Bactericidal antibody responses elicited by a meningococcal outer membrane vesicle vaccine with overexpressed factor H-binding protein and genetically attenuated endotoxin

Abstract

Background: Outer membrane vesicle (OMV) vaccines from mutant Neisseria meningitidis strains engineered to overexpress factor H-binding protein (fHbp) have elicited broadly protective serum antibody responses in mice. The vaccines investigated were not treated with detergents to avoid extracting fHbp, which is a lipoprotein. Because of their high endotoxin content, the vaccines would not be safe to administer to humans.

Methods: We prepared a native OMV vaccine from a strain engineered to overexpress fHbp and in which the gene encoding LpxL1 was inactivated, which reportedly decreases endotoxin activity.

Results: The OMV vaccine from the mutant had a similar or lower ability to induce the expression of proinflammatory cytokines by human peripheral blood mononuclear cells, compared with a detergent-extracted wild-type OMV, and 1000-10,000-fold lower activity than a native wild-type OMV. In mice, the OMV vaccine from the mutant elicited higher serum bactericidal antibody responses to a panel of heterologous N. meningitidis strains than did a control multicomponent recombinant protein vaccine or a detergent-extracted OMV vaccine that has been demonstrated to confer protection against meningococcal disease in humans.

Conclusions: The data illustrate the potential to develop a broadly immunogenic native OMV vaccine that has decreased endotoxin activity and is potentially suitable for testing in humans.

Figure 1

Panel A. fHbp v.1 in OMV preparations as detected by Western-blot. WT, native OMV prepared from H44/76 wildtype strain; fHbp KO, OMV from mutant of H44/76 in which the gene encoding fHbp was inactivated; LpxL1 KO OE fHbp, OMV from mutant of H44/76 with inactivated LpxL1 and over-expressed fHbp; rfHbp, recombinant v.1 protein. Primary antibody was anti-rfHbp monoclonal antibody JAR3. Panel B. Silver stained SDS-PAGE of LOS in OMV preparations from strain H44/76. Amounts of vesicles loaded in each lane were standardized based on total protein content.

Figure 2

Release of proinflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α after incubation of PBMCs with different concentrations of OMV for 4 hours. Left, PBMCs from Donor 1, experiment 1. Right, PBMCs from Donor 2, experiment 2. OMV vaccines tested were native OMV prepared from the H44/76 wildtype (WT native, open squares with solid line) or a H44/76 mutant with inactivated LpxL1 and over-expressed fHbp (mutant, native, closed squares with solid line), or a detergent-extracted OMV from H44/76 wildtype strain (WT extracted, open circles with dashed lines).

Figure 3

(Supplementary for website). Release of cytokines in response to incubation of human PBMCs for four hours with different concentrations of OMV. Of 27 cytokines tested, 10 were stimulated above background levels: four proinflammatory cytokines (Figure 2) and the remaining six, IL-1ra, G-CSF, IFN-γ, MCP-1, MIP-1α and MIP-1β, are shown in this figure. Left, data from PBMCs of Donor 1, experiment 1. Right, PBMCs from Donor 2, experiment 2. Symbols for OMV vaccines tested are as described for Figure 2.

Figure 4

Serum antibody responses to fHbp (v.1), NadA. GNA2132 and LOS (LpxL1 KO mutant) as measured by ELISA. Left to right, mice immunized with aluminium hydroxide (Bar 1); the Norwegian OMV vaccine (OMV, Norway, Bar 2); a recombinant protein vaccine (Bar 3); or native OMV prepared from H44/76 with inactivated LpxL1 and increased expression of fHbp (OMV, mutant, Bar 4). The error bars show the 95% confidence intervals of the geometric mean titers.

Figure 5

Effect of temperature on measurement of antibody titers to LOS and fHbp (v.1) by ELISA in sera from mice immunized with mutant OMV. Sera were incubated for 2 hrs at 37°C, or for 18 hrs at 4°C. After washing the plates, bound Ig was measured by goat-anti-mouse IgG+A+M conjugated to alkaline phosphatase.

Figure 6

Serum bactericidal activity (GMT) of immunized mice as measured against N. meningitidis strain H44/76 (used to prepare the OMV vaccines, see text) and six additional strains with heterologous PorA molecules to that of the vaccine strain and expressing subvariants of v.1 fHbp (see Table 1). Bars represent GMT of assays of 3 serum pools from each vaccine group (serum samples from 5 mice per pool). There were 2 serum pools for the Norwegian OMV vaccine group and the bars represent the GMT and range of the respective values. Symbols are identical to those used in Figure 4.