Table 1.
Characteristics of strains used in this study.
Figure 1.
Binding of human fH or control mAbs to mutant vaccine strains and NOMV vaccines.
The upper panels (A–C) show binding to live bacteria by indirect immunofluorescence flow cytometry. The lower panels (D–F) show binding to NOMV vaccines by ELISA. A and D, binding of fH; B and E, binding of anti-fHbp mAb JAR 5; C and F, binding of a control anti-PorA mAb, P1.7. WT, H44/76ΔLpxL1 bacteria or NOMV vaccine with over-expressed wild-type fHbp (blue open circles with solid blue line); R41S, H44/76ΔLpxL1 bacteria or NOMV vaccine with over-expressed R41S mutant fHbp (open squares with dashed black line); KO, H44/76ΔLpxL1ΔfHbp bacteria or corresponding NOMV vaccine with no detectable fHbp (dotted orange line (Panels A–C) or inverted orange triangles (Panels D–F)). For the flow cytometric analysis, cells with negative binding (fluorescence background values close to zero) are displayed as bi-exponential or “logical” negative values on the X axis, as described [42].
Figure 2.
Characterization of native outer membrane vesicle (NOMV) vaccines prepared from H44/76 mutant strains.
A, Western blot probed with an anti-fHbp mAb, JAR 5. Lane 1, molecular mass standards (Magic Mark; Invitrogen); Lane 2, purified, recombinant fHbp ID 1 (0.1 µg); Lanes 3 to 5, NOMV (0.25 µg) from H44/76 mutants. Lane 3, genetic knock-out of fHbp; Lane 4, over-expressed WT fHbp ID 1; Lane 5, over-expressed mutant R41S fHbp. B, SDS-polyacrylamide gel stained with Coomassie blue. Lane 1, Molecular mass standards (Kaleidoscope Prestained; Bio-Rad); lanes 2 through 5 contain the same respective samples as in Panel A except that there is 2 µg of recombinant fHbp (Lane 2) or 10 µg of NOMV (Lanes 3–5).
Figure 3.
Serum antibody responses of wild-type mice immunized with NOMV vaccines.
Each symbol represents the reciprocal serum titer of an individual mouse, and the horizontal line represents the geometric mean titer. OE WT, OE R41S or KO, NOMV vaccines with over-expressed wild-type fHbp, R41S mutant fHbp, or fHbp knock-out, respectively. Alum, aluminum hydroxide adjuvant only. rfHbp, recombinant fHbp. A, Serum IgG anti-fHbp antibody responses measured by ELISA (1/GMT of OE WT vs. OE R41S, P = 0.001; OE WT or OE R41S vs. rfHbp, P≤0.0002). B, Serum bactericidal antibody responses against strain Cu385, which has a mismatched PorA type compared to the vaccine strains and expresses fHbp ID 1 that matches the vaccine fHbp antigen (1/GMT of OE WT vs. OE R41S, P = 0.003).
Figure 4.
Effect of depletion of serum anti-fHbp antibodies on bactericidal activity.
A, IgG anti-fHbp antibody as measured by ELISA. Representative data are from one of the two serum pools from wild-type mice immunized with the NOMV with over-expressed WT fHbp. The serum pool either was not adsorbed (“none,” open triangles with dotted line), or adsorbed on a human albumin column (“mock,” filled circles with solid line), or on an fHbp column (“fHbp,” open circles with solid line). The adsorbed serum samples were adjusted to the original volume applied to the column. B, IgG anti-NOMV antibody measured by ELISA with the NOMV vaccine prepared from fHbp knock-out strain as the antigen in the wells. Data are from the same serum pool as shown in Panel A. For panels A and B, similar respective data were obtained with each of the other three serum pools. C, Serum bactericidal antibody responses against strain Cu385, which has a fHbp variant group 1 antigen matched to that of the vaccines and a mismatched PorA (see Table 1). Two serum pools for each vaccine group were assayed (NOMV with over-expressed WT fHbp (“OE WT”) or R41S mutant fHbp (“OE R41S”) following depletion on mock or fHbp columns. D. Serum bactericidal antibody titers measured against an isogenic mutant of strain H44/76 with matched PorA to that of the vaccine strains and a mismatched fHbp in variant group 3. The respective serum pools were the same as in Panel C.
Figure 5.
Concentrations of human fH in serum samples obtained before immunization of transgenic mice with NOMV vaccines.
OE WT, mice assigned to group immunized with NOMV with over-expressed wild-type fHbp; OE R41S, mice assigned to group immunized with NOMV with over-expressed mutant R41S fHbp; KO, control mice that were immunized with NOMV from the fHbp knock-out; Alum, control mice that were immunized with aluminum hydroxide adjuvant only. Human fH concentrations of mice that were removed from the study before completion of vaccination are represented as gray filled symbols.
Figure 6.
Serum antibody responses of human fH transgenic mice immunized with NOMV vaccines.
Each symbol represents the reciprocal serum titer of an individual mouse, and the horizontal line represents the geometric mean titer. A, Serum IgG anti-fHbp antibody responses measured by ELISA. Comparing 1/GMT of OE R41S vs. OE WT, P = 0.002. B, Serum bactericidal antibody responses against strain Cu385 with fHbp from variant group 1. Comparing 1/GMT of OE R41S vs. OE WT, P = 0.001.