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. 2026 Mar 26;15(1):2651468. doi: 10.1080/22221751.2026.2651468

Abolishing PorB-induced mitophagy enhances gonococcal outer membrane vesicle vaccine efficacy

Haoyu Ge a,*, Yuling Qin b,*, Shuaijie Song a,*, Dailin Yuan b, Shuai Gao a, Xu'ai Lin a, Stijn van der Veen a,b,c,CONTACT
PMCID: PMC13063334  PMID: 41883071

ABSTRACT

The emergence and global spread of ceftriaxone-resistant Neisseria gonorrhoeae underscores the urgent need for an effective vaccine. Gonococcal outer membrane vesicles (OMVs) are promising as vaccine platform, but their efficacy is potentially compromised by the immunomodulatory properties of gonococcal surface-expressed proteins, particularly the essential outer membrane porin PorB. Our previous work identified that gonococcal OMVs induce epithelial cell mitophagy via PorB, dependent on lysine residues 117 and 171. Given the critical role of dendritic cells (DCs) in initiating adaptive immunity, this study investigated whether PorB-mediated mitophagy in DCs impacts OMV vaccine efficacy. Here, we demonstrated that gonococcal OMVs induce DC mitophagy in a PorB-dependent manner, a process abolished in OMVs expressing the mitophagy-deficient PorB mutant PorBK117Q/K171Q. OMVs expressing PorBK117Q/K171Q demonstrated significantly enhanced DC activation, as shown by increased CD86 and MHC-II expression, and promoted a Th1-skewed T cell response with elevated IFN-γ and TNF-α secretion. In immunized mice, OMVs containing PorBK117Q/K171Q elicited significantly higher total IgG and IgG2a antibody titres against PorB compared with OMVs expressing wild-type PorB, with antibodies displaying enhanced bactericidal activity, including against a strain associated with the high-level ceftriaxone-resistant FC428 clone. Importantly, the OMV PorBK117Q/K171Q vaccine provided enhanced protection in a mouse vaginal colonization model, accelerating bacterial clearance and reducing overall bacterial burden. Therefore, our results identify PorB-induced mitophagy in DCs as a potential immune evasion mechanism that may dampen adaptive immunity. Engineering OMV vaccines to circumvent this process represents a rational strategy to explore for enhancing gonococcal vaccine efficacy.

KEYWORDS: Neisseria gonorrhoeae, OMV, mitophagy, vaccine, dendritic cells

Introduction

Neisseria gonorrhoeae is a multidrug-resistant bacterial pathogen that causes the second most prevalent bacterial sexually transmitted disease gonorrhea, with a global estimated incidence of 82 million new cases annually [1–3]. Ceftriaxone is currently the last-remaining recommended antimicrobial therapy in most countries [1–3]. However, in recent years, strains associated with the high-level ceftriaxone-resistant FC428 clone has shown global transmission [4–9], and these strains have become widespread in the Asia Pacific region [7,10–16], complicating future ceftriaxone-based therapy [17]. Vaccination is an important long-term, sustainable strategy for controlling gonorrhea [18]. However, vaccine development against N. gonorrhoeae has proven unsuccessful thus far, largely due to the extreme variability of surface-expressed proteins and the lack of understanding on the type of immune response required to obtain protective immunity [18–20]. Consequently, vaccine development for gonorrhea is still at the level of antigen discovery and evaluation of immune responses in mice, although several promising candidates have been discovered [21–25].

In recent years, there has been some excitement about the discovery that the Neisseria meningitidis outer membrane vesicle (OMV) vaccine MenZB displayed approximately 30% effectiveness against N. gonorrhoeae in the New Zealand population [26], which sparked several follow-up studies demonstrating 30%−50% effectiveness for the meningococcal 4CMenB vaccine that contains the MenZB OMVs as one of the vaccine components [27–29]. This cross-protection has revitalized interest in OMV-based vaccine platforms for gonorrhea.

