Experimental vaccine induces Th1-driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model

Abstract

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4⁺ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Fig. 1

I.vag immunization with gonococcal OMV plus IL-12/ms induced resistance to genital infection with N. gonorrhoeae, and generated an immune response. a: Mice were immunized 3 times at 7-day intervals with OMV (40µg protein) from strain FA1090 plus control (blank) ms or IL-12/ms (1µg IL-12); control mice were sham-immunized with either blank ms, or with IL-12/ms alone. Two weeks after the last immunization, all mice were challenged by i.vag. inoculation with N. gonorrhoeae strain FA1090 (5 × 10⁶ CFU), and infection was monitored by vaginal swabbing and plating. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), * P <0.01 (ANOVA); right panel: % of animals remaining infected at each time point, P <0.01 (Kaplan-Meier analysis, log-rank test, OMV plus IL-12/ms vs. OMV plus blank ms). b: Vaginal wash (left) and serum (right) antibodies against strain FA1090 in samples collected after termination (day 15), shown as mean ±SEM, N=5 samples; # P <0.05, * P <0.01, Student’s t. c: Intracellular cytokine staining in CD4⁺ cells recovered from ILN at termination (day 15), shown as mean ±SEM, N=3 samples, % of CD4⁺ staining for each cytokine; * P <0.01 Student’s t. d: Mice were immunized twice at a 14-day interval with gonococcal (Ngo) OMV (40µg protein) plus blank ms or IL-12/ms (1µg IL-12); control mice were sham-immunized with blank ms alone or with NTHI OMV (40µg protein) plus IL-12/ms (1µg IL-12). Two weeks later, all mice were challenged with N. gonorrhoeae FA1090 (5 × 10⁶ CFU). Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), * P <0.01 (ANOVA, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms); right panel: % of animals remaining infected at each time point, P <0.0001 (Kaplan-Meier analysis, log-rank test, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms).

Fig. 2

Antibody responses generated by immunization with gonococcal OMV plus IL-12/ms, prior to gonococcal challenge. a: Vaginal wash (left panel) and serum (right panel) antibodies assayed by ELISA 2 weeks after the last immunization with 1, 2, or 3 doses of gonococcal OMV (40µg protein) plus IL-12/ms (1µg IL-12). Control samples were obtained from mice sham-immunized with blank ms (3 doses); additional mice were immunized 3x with gonococcal OMV plus blank ms. Data shown as mean ±SEM, N=5 samples, # P <0.05, * P <0.01 relative to control samples (ANOVA). Duration of vaginal wash (b) and serum (c) antibodies in mice immunized with 2 doses of FA1090 OMV plus blank ms or IL-12/ms; data shown as mean ±SEM, N=5 samples; C, control samples from unimmunized mice.

Fig. 3

a: T cell cytokine responses in ILN cells induced by immunization with gonococcal OMV plus IL-12/ms 2 weeks after the last immunization with 1, 2, or 3 immunizations with gonococcal OMV (40µg protein) plus IL-12/ms (1µg IL-12). Control ILN were obtained from mice sham-immunized with blank ms (3 doses) and additional mice were immunized 3x with gonococcal OMV plus blank ms. Data shown as mean ±SEM, N=3 samples, % of CD4⁺ or CD8⁺ cells staining for each cytokine. * P < 0.01 (Student’s t test) comparing immunization with IL-12/ms vs. blank ms. b: Duration of IFNγ responses in CD4⁺ ILN cells 1–6 months after two immunizations with gonococcal OMV plus IL-12/ms or with OMV plus blank ms. Data shown as mean ±SEM, N=3 samples, % of CD4⁺ cells staining for IFNγ; C, control ILN from unimmunized mice.

Fig. 4

Resistance to gonococcal (FA1090) challenge persisted for at least 6 months after immunization with two doses of gonococcal (FA1090) OMV plus IL-12/ms. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), * P <0.01 (ANOVA, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms); right panel: % of animals remaining infected at each time point, P <0.001 (Kaplan-Meier analysis, log-rank test, gonococcal OMV plus IL-12/ms vs. gonococcal OMV plus blank ms).

