Additive and synergistic bactericidal activity of antibodies directed against minor outer membrane proteins of Neisseria meningitidis

Abstract

Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking children's PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection.

FIG. 1.

Schematic representation of the pCMK vectors used to deliver genes, operons, and/or expression cassettes in the genome of N. meningitidis. MCS, multiple cloning site.

FIG. 2.

Overproduction of Omp85, Hsf, TbpA, and NspA. (A) Impact on the content of proteins in MenB OMVs. OMVs purified from different N. meningitidis strains were separated by SDS-PAGE and stained with Coomassie brilliant blue. The strains used are wild-type strain H44/76 (lanes 1 and 7), a galE porA mutant derivative of H44/76 (lane 2), and galE porA mutants overexpressing Omp85 (lane 3), Hsf (lane 4), TbpA (lane 5), Hsf and TbpA simultaneously (lane 6), or NspA (lane 8). In lane 8, bands corresponding to denatured and nondenatured NspA are present. (B) Impact on the induction of antibodies against minor OMPs in mice immunized with OMVs purified from either overproducing strains or control strains or in mice immunized with adjuvant alone (AS04) (20 mice per group). Overproduction was achieved by either the promoter replacement (PR) or gene delivery (GD) strategy. ELISA titers are expressed as the reciprocal dilutions of pooled sera required to obtain an OD₄₉₀ of 0.5, using purified recombinant TbpA, Hsf, NspA, or Omp85.

FIG. 3.

Bactericidal activity against N. meningitidis strains of serum antibodies from mice immunized with OMV vaccines. Sera from 20 mice (see Table 4) were pooled, and two to four pools were combined. Bactericidal assays with (A) H44/76 and (B) Cu385 were performed using pooled sera from mice immunized with OMVs from either TbpA-, Hsf-, or Tbp/Hsf-overproducing strains or with a 1:1 (vol/vol) mixture of serum pools from mice immunized with OMVs from TbpA- and Hsf-overproducing strains. Bactericidal activity is expressed as the reciprocal antibody titer. (C) Bactericidal assays with strain M97-250687 were performed using pooled sera from mice immunized with OMVs from either Omp85-, NspA-, or TbpA/Hsf-overproducing strains or a combination thereof. The data are the means of three different mixing experiments performed using the same serum samples and are expressed as the reciprocal bactericidal titers.

FIG. 4.

Schematic representation of the impact of the antigen density on the bactericidal activity of antibodies on the surface of coccal MenB strains. A defined density is required to induce significant bactericidal antibody killing mediated by complement. This threshold density is reached when at least two minor OMPs (Ag 1 and Ag 2) are targeted by antibodies. For an efficacious OMV vaccine, at least two minor OMPs must be overproduced, resulting in vaccine-induced bactericidal antibody killing. Ag, antigen; Abs, antibodies; SBA, serum bactericidal activity. The first step of the classical pathway for activation of the complement cascade is binding of the C1q factor to antibodies, leading to the formation of the membrane attack complex.