The advantage of using gonococcal OMVs for immunization is the potential to elicit immune responses against multiple surface components displayed in a natural conformation. Immunization with N. gonorrhoeae MS11 OMVs successfully protected mice against a gonococcal challenge with the homologous MS11 strain [30]; however, these results were not reproducible for some other strains [20]. An important challenge for a successful gonococcal OMV-based vaccine is to overcome the immunosuppressive and immunomodulatory abilities of gonococcal surface-expressed proteins. The outer membrane porin PorB is a dominant immunomodulatory surface expressed protein [31]. However, PorB is an essential protein for gonococcal viability and therefore cannot be deleted. PorB has shown interactions with human complement factor H [32], C4b binding protein [33] and Toll-like receptor 2 [34,35], while for the closely related N. meningitidis it has been demonstrated that interactions between the vaccine antigen fHbp and complement factor H impair protective antibody responses [36]. For N. gonorrhoeae, it has previously been shown that OMVs are able to deliver PorB to macrophage mitochondria to induce apoptosis [37,38]. Furthermore, properly folded PorB as part of OMVs is able to suppress dendritic cell (DC)-induced T cell proliferation upon antigen stimulation [34]. These immune-modulating properties of gonococcal OMVs and PorB likely impact their efficacy as putative gonococcal vaccines. Indeed, replacement of N. gonorrhoeae PorB with N. meningitidis PorB enhances immunogenicity of an N. gonorrhoeae OMV vaccine [39].

Our previous work has demonstrated that gonococcal OMVs induce epithelial cell mitophagy, a mitochondria-selective autophagy process, through PorB-dependent dissipation of the mitochondrial membrane potential (MMP) and K63-linked polyubiquitination of PorB [40–42]. Specifically, we identified that lysine residues 117 and 171 of PorB are critical for this process, as OMVs expressing a PorB K117Q/K171Q mutant failed to induce mitophagy. Given the critical role of DCs in initiating adaptive immunity, we hypothesized that PorB-mediated mitophagy in DCs could be an unappreciated mechanism that compromises vaccine efficacy. Therefore, in the current study we investigated whether preventing PorB-mediated mitophagy could enhance the immunogenicity and protective efficacy of a gonococcal OMV vaccine.

Materials and methods

OMV purification

Isogenic N. gonorrhoeae ATCC 49226 strains expressing wild-type PorB1a (PorBWT) and the PorB1a K117Q/K171Q mutant (PorBK117Q/K171Q) were generated in our previous study [40]. Strains were routinely cultured overnight at 37 °C and 5% CO2 on GC Agar Base (Oxoid) with 1% Vitox (Oxoid). OMVs were purified by ultracentrifugation methods as described previously [40].

Animal ethics statement

BALB/c and C57BL/6 mice were obtained from the Shanghai SLAC Laboratory Animal Company and maintained for the duration of the experiments in a biosafety level 2 facility using a 24-cage individually ventilated caging system (Suzhou Suhang Technology). Mice were fed ad libitum, anesthetized by 4% isoflurane and euthanized by exsanguination or cervical dislocation. Mice were randomly assigned to treatment groups. All animal experiments were performed in compliance with the guidelines of Administration of Affairs Concerning Experimental Animals of the People’s Republic of China and are in accordance with the principles of the Declaration of Helsinki. Animal experiments were approved by the Zhejiang University Animal Care and Use Committee under license numbers 17324 and AIRB-2025-0127.

Mouse immunization and gonococcal challenge experiments

For vaccine immunogenicity analysis, groups of 8 five-week-old female BALB/c mice were immunized intraperitoneally on days 0, 14 and 28 with 50 μg doses of OMVs expressing PorBWT (OMV-PorBWT) or PorBK117Q/K171Q (OMV-PorBK117Q/K171Q) resuspended in 100 μL PBS or with a PBS-only Mock control. Sera were collected on day 35. For vaccine efficacy analysis, groups of 10 five-week-old female BALB/c mice were immunized intraperitoneally on days −35, −21 and −7 with 50 μg doses of OMV-PorBWT or OMV-PorBK117Q/K171Q in 100 μL PBS or with a PBS-only Mock control. On days −2, 0 and 2 mice were administered subcutaneously with 0.1 mg of β-estradiol (Aladdin) and from days −2–2 mice received daily intraperitoneal doses of 0.6 mg vancomycin (Meilunbio) and 1.2 mg streptomycin (BBI). Furthermore, starting at day −2 the drinking water was spiked with 0.4 g/L trimethoprim (Meilunbio). On day 0, mice were challenged intravaginally with N. gonorrhoeae strain ATCC 49226 at a dose of 2 × 107 CFU. Bacterial loads in the mouse vaginal tracts were monitored from day 1 onwards by daily swabbing and plating on GC-Vitox agar containing vancomycin, colistin (Meilunbio), nystatin (Meilunbio), trimethoprim and streptomycin. Mice that were not colonized successfully on day 1 based on CFU counts <1,000 were excluded for further analyses. For the two OMV groups, 8 mice were successfully colonized on day 1 and for the Mock control group 7 mice were successfully colonized.