Fig. 5

Resistance to heterologous gonococcal challenge. a: One month after immunization with FA1090 OMV plus IL-12/ms or blank ms, mice were challenged with N. gonorrhoeae strain FA1090 (homologous challenge) or strain MS11 (heterologous challenge). Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), * P <0.001 (ANOVA, for comparisons shown); right panel: % of animals remaining infected at each time point, P <0.02 for FA1090 challenge, IL-12/ms vs. blank ms; P <0.001 for MS11 challenge, IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). b: Mice immunized with MS11 OMV were resistant to challenge with N. gonorrhoeae FA1090. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), P <0.01 (ANOVA); right panel: % of animals remaining infected at each time point), P <0.01 (Kaplan-Meier analysis, log-rank test). c: Mice immunized with FA1090 OMV were resistant to challenge with N. gonorrhoeae FA19. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), * P <0.01 (ANOVA, for comparisons shown); right panel: % of animals remaining infected at each time point, P <0.01, IL-12/ms vs. blank ms for FA1090 challenge; P <0.0001, IL-12/ms vs. blank ms for FA19 challenge (Kaplan-Meier analysis, log-rank test), N=8 mice. d: Mice immunized with FA19 OMV were resistant to challenge with N. gonorrhoeae FA1090. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), P <0.01 (ANOVA); right panel: % of animals remaining infected at each time point), P <0.01 (Kaplan-Meier analysis, log-rank test). e: Mice immunized with FA1090 OMV were resistant to challenge with clinical isolate GC68. Left panel: recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), P <0.01 (ANOVA); right panel: % of animals remaining infected at each time point), P <0.01 (Kaplan-Meier analysis, log-rank test).

Fig. 6

Immunoproteomics of gonococal OMV. a: SDS-PAGE of OMV preparations from N. gonorrhoeae strains FA1090, MS11, and FA19, stained with Coomassie blue. b: Western blot analysis of mouse sera tested on gonococcal OMV preparations separated by SDS-PAGE. Lane 1, control serum from a mouse immunized with FA1090 OMV plus blank ms, tested against FA1090 OMV; lanes 2–4, serum #1 from a mouse immunized with FA1090 OMV plus IL-12/ms, tested against OMV from FA1090 (lane 2), MS11 (lane 3), or FA19 (lane 4); lane 5, serum #2 from a mouse immunized with FA1090 OMV plus IL-12/ms, tested against OMV from FA1090; lane 6, antibody H5 (anti-porin PIB3) tested against FA1090 OMV. c–e: proteome maps of gonococcal OMV derived from FA1090 (c), MS11 (d), and FA19 (e) revealed by 2D electrophoresis and Flamingo fluorescent staining (left panels) and their corresponding immunoblots (right panels) obtained by probing with mouse serum #2. Immunoreactive spots subjected to MS/MS analysis are labeled as spots 1 and 2 (arrows). Molecular mass marker (kDa) indicated on the left.

Fig. 7

Resistance to challenge induced by immunization with gonococcal OMV plus IL-12/ms depended on IFNγ and B cells. a: Course of infection (FA1090) in IFNγ-ko vs wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), • P <0.01 (ANOVA); right panel, % of animals remaining infected at each time point, P <0.0001 for wild-type mice, IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). b: Course of infection (FA1090) in µMT vs. wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), • P <0.01 (ANOVA); right panel, % of animals remaining infected at each time point, P <0.0001 for wild-type mice, IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). Vaginal wash (c) and serum (d) antibody responses in IFNγ-ko vs wild-type (mean ±SEM, N=5 samples) assayed at termination (day 13). IgA and IgG responses in vaginal wash and serum were significant (P <0.05, Student’s t, OMV plus blank ms vs. OMV plus IL-12/ms) for wild-type mice, but not for IFNγ-ko mice. e: T cell cytokine responses in µMT vs. wild-type mice (mean ±SEM, N=3 samples) assayed at termination (day 13). IFNγ response to immunization with OMV plus blank ms vs. OMV plus IL-12/ms was significant (P <0.01) for both wild-type and µMT mice (ANOVA). f: Course of infection (FA1090) in CD4-ko vs wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), # P <0.05, • P <0.01 (ANOVA) for comparisons shown; right panel, % of animals remaining infected at each time point, P <0.001 for wild-type mice IL-12/ms vs. blank ms, P <0.01 for CD4-ko mice IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test). g: Course of infection (FA1090) in CD8-ko vs wild-type mice immunized with FA1090 OMV; left panel, recovery (CFU) of N. gonorrhoeae (mean ±SEM, N=8 mice), # P <0.05, • P <0.01 (ANOVA) for comparisons shown; right panel, % of animals remaining infected at each time point, P <0.001 for wild-type mice IL-12/ms vs. blank ms, P <0.02 for CD8-ko mice IL-12/ms vs. blank ms (Kaplan-Meier analysis, log-rank test).