Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines

Abstract

A unique multicomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ≥80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if one antigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria.

Fig. 1.

Schematic of the MATS ELISA method. (A) MenB bacteria are grown overnight on chocolate agar. (B) A suspension of bacteria taken from the plate is prepared to a specified OD₆₀₀. (C) Detergent is added to the suspension to extract the capsule and expose the antigens. (D) Serial dilutions of extract are tested in the MATS ELISA. A specific capture antibody (yellow) binds one of the antigens (example: fHbp, blue) from the extract, which is then detected with a specific biotin-labeled antibody (yellow and purple) and a streptavidin–enzyme conjugate (green and gold). (E) Plates are read at 490 nm in an ELISA reader. (F) Results are calculated by comparing the curve of OD₄₉₀ vs. dilution obtained with the serially diluted unknown strain to a serially diluted reference strain tested in the same ELISA plate.

Fig. 2.

(A–C) Frequency distribution of relative potency (RP) of NHBA, NadA, and fHbp antigens in 124 serogroup B strains. (Upper) Reverse cumulative distributions of the proportion of strains with antigen RP greater than the values indicated on the x axis. (Lower) Histograms of frequency distribution of strains with different values of antigen RP. (A) Antigen RP of different strains for fHbp. Genetic variants of fHbp (variants 1, 2, or 3, with subvariants denoted by decimals) are indicated by brackets. (B) Antigen RP of different strains for NHBA. (C) Antigen RP of NadA in different strains. Brackets denote strains with low expression and higher expression of NadA. Eighty-eight percent of the strains not quantifiable by the test are PCR negative for the NadA gene. rel. pot., relative potency for each antigen determined by MATS in comparison with the reference strains H44/76 (fHbp), 5/99 (NadA), and NGH38 (NHBA). LLOQ, lower limit of quantitation, lowest antigen RP values with between-assay CV ≤20%, 27%, and 34% for fHbp, NHBA, and NadA, respectively.

Fig. 3.

MATS relative potency (RP) of NHBA, NadA, and fHbp determined by the MATS ELISA in antigen-specific subpanels (strains mismatched for the other three antigens, A, B, and C, respectively) and in the 57-strains panel (D, E, and F, respectively). The strains are in ascending order of MATS RP. Strains covered (as determined by killing in the SBA with pooled postimmune infant sera) are shown with solid red circles, and those not covered are shown with solid blue circles. The dashed line represents the positive bactericidal threshold (PBT) for each antigen; this threshold is the relative potency above which at least 80% of MenB strains are killed in SBA with serum from vaccinated subjects. Error bars represent 95% confidence limits for each measurement, deduced from the intermediate precision of the method.

Fig. 4.

Phylogenetic reconstruction of the MenB strains used in this study. MLST-based phylogenetic reconstruction is shown of the 124 strains that were MATS typed in this study. Color coding shows antigens at or above the PBT. Strains killed in the SBA with postimmune serum from adult vaccinees are coded by a thick black border. Major clonal complexes (CC) are indicated with the founder CC. The phylogenetic tree was obtained with the Neighbor program from the PHYLIP package, with branch lengths computed from the Kimura two-parameter distances.