Dendritic cell activation and T cell stimulation experiments

DC activation and T cell stimulation experiments were performed as described previously [43]. Shortly, bone marrow-derived DCs were obtained from the femurs and tibiae of 6- to 8-week-old female C57BL/6 mice and cultured for 7 days in RPMI-1640 medium (Biological Industries) containing 10% fetal bovine serum (FBS; Gibco) and 20 ng/mL GM-CSF (PeproTech). Mature DCs (1 × 10⁶ cells/well) were stimulated for 20 h with 20 μg of OMV-PorBWT or OMV-PorBK117Q/K171Q or with a PBS-only Mock control. for 20 h. Expression of CD86 and MHCII was analyzed by flow cytometry on CD11c-positive cells (FITC-labeled anti-mouse CD11c; Elabscience, #E-AB-F0991C) using PE-labeled anti-mouse CD86 (Elabscience, #E-AB-F0994D) and APC-labeled anti-mouse MHCII (Elabscience, #E-AB-F0990E). Naïve T cells were isolated from splenocytes of C57BL/6 mice using the EasySep Mouse CD4 + CD62L + T Cell Isolation Kit (Stemcell Technologies). The isolated T cells were co-cultured with pre-stimulated DCs for 72 h. IFN-γ, TNF-α, IL-4, IL-2, IL-10 and IL-17A cytokine levels in the co-culture supernatants were quantified using cytokine-specific ELISA kits (HuaBio).

Mitophagy analysis in dendritic cells

For co-localization analysis between mitochondria and LC3 or lysosomes, mature DCs (1 × 10⁶ cells per group) were stimulated for 4 h with 20 μg of OMV-PorBWT or OMV-PorBK117Q/K171Q or with a PBS-only Mock control and subsequently fixed with 4% paraformaldehyde (Biosharp). Fixed cells were blocked and permeabilized with QuickBlock buffer (Beyotime), transferred onto poly-lysine-coated slides (Solarbio), and incubated overnight at 4 °C with mouse anti-HSP60 (Santa Cruz, #sc-13115), rabbit anti-LC3B (Huabio, #ET1701-65), rabbit anti-LAMP1 (Huabio, #HA722302) or mouse anti-TOM20 (Huabio, #HA601454). Subsequently, slides were incubated for 1 h at room temperature with Alexa Fluor 488 goat anti-mouse (Proteintech, #RGAM002) or Alexa Fluor 594 goat anti-rabbit (Lablead, #Y1007) and nuclei were counterstained with 10 μg/mL DAPI (Solarbio). Images were acquired using a Zeiss LSM880 confocal microscope. For Western analysis, mature DCs (1 × 10⁶ cells per group), untreated (Mock) or pretreated for 4 h with Mdivi-1 (Selleck), were stimulated for 4 h or 20 h with 10 μM CCCP (Selleck), 20 μg of OMV-PorBWT or OMV-PorBK117Q/K171Q, or with a PBS-only Mock control. Cells were heated at 95 °C for 10 min in SDS-PAGE loading buffer (Beyotime), separated by 12% SDS-PAGE and subsequently transferred onto PVDF membranes (Biorad), which were blocked with QuickBlock Buffer (Beyotime). Membranes were incubated overnight at 4°C with rabbit anti-LC3A/B (CST, #4108), mouse anti-TOM20 (Huabio, #HA601454), rabbit anti-TIM23 (Proteintech, #11123-1-AP) or rabbit anti-GAPDH (Huabio, #ET1601-4) and subsequently for 2 h at room temperature with HRP-conjugated goat anti-rabbit (ABclonal, #AS014) or HRP-conjugated goat anti-mouse (ABclonal, #AS003). Finally, Western bands were visualized on a ChemiDoc Touch Imaging System (Bio-Rad). For apoptosis analysis, mature DCs (1 × 10⁶ cells per group) were stimulated for 12 h with 20 μg of OMV-PorBWT or OMV-PorBK117Q/K171Q, or with a PBS-only Mock control. Staurosporine (1 μg/mL) was used as positive control. Induction of apoptosis was analyzed by flow cytometry using the Annexin V, FITC Apoptosis Detection Kit (Dojindo) according to manufacturer’s instructions.

