Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model

Abstract

Background: Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea.

Methods: We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets.

Results: Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB.

Conclusions: Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Keywords: Neisseria gonorrhoeae; Neisseria meningitidis; OMV; gonococcus; meningococcus; vaccine.

Figure 1.

Bactericidal activity of B5, B7, and B14 serum antibodies from rabbits immunized with meningococcal wild-type (WT), OCh, or ΔABR detoxified outer membrane vesicle vaccines against 3 gonococcal strains, FA19, MS11, and F62; B8 antiserum from an Alum-immunized rabbit was also tested as a negative control. Unabsorbed sera (blue squares) and sera preabsorbed against meningococcal strain MC58 (green circles) were diluted 1:5 through 1:160 and tested for induction of bacteriolysis in the presence of active (closed symbols) or heat-inactivated (HI; open symbols) human complement. Hashed lines represent Titer₅₀, Titer₃₀, and Titer₁₀ values. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001 represent significant differences between unabsorbed and absorbed sera by 2-tailed unpaired Student t test.

Figure 2.

Dynamics of gonococcal clearance in response to vaccination with meningococcal detoxified outer membrane vesicles. Percent of colonized mice (left panel), percent of mice cleared at the end of the time course (middle panel), and the average bacterial burden (right panel) are shown for the first (A) and second (B) challenge studies. For A, the number of infected mice included 18 for the wild-type (WT), OCh, and Alum groups, 17 for the ΔABR group, and 16 for the unimmunized (Unim.) group. For B, the number of infected mice included 18 for the ΔABR and unimmunized groups, 17 for the WT and OCh groups, and 16 for the Alum group. P_WT, P_OCh, and PΔ ABR represent P values of WT-, OCh-, and ΔABR dOMV-immunized groups relative to Alum-immunized (black text) and unimmunized (blue text) control groups, respectively, as determined by the log-rank (Mantel–Cox) test (percent clearance) and 2-way analysis of variance with Bonferroni correction (mean bacterial burden). Hashed line represents the lower limit of detection (20 colony-forming units [CFU]/mL of vaginal lavage) for colony counts.

Figure 3.

F62-specific vaginal and serum total immunoglobulin G (IgG) antibody titers. Total IgG titers in vaginal lavages at day 24 (top panel) and sera at day 24 (middle panel) and day 38 (bottom panel) from each independent challenge study are shown. Purple, red, blue, green, and black symbols represent antibody titers of individual mice that cleared infection on days 1, 3, 5, 7, and ˃7, respectively. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001 by Kruskal–Wallis test with Dunn multiple comparison. Abbreviations: IgG, immunoglobulin G; Unim., unimmunized; WT, wild-type.

Figure 4.

Serum immunoglobulin G antibodies induced by immunization with wild-type (WT), OCh, and ΔABR detoxified outer membrane vesicle (dOMV) vaccines cross-react with FA19, FA1090, MS11, and F62 gonococcal lysates. Binding of antibodies to the parental meningococcal vaccine strain MC58 is also shown. Serum dilutions for samples from the (A) first and (B) second challenge studies are as follows: WT and OCh, 1:40 000; ΔABR, 1:80 000; Alum, 1:10 000. Closed arrows indicate immunogenic antigens unique to OCh or ΔABR dOMV immunization relative to WT dOMV-specific antisera. Open arrows indicate binding of anti-RmpM antibodies. No bands were observed upon probing with the Alum-specific serum pool at comparable dilutions.

Figure 5.

Bactericidal antibody and murine polymorphonuclear leukocyte (PMN) responses. A, Pooled sera from mice immunized with wild-type, OCh, and ΔABR vesicle vaccines or Alum alone in the first challenge study were tested for bactericidal activity at a titer of 1:5 against gonococcal strains FA1090, MS11, and F62. Closed and open circles indicate percent survival upon incubation with active and heat-inactivated complement, respectively. B, Vaginal PMN influx over the course of infection of mice that cleared F62 or remained infected by the day 7 timepoint. Datasets from the first (upper panel) and second (middle panel) challenge studies and the combined dataset (bottom panel) are shown. Bars represent median ± 95% confidence interval. **P ≤ .01 by 2-tailed unpaired Student t test. Abbreviations: AUC, area under the curve; HI, heat-inactivated; PMN, polymorphonuclear leukocyte; WT, wild-type.