evident because the differences between the respective replicate titers were small. Diagonal hatched bars, unadsorbed sera; open bars, sera adsorbed with irrelevant antigen (recombinant human albumin); black bars, adsorbed with NHba; gray bars, adsorbed with fHbp. The antibody concentrations in unadsorbed sera obtained before vaccination were <100 U/ml.

Figure 2

Human complement-mediated serum bactericidal antibody titer against group B strain H44/76, which expressed fHbp in the variant 1 group that matched the amino acid sequence of the vaccine antigen. The titers and error bars represent the respective mean and range of values measured in two to three independent assays. Pre-immune serum, bar with horizontal hatching; symbols for bars indicating respective post-immunization unadsorbed and adsorbed sera are described in legend to Figure 1.

Figure 3

Bactericidal activity against group B strain M4407. Panels A-D show data for subjects 1–4, respectively. Left panels, mean bactericidal titers of unadsorbed pre-immunization sera (horizontal hatched bars) and the respective post-immunization sera: unadsorbed (bars with diagonal hatching), adsorbed two times with the negative control rHA-coupled immunoadsorbent (open bars) and adsorbed with both anti-NHba and anti-fHbp antibodies (green bars). The error bars represent the respective ranges of values measured in two to three independent assays. On some bars, the ranges are not evident because the respective ranges were small. Right panels, percent survival after incubation of the test strain for 60 minutes with human complement and dilutions of post-immunization test sera before (X with solid line) or after depletion of antibodies to fHbp (yellow circles, dashed lines), NHba (blue circles, solid lines), or recombinant human albumin (negative control, open triangles, dashed lines). Data shown are from a representative experiment, which were confirmed in at least two or three independent experiments.

Figure 4

Percent survival of group B strain M4407 when incubated for 60 mins with human complement and dilutions of serum pools from immunized mice. Open circles with solid line, serum pool from mice immunized with recombinant fHbp variant 1 vaccine; X’s with dotted lines, serum pool from negative control mice immunized with aluminum hydroxide adjuvant alone; filled triangles with solid line, 1:1 mixture of the serum pool from mice immunized with the recombinant NHba vaccine and the pool from negative control mice immunized with aluminum hydroxide; open triangles with dashed line, 1:1 mixture of the serum pool from mice immunized with recombinant NHba vaccine and the pool from mice immunized with the recombinant fHbp variant 1 vaccine. The error bars represent the range at each dilution for independently diluted serum samples. The respective results were confirmed in a second independent experiment.