Expression and purification of recombinant MtrE and PorB

PorB1a and MtrE were expressed in Escherichia coli BL21 (DE3) using vectors pET21 and pEASY-E2 (Transgen Biotech), respectively. Recombinant MtrE was expressed and purified as described in details previously [24]. For expression and purification of PorB1a, exponentially growing cultures (1 L) were induced with 1 mM IPTG (Biosharp) and incubated overnight at 16°C and 200 rpm. Bacteria were pelleted (8,000 × g, 10 min, 4 °C), resuspended in PBS and lysed using an Ultrasonic Homogenizer (Scientz). The lysate was centrifuged (12,000 × g, 30 min, 4°C) and the PorB1a inclusion body pellet was washed repeatedly (0.5% Triton-X100, 50 mM Tris pH8.0, 300 mM NaCl, 10 mM EDTA, 10 mM DTT) and solubilized (6 M Guanidine-HCl, 10% Glycerin, 50 mM Tris pH8.0, 100 mM NaCl, 10 mM EDTA,). For refolding, clarified supernatant (12,000 × g, 20 min, 4°C) was dripped into refolding buffer (100 mM Tris pH8.0, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM GSH, 0.5 mM GSSG) under gentle agitation. The refolded PorB1a solution was subsequently concentrated using Amicon Ultra 10 kDa MWCO centrifugal filters (Millipore).

Determination of antibody titres

Antibody titres in immune sera were determined by ELISA as described previously [24,43]. Shortly, 96-well microtiter plates (NUNC, Maxisorp) were coated with 150 ng recombinant PorB1a or MtrE or for whole-cell ELISA with 5 × 107 CFU of paraformaldehyde-fixed N. gonorrhoeae strain ATCC 49226 or the FC428-associatedd strain SRRSH240 (expressing PorB1b) [12]. Plates were blocked with 4% BSA (Biosharp), incubated for 1 h at 37°C with twofold dilution series of immune sera, and finally incubated for 1 h at 37°C with HRP-labeled goat anti-mouse IgG (Beyotime, #A0216), HRP-conjugated goat anti-mouse IgG1 (Southern Biotech, #1070-05) or HRP-conjugated goat anti-mouse IgG2a (Southern Biotech, #1080-05). Finally, TMB substrate (Beyotime) was used to determine HRP activity.

Serum bactericidal activity assays

Complement-dependent serum bactericidal activity (SBA) assays were performed as described previously [24,43] using 2.5% (v/v) baby rabbit serum (Cedarlane) or 25% (v/v) normal human serum from healthy volunteers as complement source. Shortly, N. gonorrhoeae strains ATCC 49226 or SRRSH240 were incubated for 1 h at 37 °C with twofold dilution series of heat-inactivated mouse sera (56 °C, 60 min) and rabbit or human complement. Reactions were spot-plated and SBA titres were determined as the highest serum dilution killing at least 50% of the bacteria compared with the no serum control.

Results

Gonococcal OMVs induce mitophagy in DCs

It has previously been demonstrated that gonococcal OMVs induce apoptosis in macrophages [37,38], which are related to DCs as both are phagocytic antigen presenting cells derived from monocytes. Since DCs are critical mediators of adaptive immune responses, we first investigated whether gonococcal OMVs are able to induce apoptosis in DCs. However, flow cytometry analysis using FITC-Annexin V/propidium iodide demonstrated that gonococcal OMVs do not induce apoptosis (Figure 1A). In contrast, Western analysis demonstrated that gonococcal OMVs expressing PorBWT (OMV-PorBWT) induce degradation of the mitochondrial marker proteins TOM20 and TIM23 after both 4 h or 20 h exposure (Figure 1B), which was prevented by pre-treatment with mitophagy inhibitor Mdivi-1. Furthermore, confocal microscopy analysis demonstrated that OMV-PorBWT induces strong co-localization between mitochondria (TOM20) and the autophagy marker protein LC3 (Figure 1C-D). Similarly, OMV-PorBWT induces strong co-localization between mitochondria (HSP60) and lysosomes (LAMP1) (Figure 1E-F). Therefore, these results demonstrate that gonococcal OMVs induce autophagic degradation of mitochondria in DCs. Importantly, induction of mitophagy was not observed for gonococcal OMVs expressing the PorBK117Q/K171Q mutant (OMV-PorBK117Q/K171Q) (Figure 1B-F), indicating that OMV-induced mitophagy in DCs is a specific PorB-dependent process, similar to the mechanisms we previously described for epithelial cells [40,41].

← Figure 1.

