A novel GMMA-based gonococcal vaccine demonstrates functional immune responses in mice

Abstract

Gonorrhea, caused by Neisseria gonorrhoeae (GC) represents a significant public health threat that may be mitigated by an effective vaccine. Vaccines containing N. meningitidis outer membrane vesicles (OMVs), such as 4CMenB, demonstrated moderate effectiveness in preventing GC infections. Here, we developed NgG, an investigational GC vaccine based on Generalized Modules for Membrane Antigens (GMMA). NgG includes genetically detoxified OMVs from the FA1090 strain, engineered to reduce endotoxin activity and limit immune interference. NgG induced a robust immune response in mice and outperformed the comparator vaccine 4CMenB in several serological and functional tests. Immunization with GMMA from a FA1090 mutant, where major oligosaccharide epitopes are incomplete or absent, revealed that NgG lipooligosaccharide plays a major role in the breadth of functional responses, with protein component also contributing in some GC strains. These results suggest that NgG has the potential to block GC infection through various mechanisms, supporting further vaccine development.

Fig. 1. Genetic detoxification assesment.

A Negative-ion mode MALDI-TOF spectra of the lipid A purified from the WT OMVs (a) and ΔlpxL1 GMMA (b). The major peaks correspond to monophosphoryl forms of (hexa or penta)acyl-lipid A (MPLA) and diphosphoryl forms of (hexa or penta)acyl-lipid A (BPLA) species. The mass difference between the corresponding peaks in the two spectra is 182.19 Da in agreement with the mass of a lauric acid chain. A signal at m/z 1572.8 corresponding to a non-identified lipid is present in both spectra. B Luciferase assay. Serial three-fold dilutions of FA1090ΔlpxL1Δrmp (Ng) GMMA, MenB OMV or FA1090 WT nOMV were added to HEK293-hTLR4 cells. The light emitted by luciferase activity was quantified. NF-κB activation of cells stimulated with the different samples was expressed as fold-increase of emitted light over the average result of PBS-stimulated control cells. C IL-6 release assay. Purified peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with serial three-fold dilutions of Ng GMMA, Shigella GMMA, MenB OMV or FA1090 WT nOMV. Released IL-6 was detected in each supernatant by an electrochemiluminescence immunoassay.

Fig. 2. Cellular-mediated immune response analysis.

CD1 mice (5 animals/group) were immunized intra-peritoneally, twice, three weeks apart with alum, 4CMenB or NgG. Fourteen days after the second immunization, the frequency of antigen-specific cytokine-secreting CD4⁺ T cells was determined by flow cytometry on splenocytes stimulated in vitro with Salmonella GMMA, FA1090 GMMA, heat-killed (HK) FA1090, SK92-679 GMMA, HK SK92-679, or MenB OMV. Antigen-specific CD4⁺ T cells are expressed as percentage of CD4⁺ T cells. T cell subsets (Th17, Th2, Th1 and Th0) were identified based on the type of secreted cytokines.

Fig. 3. Serum and vaginal antibody response analysis.

CD1 mice (10 animals/group) were immunized three times intra-peritoneally on Days 1, 29 and 57 with alum, 4CMenB or NgG (three different batches, NgG1, NgG2 and NgG3). Two weeks after the third immunization (2wp3), sera and vaginal washes were collected and the specific anti-Ng GMMA antibody responses were measured by Luminex assay. A Individual IgG serum titers for each immunization group are shown, with mean and 95% confidence intervals. B The statistical analysis for individual IgG serum titers is shown as geometric mean ratio (GMR) comparison (with 95% confidence intervals) for each batch versus 4CMenB. C Individual IgG vaginal titers for each immunization group are shown, with mean and 95% confidence intervals. D The statistical analysis for individual IgG vaginal titers is shown as geometric mean ratio (GMR) comparison (with 95% confidence intervals) for each batch versus alum and 4CMenB. E Individual IgA vaginal titers for each immunization group are shown, with mean and 95% confidence intervals. F The statistical analysis for individual IgA vaginal titers is shown as geometric mean ratio (GMR) comparison (with 95% confidence intervals) for each batch versus alum and 4CMenB. For all statistical results, the dotted lines at x = 2 indicate the threshold for the Lower Limit of GMR beyond which the differences are significant.

Fig. 4. Human serum bactericidal assay (hSBA) results.

Mice were immunized with alum, 4CMenB or NgG (three different batches called NgG1, NgG2, or NgG3). Sera were collected two weeks after the third immunization and functional antibodies were measured by hSBA against the homologous FA1090 GC strain and the heterologous BG27, BG8, F62, GC14, MS11, SK-92-679, WHO-F, WHO-G, WHO-M and WHO-N strains, using normal human serum from healthy donors as complement source. On the left side of the panel, individual serum titers are shown for each GC strain. On the right are shown statistical results as geometric mean titers (GMR) comparison (with 95% confidence intervals) between each strain and alum (upper part) and each strain and 4CMenB (lower part). The dotted lines at y = 1 indicate the threshold for the Lower Limit of GMR beyond which the differences are significant. Non statistically superior results are shown in red.

Fig. 5. Bacterial adhesion inhibition assay (BAI).

In this assay, the capacity of alum-, 4CMenB- and NgG-immunized murine sera to inhibit the adhesion of homologous FA1090 and heterologous SK92-679 GC strains to epithelial cells was assessed. Two different epithelial cell types were used: SV-HUC1 human ureteral cells and Ect1 human ectocervical. Briefly, GC strains (FA1090 or SK92-679) were labeled with Oregon Green dye before incubation with serially diluted murine sera (pooled 2wp3 sera). Bacteria-sera complexes were then added to the cultured epithelial cells. The percentage of adhesion inhibition was evaluated after one hour. The dotted lines at 30% indicate the blank limit.

Fig. 6. Role of anti-LOS antibodies in the functional response to the NgG vaccine.

Mice were immunized with alum, NgG or NgGΔlgtF. A Sera were collected two weeks after the third immunization and functional antibodies were measured by hSBA against the homologous FA1090 strain and the heterologous BG27, BG8, F62, GC14, MS11, SK92-679, WHO-F, WHO-G, WHO-M and WHO-N strains, using normal human serum from healthy donors as complement source. Titers obtained with pooled sera are shown for each GC strain. B The capacity of alum-, NgG- and NgGΔlgtF-immunized murine sera to inhibit the adhesion of homologous FA1090 and heterologous SK92-679 strains to SV-HUC-1 epithelial cells was assessed. Briefly, GC strains (FA1090 or SK92-679) were labeled with Oregon Green dye before incubation with serially diluted murine sera (pooled 2wp3 sera). Bacteria-sera complexes were then added to the cultured epithelial cells. The percentage of adhesion inhibition was evaluated after one hour. The dotted lines at 30% indicate the blank limit.