OpcA and PorB are novel bactericidal antigens of the 4CMenB vaccine in mice and humans

Abstract

The ability of Neisseria meningitidis Outer Membrane Vesicles (OMV) to induce protective responses in humans is well established and mainly attributed to Porin A (PorA). However, the contribution of additional protein antigens to protection remains to be elucidated. In this study we dissected the immunogenicity of antigens originating from the OMV component of the 4CMenB vaccine in mice and humans. We collected functional data on a panel of strains for which bactericidal responses to 4CMenB in infants was attributable to the OMV component and evaluated the role of 30 OMV-specific protein antigens in cross-coverage. By using tailor-made protein microarrays, the immunosignature of OMV antigens was determined. Three of these proteins, OpcA, NspA, and PorB, triggered mouse antibodies that were bactericidal against several N. meningitidis strains. Finally, by genetic deletion and/or serum depletion studies, we demonstrated the ability of OpcA and PorB to induce functional immune responses in infant sera after vaccination. In conclusion, while confirming the role of PorA in eliciting protective immunity, we identified two OMV antigens playing a key role in protection of infants vaccinated with the 4CMenB vaccine against different N. meningitidis serogroup B strains.

Fig. 1. The OMV component of 4CMenB mediated bactericidal responses on vaccine heterologous strains.

a Infant sera were collected and pooled (25 subjects each group) after the fourth vaccination dose and they were assayed in SBA with human complement. Solid bars indicate hSBA titers obtained against the 12 MenB strains using post-immunization rMenB + OMV (dark green), rMenB + 1/4 OMV (light green), rMenB (yellow-green) antisera. Bactericidal titers of preimmune sera were <2 for all groups against all tested strains. Titers ≥4 (dashed line) are considered protective. b rSBA titers were obtained using anti-OMV mouse sera against the panel of 12 MenB strains under investigation. Eleven groups of eight mice each deriving from distinct immunization schemes were pooled (n = 88) and used in the analysis. Serum bactericidal titers, indicating the dilution of the pooled mouse sera at which 50% of killing is reached, were determined using rabbit complement as source of complement. Titers ≥16 (dashed line) are considered positive as this dilution represents a fourfold increase respect to the lower limit of the assay corresponding to the first dilution tested (1:4).

Fig. 2. The immunosignature of human and mouse antisera revealed the immunodominant OMPs of OMV.

a Infant sera collected before and after the fourth dose of different vaccines were assayed as pool on a microarray containing exclusively recombinant proteins. b Pre- and post-immunization mouse sera were pooled (preimmune pool n = 80, rMenB pool n = 10, 4CMenB pool n = 54, OMV pool n = 88) and probed onto a microarray spotted with engineered E. coli GMMA. Samples are ranked according to the relative abundance of antigens in OMV as shown by the triangle. Each tinted block represents the averaged reactivity of the replicated spots for each serum screened, and results are expressed as MFI values. Signals were considered positive when their MFI were greater than 5000, corresponding to the MFI of control protein spots after detection with fluorescent-labeled secondary antibodies, plus ten times the standard deviation. Three arbitrary MFI thresholds were also assigned for low (5000 ≤ MFI < 15,000), medium (15,000 ≤ MFI < 30,000 MFI) and high (MFI ≥ 30,000) reactivities. Color scale of signal intensity is reported on the right of the heatmap.

Fig. 3. PorA, OpcA, PorB, and NspA antibodies showed strain-specific bactericidal killing.

rSBA titers obtained using GMMA-PorA (a), GMMA-OpcA (b), rPorB (c), and GMMA-NspA (d) antisera are represented by histograms. Filled bars represent natural strains, while stripped bars indicate knockout mutants used as negative controls. rSBA titers ≥16 are considered positive (dashed lines). Sera used in all the analyses are pool of eight mice.

Fig. 4. Evaluation of the expression levels and surface-localization of PorA, OpcA, PorB, and NspA in the panel of OMV-specific strains.

OMV and meningococcal crude lysates, prepared from bacteria culture grown in SBA-like conditions, were resolved in SDS-PAGE prior to western blotting and probing with GMMA-PorA (a), GMMA-OpcA (c), rPorB (e), and GMMA-NspA (g) sera. Specific bands are indicated by arrows. Asterisk refers to the unspecific band used as loading control among strain lysates. The binding of polyclonal antisera raised by PorA (b), OpcA (d), PorB (f), and NspA (h) antigens was assessed by flow cytometry against four MenB strains (the vaccine strain NZ98/254, two representative heterologous strains for each antigen and the specific negative control strain). The whole panel of strains assayed in FACS surface staining is represented in Supplementary Fig. 8. Shaded gray profiles represent bacterial cells exclusively stained with secondary antibodies, while non-shaded dark gray profiles indicate sera raised against GMMA empty ΔtolR and ΔompA (negative controls). Non-shaded orange profiles show the reaction with specific immune sera. Ab II secondary antibody. The uncropped and unprocessed scans are reported in Supplementary Fig. 9.

Fig. 5. Anti-PorA, -OpcA, and -PorB functional antibodies were raised by the OMV component of the 4CMenB vaccine in humans.

a–c rSBA titers obtained with pooled OMV mice sera against wild-type and knockout strains are represented by columns. Dashed lines represent the minimal threshold of functionality observed in the presence of baby rabbit complement. d The ability of PBS buffer alone or purified recombinant PorB protein to inhibit the killing of M09929 in the presence of OMV pooled sera was tested by competitive SBA and the resulting rSBA titers are illustrated as filled or striped histograms, respectively. Competitor is indicated in brackets. rSBA ≥16 are considered positive (dashed lines). e–g The ability of pooled (n = 25) infant immune sera collected from infants who received four doses of 4CMenB to kill wild-type and knockout N. meningitidis strains was tested in hSBA. Bactericidal titers are described as columns. hSBA ≥4 are considered protective (dashed line). h Competitive hSBA was performed on M09929 using PBS or PorB protein as a competitor (filled and striped bars, respectively).