Protection against N. gonorrhoeae induced by OMV-based meningococcal vaccines are associated with cross-species directed humoral and cellular immune responses

Abstract

Introduction: Limited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear.

Methods: To study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae, 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 ΔABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model.

Results: Mice immunized with 4CMenB or MC58 ΔABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58ΔABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae. Further, splenocytes in 4CMenB and MC58 ΔABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine.

Discussion: Our findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

Keywords: Neisseria gonorrhoeae; Neisseria meningitidis; correlates of protection; outer membrane vesicle (OMV); vaccine.

Figure 1

A mouse vaccination followed by vaginal challenge with N. gonorrhoeae experimental schema was executed to test whether common immunologic responses to different N. meningitidis OMV vaccines that correlated with clearance of infection could be identified. Two identical experiments were performed in which mice were assigned to one of three vaccine groups: Alum (the adjuvant alone control group), MC58 ΔABR OMV vaccine, or 4CMenB vaccine. After the indicated vaccines were administered, an intravaginal challenge with N gonorrhoeae strain F62 was conducted and vaginal N. gonorrhoeae and PMN influx was assessed over 7 days. Blood, vaginal wash fluid and splenocytes were collected from the mice at the indicated times for the indicated battery of immunologic tests were conducted. The immune responses were tested for correlation to reduced bacterial burden during the period of infection. Created in BioRender. Zhu, W (2025). https://BioRender.com/m71q724.

Figure 2

Vaccination with MC58 ΔABR and 4CMenB enhance N. gonorrhoeae clearance from lower genital tract in mice (A) Fraction of mice remaining infected over time with N. gonorrhoeae strain F62 after intravaginal inoculation are shown for mice vaccinated with Alum (adjuvant alone control), MC58 ΔABR OMV, and 4CMenB as indicated. Differences between infection persistence were compared using a log-rank (Mentel-Cox) test; P were 0.03 or <0.001 when MC58 ΔABR OMV and 4CMenB were compared to the Alum control. (B) Vaginal swab specimens were quantitatively cultured to determine the bacterial burden (number of CFU per milliliter) on days 1,3,5, and 7, the total N. gonorrhoeae bacterial burden (AUC vaginal N. gonorrhoeae recovered) for the infection course was determined for each individual mouse by taking the area under the curve of the log of the recovered CFU plotted against day post infection. The AUC are plotted for mice in each immunization group and groups are split into those mice that cleared infection by day 7 and those that had persistent infection at day 7. Statistical significance was performed using 2-way ANOVA followed by Bonferroni Post-hoc multiple comparisons to compare immunization groups. (C) Neutrophils (PMN) recovered on vaginal swabs collected on indicated days were quantified and plotted for individual mice in each vaccination group on each day (top panel) and as the mean for the group (bottom panel). (D) The total neutrophil recovery for the course of the infection was determined for each individual mouse by determining the area under the curve of the plotted recovered neutrophils over time and the AUC of Vaginal PMN recovery was plotted against the N. gonorrhoeae burden (AUC of Vaginal N. gonorrhoeae recovery) for each individual mouse, the Pearson Correlation Coefficient was determined for each immunized group of mice to assess for significant correlations between vaginal PMN and recovered bacteria. Created in BioRender. Zhu, W (2025). https://BioRender.com/q33k508.

Figure 3

Vaccination with MC58 ΔABR and 4CMenB elicited cross-reactive anti-Ng-OMV antibodies in serum and vaginal fluid. N. gonorrhoeae OMV reactive antibody levels were measured by ELISA as described in Materials and Methods in sera collected at day 52, two weeks after final vaccination (A), and at terminal sera collection after N. gonorrhoeae challenge (B), and in terminal vaginal wash after N. gonorrhoeae challenge (D). The dilution of sera resulting in 50% maximal signal (EC50) and the greatest dilution of vaginal wash fluid resulting in signal above the baseline (Endpoint titer) were determined for total IgG, IgG1 or IgG2a isotype using immunoglobulin subclass specific secondary antibodies. (C) the fold change in each indicated N. gonorrhoeae OMV-directed immunoglobulin level between serum collected before challenge and after N. gonorrhoeae challenge was also determined. Levels from Alum (black), MC58 ΔABR (blue) and 4CMenB (Red) immunized mice are reported. Data were represented at log scale. Statistical significance was performed using one-way ANOVA followed by no paring Šidák’s multiple comparison test, with a single polled variance. Created in BioRender. Zhu, W (2025). https://BioRender.com/h21e029.

Figure 4

Anti-gonoccoccal IgG2A levels measured by ELISA in serum correlate with reduced gonococcal burden during infection in MC58 ΔABR OMV- immunized mice but not 4CMenB-immunized mice. Mice were immunized with MC58 ΔABR OMV or 4CMenB and subsequently intravaginally challenged with N. gonorrhoeae (as described in Figure 1 ), serum antibody levels of total anti-gonococcal OMV IgG, IgG1, IgG2a and IgG3 were measured by ELISA before and after gonococcal challenge and, levels expressed as log₂ (EC₅₀). The change in levels for each immunoglobulin between levels determined before and after gonococcal challenge were determined as described in Figure 3 . Mouse vaginal total anti-gonococcal IgG, IgG1, IgG2a and IgG3 was measured after gonococcal challenge only and measured by ELISA, expressed as log₂ (endpoint titers). The mean change in bacterial burden (AUC of log₁₀ CFU) with respect to serum antibody pre-challenge levels (A), post-challenge levels (B), and fold change (C) and vaginal wash antibody levels (D) for each mouse was assessed with simple linear regression and 95% confidence intervals are being reported.