Six visuals: flow cytometry plots, Western blot strips, two microscopy image series, and two bar charts comparing OMV-PorB treatments. The figure shows six visuals that compare effects of different gonococcal outer membrane vesicle PorB variants on dendritic cells. A set of four flow cytometry scatter plots labeled OMV-PorBWT, OMV-PorBK117Q/K171Q, Mock, and Positive displays Annexin V fluorescein isothiocyanate on the horizontal axis and propidium iodide on the vertical axis, each divided into quadrants with percentages printed in the corners. A series of Western blot strips is arranged in two horizontal blocks labeled OMVs 4 hours and OMVs 20 hours. Within each block, rows are labeled on the left as TOM20, TIM23, LC3 and GAPDH. Lanes across each row are labeled at the top as Mock, CCCP, DMSO, Mdivi 1, and DMSO, Mdivi 1 for PorBWT and PorBK117Q/K171Q. TOM20 and TIM23 bands vary in intensity between conditions, LC3 shows two bands labeled LC3 I and LC3 II, and GAPDH bands appear uniform. Two grids of confocal microscopy images depict single dendritic cells stained for TOM20 with LC3 or HSP60 with LAMP1, each accompanied by a nuclear stain and a merged image. Rows in both grids are labeled Mock, OMV-PorB WT, and OMV-PorB/K117Q/K171Q. In the TOM20 and LC3 grid, overlap in the merged images differs between rows; in the HSP60 and LAMP1 grid, overlap also differs between rows. To the right of each grid, a vertical bar chart summarizes colocalization percentages. The upper chart is labeled on the horizontal axis OMV-PorB with categories WT, K117Q/K171Q, and Mock and on the vertical axis LC3 colocalized with TOM20 percent from 0 to 120 with 20 unit intervals. The lower chart shows the same categories on the horizontal axis and LAMP1 colocalized with HSP60 percent on the vertical axis from 0 to 120 with 20 unit intervals. Each bar is composed of individual scatter points, and horizontal lines with labels P less than 10 to the power of 15 indicate comparisons between groups. All data are approximate.

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Gonococcal OMVs induce dendritic cell mitophagy. Mature murine bone marrow-derived dendritic cells (BMDCs) were stimulated with OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control. (A) Detection of apoptosis in OMV-stimulated DCs by flow cytometry using Annexin V-FITC/propidium iodide. (B) Representative Western blots of the mitochondrial marker proteins TOM20 and TIM23, and autophagy marker protein LC3 in 4 h or 20 h OMV-stimulated DCs with or without pretreatment with mitophagy inhibitor Mdivi-1. CCCP treatment was included as positive control for mitophagy activation. GAPDH was included as loading control. (C) Representative confocal microscopy images of antibody-stained TOM20 (green) and antibody-stained LC3 (red) in 4 h OMV-stimulated DCs. Scale bar, 5 μm. (D) Quantification of TOM20-LC3 co-localization in OMV-stimulated DCs. Graph represents the mean of 60 cells from three biological repeats. Significant differences were identified by one-way ANOVA (GraphPad Prism). (E) Representative confocal microscopy images of antibody-stained mitochondrial marker protein HSP60 (green) and antibody-stained lysosomal marker protein LAMP1 (red) in 4 h OMV-stimulated DCs. Scale bar, 5 μm. (F) Quantification of HSP60-LAMP1 co-localization in OMV-stimulated DCs. Graph represents the mean of 60 cells from three biological repeats. Significant differences were identified by one-way ANOVA (GraphPad Prism).

Preventing OMV-induced mitophagy enhances DC activation and T cell differentiation

Since mitophagy might be an undesired process for DC functionality, we investigated the impact of OMV-induced mitophagy in DC activation and T cell differentiation assays. Stimulation of DCs with OMV-PorBWT or OMV-PorBK117Q/K171Q both significantly upregulated expression levels of DC activation markers CD86 and MHC-II compared with the Mock control (Figure 2A-D). However, DCs stimulated with OMV-PorBK117Q/K171Q showed significantly enhanced CD86 and MHC-II levels compared with OMV-PorBWT, indicating that OMV-induced mitophagy reduces DC activation. Subsequently, stimulated DCs were co-cultured with naïve T cells and cytokine secretion was investigated by ELISA. OMV-PorBWT and OMV-PorBK117Q/K171Q both significantly induced secretion of IL-2, TNF-α, IFN-γ, IL-4, IL-10 and IL-17A compared with the Mock control (Figure 2E-J). However, secretion of the T cell activation and proliferation cytokine IL-2 was significantly enhanced for cells stimulated with OMV-PorBK117Q/K171Q compared with OMV-PorBWT (Figure 2E), indicating that OMV-induced mitophagy in DCs dampens subsequent T cell activation. Similarly, secretion of IL-10 was significantly enhanced for OMV-PorBK117Q/K171Q stimulated cells (Figure 2F), which is closely linked to regulatory T cell activity and important for balancing immunity by limiting inflammation. Importantly, while no significant differences in secretion of the Th2 cytokine IL-4 were observed between OMV-PorBWT and OMV-PorBK117Q/K171Q (Figure 2G), secretion of the Th1 cytokines TNF-α and IFN-γ was enhanced for cells stimulated with OMV-PorBK117Q/K171Q (Figure 2H-I). Therefore, these results suggest that OMV-induced mitophagy in DCs negatively impacts subsequent T cell differentiation towards a Th1 response. No differences in secretion of the pro-inflammatory Th17 cytokine IL-17A were observed between OMV-PorBWT and OMV-PorBK117Q/K171Q (Figure 2J).

