Mutant Native Outer Membrane Vesicles Combined with a Serogroup A Polysaccharide Conjugate Vaccine for Prevention of Meningococcal Epidemics in Africa

Abstract

Background: The meningococcal serogroup A (MenA) polysaccharide conjugate vaccine used in Sub-Saharan Africa does not prevent disease caused by MenW or MenX strains, which also cause epidemics in the region. We investigated the vaccine-potential of native outer membrane vesicles with over-expressed factor H-binding protein (NOMV-fHbp), which targeted antigens in African meningococcal strains, and was combined with a MenA polysaccharide conjugate vaccine.

Methodology/principal findings: The NOMV-fHbp vaccine was prepared from a mutant African MenW strain with PorA P1.5,2, attenuated endotoxin (ΔLpxL1), deleted capsular genes, and over-expressed fHbp in variant group 1. The NOMV-fHbp was adsorbed with Al(OH)3 and used to reconstitute a lyophilized MenA conjugate vaccine, which normally is reconstituted with liquid MenC, Y and W conjugates in a meningococcal quadrivalent conjugate vaccine (MCV4-CRM, Novartis). Mice immunized with the NOMV-fHbp vaccine alone developed serum bactericidal (human complement) activity against 13 of 15 African MenA strains tested; 10 of 10 African MenX strains, 7 of 7 African MenW strains, and 6 of 6 genetically diverse MenB strains with fHbp variant group 1 (including 1 strain from The Gambia). The combination NOMV-fHbp/MenA conjugate vaccine elicited high serum bactericidal titers against the two MenA strains tested that were resistant to bactericidal antibodies elicited by the NOMV-fHbp alone; the combination elicited higher titers against the MenA and MenW strains than those elicited by a control MCV4-CRM vaccine (P<0.05); and high titers against MenX and MenB strains. For most strains, the titers elicited by a control NOMV-fHbp knock out vaccine were <1∶10 except when the strain PorA matched the vaccine (titers >1∶000).

Conclusion/significance: The NOMV-fHbp/MenA conjugate vaccine provided similar or higher coverage against MenA and MenW strains than a quadrivalent meningococcal conjugate vaccine, and extended protection against MenX strains responsible for epidemics in Africa, and MenB strains with fHbp in variant group 1.

Figure 1. Schematic representation of the genetic strategy to over-express fHbp ID 9 R41S.

Panel A: Sequences of natural porA and nadA promoters and the engineered (Hybrid) promoter where the sequence between the −10 and −35 boxes of the porA promoter containing the poly G tract was replaced with the sequence from the Neisserial nadA promoter to eliminate slip-strand miss-pairing. Although not shown, in the hybrid promoter the porA promoter sequence (−164 to −35 and −10 to +57) was left intact. Panels B-C: Strategy for replacement of the native fHbp promoter by the engineered promoter sequence, and introduction of the R41S mutation in fHbp to decrease fH binding (see text for additional details). Panel B shows the genomic environment of the fHbp gene (nmb1870) and the plasmid RP-EH used. CAT, represents the chloramphenicol resistance cassette used for selection of the recombinants. The yellow R41S box represents the first 255 base pairs of the fHbp ID 9 gene containing the mutation that leads to the R41S change in the mature mutant ID 9 fHbp. Panel C represents the organization of the region after the recombination.

Figure 2. Surface-accessible fHbp on live bacteria of the mutant vaccine strain Sudan 1/06 with over-expressed fHbp as measured by flow cytometry.

A. Binding of anti-fHbp mAb, JAR 5. WT, parental strain with wildtype fHbp expression; KO, mutant with fHbp gene inactivated; OE, strain with the engineered promoter and mutant gene encoding fHbp ID 9 with the R41S amino acid substitution. Panel B: Detection of MenW using anticapsular mAb JW-W1 as a control.

Figure 3. Detection of fHbp in NOMV vaccines by Western blot using anti-fHbp mAb JAR 5.

rfHbp, recombinant fHbp ID 9. OE, NOMV-fHbp vaccine with over-expressed R41S mutant fHbp prepared from the mutant vaccine strain with Δlpxl1 and Δcapsule; KO, NOMV prepared from a triple knockout (ΔfHbp, Δlpxl1, and Δcapsule); WT, NOMV vaccine prepared from the wildtype strain. A total of 0.25 µg of NOMV was loaded in each lane.

Figure 4. Characterization of NOMV vaccines by ELISA.

Panel A. fHbp was measured using anti-fHbp mAb, JAR 5. Panel B. Binding of human fH to the NOMV vaccines was performed as previously described . Panel C. PorA was measured using anti-PorA mAbs specific for P1.2 . Panel D. MenW capsular polysaccharide was measured using anticapsular mAb, JW-W1.

Figure 5. Serum bactericidal antibody responses of mice immunized with NOMV-fHbp vaccine.

Panels A, B and C, MenA, W or X isolates, respectively, from cases of disease isolated during epidemics in sub-Saharan Africa. Panel D, MenB isolates from industrialized countries. The MenA, X and B strains had fHbp amino acid sequence variants in variant group 1 (See Table 1). The MenW stains had fHbp in variant group 1 (ID 9, ID 350) or variant group 2 (ID 23, ID 22). Bars represent the reciprocal GMT and ranges of three serum pools per vaccine group, and two pools for the adjuvant alone (four individual sera in each pool). Black bars, control mice immunized with aluminum hydroxide without a vaccine antigen; orange bars, control mice immunized with a NOMV-fHbp KO vaccine; white bars, mice immunized with the NOMV-fHbp vaccine.

Figure 6. Serum IgG antibody responses to vaccination as measured by ELISA.

Panel A. Anti-fHbp ID 9 titers. Panel B. Anticapsular antibody titers to MenA polysaccharide. Each symbol represents the reciprocal titer of an individual immunized mouse. Horizontal lines denote reciprocal geometric mean titers. There were no significant differences in the IgG anti-fHbp antibody titers elicited by the NOMV-fHbp or combination NOMV-fHbp/MenA conjugate vaccine (P>0.5). The serum IgG anticapsular titers were higher in mice immunized with the combination NOMV-fHbp/Men A conjugate vaccine than the control MCV4-CRM conjugate vaccine (P = 0.001).

Figure 7. Serum bactericidal antibody responses of mice immunized with a combination NOMV-fHbp/MenA conjugate vaccine.

Test strains are described in Table 1, and strain selection criteria are described in the text. Panels A and B, MenA strains A3 and A9 with fHbp ID 5, respectively in variant group 1. Panel C, MenW strain, W2, with fHbp ID 23 in variant group 2. Panels D and E, MenX strains, X2 or X3, respectively, with fHbp ID 74 in variant group 1. Panel F, MenB strain, B6, from The Gambia, with fHbp ID 347 in variant group 1. Each symbol represents the reciprocal titer of an individual mouse; horizontal bars represent 1/GMT. The respective serum bactericidal titers elicited by the combination NOMV-fHbp/MenA vaccine were higher than the NOMV-fHbp alone against strains A3 and A9 (P<0.0001) and strain W2 (P = 0.03). For these three strains, the serum bactericidal titers elicited by the combination NOMV-fHbp/MenA conjugate vaccine also were higher than the respective titers elicited by control MCV4-CRM vaccine (P<0.03). For the two MenX strains and the MenB strain, the respective titers elicited by the combination NOMV-fHbp/MenA conjugate vaccine were not significantly different from those elicited by the NOMV-fHbp vaccine alone (P>0.14).