Figure 5

Immunoblot reactivity against N. gonorrhoeae OMV proteins correlates with increased N. gonorrhoeae burden in MC58 ΔABR OMV-immunized mice and with decreased N. gonorrhoeae burden in 4CMenB-immunized mice. (A) Proteins from crude outer membrane vesicle (cOMV) preparations from N. gonorrhoeae strain F62 were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. The separated N. gonorrhoeae proteins were blotted with individual mouse serum from mice immunized with either MC58 ΔABR OMV (top panel) or 4CMenB (bottom panel) collected after N. gonorrhoeae challenge used at 1:1500 dilution in a slot blotting apparatus. Sera from alum-immunized control mice was pooled from each of the two experiments performed and these two pools were used in the indicated slots on each immunoblot as non-vaccine exposed control serum. The order of the analyzed serum specimens from each immunized group was arranged by recovered N. gonorrhoeae burden of each mouse and the relative burden of recovered N. gonorrhoeae is noted below slot lane for each mouse. The relative antibody reactivity in each slot lane was assessed by densitometric analysis of total peak area for the peak area of each of the 4 most reactive bands common to each immunization type. (B) The relationship between immunoblot signal intensity (assessed by densitometry) and the N. gonorrhoeae burden in the vaginal tract of each mouse over the course of infection, expressed as the area under the curve AUC of log₁₀ (CFU) recovered from all culture-positive days, was assessed using simple linear regression and 95% confidence intervals.

Figure 6

Immunization with MC58 ΔABR OMV and 4CMenB induce N. gonorrhoeae antigen induced cellular immune responses. Splenocytes were collected from mice immunized with Alum, MC58 ΔABR, or 4CmenB and subsequent N. gonorrhoeae vaginal challenge. Cells were cultured without stimulation or were stimulated ex vivo with either N. gonorrhoeae OMV (A) or 4CMenB (B). After 48 hours, cell culture supernatant was collected and the indicated secreted cytokines were measured using a multiplexed bead array based assay. The antigen-induced response is reported as fold increase in cytokine production in supernatant from antigen stimulated cells and unstimulated cells. Statistical significance was performed using one-way ANOVA followed by no paring Šidák’s multiple comparison test, with a single polled variance. Created in BioRender. Zhu, W (2025). https://BioRender.com/p88e324.

Figure 7

Assessment of the relationship between bacterial burden and cytokine secretion profile of the post-vaccination mouse splenocytes reveals 4CMenB stimulated IL-10 and IL-2 is associated with increased N. gonorrhoeae burden in 4CMenB immunized mice. The association between bacterial burden and post-vaccination unstimulated splenocyte cytokine secretion (A), gonococcal-specific response when splenocytes were stimulated ex vivo with N. gonorrhoeae OMV (B), and 4CMenB-induced cytokine secretion when splenocytes were stimulated ex vivo with 4CMenB as antigen (C) was examined. The effect of each measured cytokine on the mean change in bacterial burden (AUC of log₁₀ CFU) was assessed with simple linear regression, the results of which are presented here as forest plots with 95% confidence intervals.

Figure 8

Principal Coordinate Analysis (PCoA) of antibody and cellular immune responses among mice vaccinated with MC58 ΔABR OMV, 4CMenB and Alum control can distinguish immunization type but does not reveal associations between multiple immunologic parameters and clearance of infection. Principal Coordinate Analysis (PCoA) was utilized in R to quantify and visualize the variation in antibody and antigen-specific cellular responses and determine if clustering correlates with vaccination status. Data was preprocessed with dplyr for filtering and transformation, and standardized using clusterSim. The PCoA function in the ape package computed principal coordinates based on a distance matrix, providing a visual representation of sample similarities in reduced dimensional space. The package ggfortify was used to visualize the clustering. The following variables were considered for the generation of the distance matrix: the pre-gonococcal and post-gonococcal serum immunoglobulin, post-gonococcal challenge and vaginal wash immunoglobulin, and the magnitude of the antigen-specific cellular immune responses (as defined by the log2 fold change in secreted cytokines measured by multiplex Luminex when the splenocytes of immunized and N. gonorrhoeae-challenged mice were co-cultured with gonococcal outer membrane vesicles, OMV). Data from mice of the three vaccine groups (MC58 ΔABR, 4CMenB, and control mice immunized with Alum adjuvant only) were used. Each point on the PCoA plots corresponds to the immune profile of an individual mouse, with respect to the variables considered. Color coding indicates the three vaccine groups. Open circles denote mice that cleared N. gonorrhoeae and closed circles denote mice that were persistently colonized with N. gonorrhoeae post-challenge. Clusters indicate relatedness among samples. The differences in immune response were characteristic of the immunization group and could clearly distinguish between the two vaccine groups and Alum control mice (A) and between the two vaccine groups (B), with 66.1% and 62.4% of the total variation in the dataset being explained by the first two principal coordinates, respectively. Within the MC58 ΔABR vaccine group (C) and 4CMenB vaccine group (D), the variation in the multivariable immune response was effectively captured by the first two coordinates (60.8% and 59.1%, respectively) but was not correlated with the ability of the mice to clear infecting gonococci, as evidence by the lack of clustering with respect to gonococcal clearance.