Figure 2.

Ten charts showing CD86, MHCII, IL2, IL10, IL4, interferon gamma, tumor necrosis factor alpha, and IL17A responses to OMV-PorB treatments. The figure shows ten visuals arranged in two rows that compare dendritic cell activation markers and T cell cytokine secretion after stimulation with three conditions: OMV-PorB wild type, OMV-PorB K117Q/K171Q, and Mock. The first row begins with three flow cytometry dot plots of CD11c on the vertical axis and CD86 on the horizontal axis, each divided into four quadrants, one plot for each treatment. To the right is a vertical bar chart with CD86 mean fluorescence intensity in arbitrary units on the vertical axis from 0 to about 160 at intervals of 20, and OMV-PorB on the horizontal axis with three grouped bars labeled WT, K117Q/K171Q, and Mock. Horizontal lines with P value labels connect bar tops. The second row starts with three flow cytometry dot plots of CD11c on the vertical axis and MHCII on the horizontal axis, again shown for OMV-PorB wild type, OMV-PorB K117Q/K171Q, and Mock. Next is a bar chart of MHCII mean fluorescence intensity in arbitrary units with a 0 to about 120 scale, grouped by the same three treatments and annotated with P values. The remaining six visuals are vertical bar charts for interleukin 2, interleukin 10, interleukin 4, interferon gamma, tumor necrosis factor alpha, and interleukin 17A, each with cytokine concentration in picograms per milliliter on the vertical axis, OMV-PorB treatment group on the horizontal axis, and bars for WT, K117Q/K171Q, and Mock, with P value brackets highlighting differences. All data are approximate.

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Impact of gonococcal OMVs on dendritic cell activation and T cell differentiation. Mature murine bone marrow-derived dendritic cells (BMDCs) were stimulated with OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control and surface expression of CD86 and MHCII was determined by flow cytometry. Stimulated BMDCs were subsequently co-cultured with naïve murine T cells and IFN-γ, TNF-α, and IL-4 cytokine levels in the culture supernatant was determined by ELISA. (A) Representative flow cytometry repeat for detection of CD86 levels at the surface of OMV-stimulated DCs. (B) Quantification of CD86 levels at the surface of OMV-stimulated DCs. (C) Representative flow cytometry repeat for detection of MHCII levels at the surface of OMV-stimulated DCs. (D) Quantification of MHCII levels at the surface of OMV-stimulated DCs. (E) Quantification of IL-2 secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. (F) Quantification of IL-10 secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. (G) Quantification of IL-4 secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. (H) Quantification of IFN-γ secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. (I) Quantification of TNF-α secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. (J) Quantification of IL-17A secretion in the culture supernatant after co-culture of T cells with OMV-stimulated DCs. Graphs represent mean ± SD of three biological repeats. Significant differences were identified by one-way ANOVA (GraphPad Prism).

OMV-PorBK117Q/K171Q vaccine elicits enhanced bactericidal immune responses in mice

The impact of OMV-induced mitophagy on the immunogenicity of OMVs was investigated by immunizing groups of eight mice with OMV-PorBWT or OMV-PorBK117Q/K171Q or with a Mock control. After 3 vaccine doses immune sera of individual mice were investigated for antibody titres against PorB, which is the most abundant outer membrane protein in N. gonorrhoeae [38]. OMV-PorBK117Q/K171Q elicited significantly higher total IgG antibody titres (1.6-fold) compared with OMV-PorBWT (Figure 3A). Interestingly, while no significant differences were observed in IgG1 antibody titres elicited by OMV-PorBWT or OMV-PorBK117Q/K171Q (Figure 3B), IgG2a titres were significantly higher for OMV-PorBK117Q/K171Q (Figure 3B), resulting in significantly higher IgG2a/IgG1 ratios (Figure 3C). These results support the observed impact on Th1 responses in the DC activation and T cell differentiation assays. In contrast, antibody responses and IgG2a/IgG1 ratios for the low abundant outer membrane protein MtrE were similar for OMV-PorBWT and OMV-PorBK117Q/K171Q (Figure 3D-F).

