Characterisation and Immunogenicity of Neisseria cinerea outer membrane vesicles displaying NadA, NHBA and fHbp from Neisseria meningitidis serogroup B

Abstract

More affordable and effective vaccines against bacterial meningitis caused by Neisseria meningitidis serogroup B are still required for global prevention. We have previously shown that modified outer membrane vesicles (mOMVs) from commensal Neisseria cinerea can be used as a platform to induce immune responses against meningococcal antigens. The aim of the present study was to use a combination of two genetically engineered mOMVs to express multiple antigens from N. meningitidis known to be involved in protective immunity to meningococcal meningitis (different variants of factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisseria Adhesin A (NadA)). Antigen expression in the mOMVs was confirmed by Western blotting; detoxification of the lipooligosaccharide (LOS) was confirmed by measuring human Toll-like receptor 4 (hTLR4) activation using in vitro cell assays. Mice immunised with a combination of two mOMVs expressing fHbp, NHBA and NadA produced antibodies to all the antigens. Furthermore, serum bactericidal activity (SBA) was induced by the immunisation, with mOMVs expressing NadA displaying high SBA titres against a nadA⁺ MenB strain. The work highlights the potential of mOMVs from N. cinerea to induce functional immune responses against multiple antigens involved in the protective immune response to meningococcal disease.

Keywords: NHBA; NadA; Neisseria cinerea; Neisseria meningitidis; fHbp; mOMVs; meningitidis; modified outer membrane vesicles.

Figure 1

Analysis of the expression of fHbp ID100 and NadA-8 in mOMV. The presence of fHbpID100 in the mOMV from N. cinerea strains were analysed by WB with (A) JAR5 and the presence of NadA-8 were analysed by WB with (B) a polyclonal α-NadA-8. Each lane was loaded with either 2.5 μg of OMVs or 0.1 μg of recombinant proteins. Recombinant fHbp_ID100 and NadA-8 were used as positive controls.

Figure 2

Analysis of the expression of fHbp ID45 and NHBA-2 in mOMV. The presence of fHbp_ID45 was analysed by WB with (A) JAR13 and the presence of NHBA-2 in the mOMV from N. cinerea strains was analysed by WB with (B) a monoclonal α-NHBA 10C3. Each lane was loaded with either 2.5 μg of OMVs or 0.1 μg of recombinant proteins. Recombinant fHbp_ID45 and NHBA-2 were used as positive controls.

Figure 3

TEM images of mOMVs stained with uranyl acetate. Scale bar = 100 m.

Figure 4

Cellular in vitro assay to assess TLR4 activation after stimulation with mOMVs. 2.5 x 10⁴ cells/well of HEK-293 hTLR4, MD2, CD14 were stimulated for 22 hours with 10-fold serial dilutions of mOMV preparations. hTLR4 activation correlates with SEAP induction, measured as per manufacturer’s protocol. Data shown are from three biological replicates, with each replicates containing technical triplicates; data are shown as mean with standard deviation (vertical error bars). LPS-EK: ultrapure lipopolysaccharide from E. coli K12; it served as positive control for the assay. HEK-Null cells (which contained the gene reporter system, but lacking hTLR4, MD2 and CD14) were used as a negative control for the assay (data not shown) and showed SEAP fold induction of less than 1.5 when stimulated with OMV or purified LPS-EK throughout the dose range used for the assays. Statistical analysis was performed using Two-Way ANOVA with Dunnett’s multiple comparisons test, where * denotes p-value of <0.05, ** denotes p-value of <0.01, and ns denotes not significant.

Figure 5

Sera responses of mice immunized with OMV-ID100 NadA-8 and/or OMV-ID45 NHBA-2, when plates were coated with their respective mOMVs. IgG antibody levels measured in sera diluted at 1:27,000 when plates were coated with A) OMV-ID100 NadA and B) OMV-ID45 NHBA-2. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison post-test, where ns denotes not significant, *** denotes p-value of <0.001 and **** denotes p-value of <0.0001.

Figure 6

Sera responses of mice immunized with OMV-ID100 NadA-8 and/or OMV-ID45 NHBA-2, when plates were coated with recombinant proteins. (A) IgG antibody levels measured in sera diluted at 1:80 when plates were coated with rID100 protein. (B) IgG antibody levels measured in sera diluted at 1:4,000 when plates were coated with rNadA-8 protein. (C) IgG antibody levels measured in sera diluted at 1:40 when plates were coated with rID45 protein. (D) IgG antibody levels measured in sera diluted at 1:40 when plates were coated with rNHBA-2 protein. Line denotes the mean of IgG antibody levels. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison post-test, where ns denotes not significant, * denotes p-value of <0.05, ** denotes p-value of <0.01, *** denotes p-value of <0.001 and **** denotes p-value of <0.0001.

Figure 7

Sera antibodies induced by immunization with mOMVs expressing meningococcal antigens cross-react with MenB. Sera from mice immunized with (A) 10 μg OMV-ID100 NadA-8 (B) 10 μg OMV-ID45 NHBA-2, with (C) 20 μg OMV-ID100 NadA-NadA-8 (D) 20 g OMV-ID45 NHBA-2 (E) 10 μg OMV-ID100 NadA-8 + 10 μg OMV-ID45 NHBA-2 and (F) Alhydrogel only were used to probe whole-cell lysates of MenB MC58, as well as N. cinerea ATCC^® 14685^TM and N. sicca ATCC^® 29259^TM.