Figure 3.

Six dot plots showing anti PorB and anti MtrE IgG, IgG1, IgG2a titres and IgG2a / IgG1 ratios for OMV-PorBWT, OMV-PorBK117Q / K171Q and Mock. The figure shows 6 dot plot graphs labelled A to F comparing antibody titres in mouse sera after immunization. Graph A plots anti PorB immunoglobulin G titre on a logarithmic y axis from less than 10 to 10 to the power of 8, with vaccine groups on the x axis: OMV-PorB wild type, OMV-PorBK117Q / K171Q, and Mock. Each mouse is a dot; solid horizontal bars mark group means and dashed dots mark the lower limit of detection. Graph B has 2 dot plots sharing the y axis anti PorB immunoglobulin G1 or immunoglobulin G2a titre, again on a logarithmic scale from less than 10 to 10 to the power of 8. The x axis shows OMV-PorB wild type, OMV-PorBK117Q / K171Q, and Mock under immunoglobulin G1 on the left and immunoglobulin G2a on the right. Graph C shows anti PorB immunoglobulin G2a or immunoglobulin G1 ratio on the y axis from 0.5 to 2.5 with OMV-PorB wild type, OMV-PorBK117Q / K171Q, and Mock on the x axis. Graphs D and E mirror A and B for anti MtrE immunoglobulin G, immunoglobulin G1, and immunoglobulin G2a, with titres from less than 10 to 10 to the power of 6. Graph F shows anti MtrE immunoglobulin G2a or immunoglobulin G1 ratio from 0.5 to 5 along the y axis. P values above brackets compare groups. All data are approximate.

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Immunogenicity of gonococcal OMV vaccines. Sera of mice immunized with OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control were analyzed for PorB- and MtrE-specific antibody responses by ELISA. (A) Total IgG reciprocal geometric mean titres (rGMTs) against recombinant PorB. (B) IgG1 and IgG2a isotype rGMTs against recombinant PorB. (C) IgG2a/IgG1 ratios of rGMTs against recombinant PorB. (D) Total IgG rGMTs against recombinant MtrE. (E) IgG1 and IgG2a isotype rGMTs against recombinant MtrE. (F) IgG2a/IgG1 ratios of rGMTs against recombinant MtrE. Significant differences were identified by one-way ANOVA (GraphPad Prism).

To further investigate whether antibody responses were able to recognize the surface of N. gonorrhoeae, we performed whole-cell ELISA with strain ATCC 49226, the strain used for generating the OMVs, and FC428-associated strain SRRSH240. Surface binding antibody titres for both strains were significantly higher for sera elicited by OMV-PorBK117Q/K171Q compared with OMV-PorBWT (Figure 4A-B). To further investigate functional activity of the antibody responses, bactericidal activity was investigated by serum bactericidal activity (SBA) assays. For both strains, OMV-PorBK117Q/K171Q elicited significantly higher SBA titres compared with OMV-PorBWT (Figure 4C-D). Similarly, SBA titres were significantly higher for OMV-PorBK117Q/K171Q when human serum was used as a more stringent complement source (Figure 4E-F). Therefore, these results implicate that OMV-induced mitophagy reduces the magnitude of bactericidal antibody responses elicited by a gonococcl OMV vaccine, which is prevented using OMV-PorBK117Q/K171Q.

Figure 4.

Six scatter plots showing antibody titres for OMV-PorBWT, OMV-PorBK117Q / K171Q, and Mock against ATCC 49226 and FC428 strains. The figure shows six scatter plots that compare antibody titres in mouse sera elicited by OMV-PorBWT, OMV-PorBK117Q / K171Q, and Mock immunizations against Neisseria gonorrhoeae strains ATCC 49226 and FC428 associated strain SRRSH240. Each scatter plot has the x axis labeled OMV-PorB with three groups: WT, K117Q / K171Q, and Mock. Each group contains multiple individual dots with a horizontal line marking the group mean. All y axes are on logarithmic scales and report titres as reciprocal values. In the top row, two whole cell enzyme linked immunosorbent assay plots are shown. The left graph is labeled ATCC 49226 and the right graph is labeled FC428. The y axis for these graphs is labeled Whole cell enzyme linked immunosorbent assay reciprocal titre and ranges approximately from less than 10 squared to 10 to the power of 8 with decade tick intervals. In both graphs, titres for the K117Q / K171Q group appear higher than for the WT group, and both are higher than the near baseline Mock group. In the middle and bottom rows, four serum bactericidal activity plots are shown. The middle row shows ATCC 49226 and FC428 with baby rabbit serum as complement source and has y axes labeled serum bactericidal activity reciprocal titre ranging from less than 512 to 10 to the power of 6. The bottom row shows ATCC 49226 and FC428 with normal human serum as complement source and has y axes ranging from less than 8 to 10 to the power of 3. Across all serum bactericidal activity graphs, K117Q / K171Q titres are highest, WT titres are intermediate, and Mock titres cluster at or below the lowest plotted value. All data are approximate.

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Functional activity of antibodies elicited by gonococcal OMV vaccines. Sera of mice immunized with OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control were analyzed for bacterial surface binding and complement-dependent bactericidal activity. (A) Reciprocal geometric mean titres (rGMTs) of sera against N. gonorrhoeae strain ATCC 49226 by whole-cell ELISA. (B) Whole-cell ELISA rGMTs against FC428-associated N. gonorrhoeae strain SRRSH240. (C) Serum bactericidal activity (SBA) rGMTs against N. gonorrhoeae strain ATCC 49226 using baby rabbit serum as complement source. (D) SBA rGMTs against N. gonorrhoeae strain SRRSH240 using baby rabbit serum as complement source. (E) SBA rGMTs against N. gonorrhoeae strain ATCC 49226 using normal human serum (NHS) as complement source. (F) SBA rGMTs against N. gonorrhoeae strain SRRSH240 using normal human serum (NHS) as complement source. Significant differences were identified by one-way ANOVA (GraphPad Prism).

OMV-PorBK117Q/K171Q vaccine provides enhanced protection in a mouse infection model

The vaccine efficacy of OMV-PorBWT and OMV-PorBK117Q/K171Q was investigated in a mouse vaginal tract infection model. Groups of 10 mice were immunized with OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control, however, for OMV-PorBWT and OMV-PorBK117Q/K171Q only 9 mice were successfully colonized, while for the Mock control group only 7 mice were colonized (Figure 5A). The percentage of colonized mice in the Mock control group showed a gradual decrease between days 6 and 11 (Figure 5B). In contrast, mice immunized with OMV-PorBWT were cleared between days 2 and 8, while mice immunized with OMV-PorBK117Q/K171Q cleared significantly more rapid between days 2 and 6, with 8 of the 9 mice being cleared already by day 3 (Figure 5B). Bacterial loads in the vaginal tract of the mice were similarly lowest for the OMV-PorBK117Q/K171Q-immunized mice (Figure 5C), which resulted in significantly lower area under the curves compared with OMV-PorBWT (Figure 5D). Overall, our results demonstrate that OMVs expressing the PorBK117Q/K171Q mutant display significantly increased vaccine efficacy compared with PorBWT, implicating that mitophagy might be an unfavourable process for OMV vaccine efficacy.

Figure 5.

Four visuals: one timeline diagram, two line graphs, and one dot plot comparing OMV-PorBWT, OMV-PorBK117Q/K171Q, and Mock outcomes. The figure shows four visuals summarizing a mouse vaginal tract infection experiment with gonococcal outer membrane vesicle vaccines. The first visual is a horizontal timeline diagram from day minus 35 to day + 12. It marks three immunizations of groups of 10 mice before day 0, a gonococcal inoculation with Neisseria gonorrhoeae ATCC 49226 at day 1, antibiotic treatments with beta estradiol, vancomycin or streptomycin, and trimethoprim in drinking water, and daily vaginal swabs for colony forming unit determination. Text on the diagram notes that 9 mice are colonized in the OMV-PorBWT group, 9 in the OMV-PorBK117Q / K171Q group, and 7 in the Mock group. The second visual is a line graph with days post infection on the horizontal axis from 0 to 12 and infected mice in percent on the vertical axis from 0 to 100 in steps of 20. Three curves for OMV-PorBWT, OMV-PorBK117Q / K171Q, and Mock all start near 100 percent and decrease over time, with OMV-PorBK117Q / K171Q reaching 0 percent earliest. The third visual is a line graph with days post infection from 2 to 12 on the horizontal axis and logarithm of colony forming units per mouse from 1 to 5 on the vertical axis in steps of 1. The OMV-PorBK117Q / K171Q line starts near 4 and drops quickly toward 1, the OMV-PorBWT line declines more slowly, and the Mock line remains higher for longer. Error bars are shown for all time points. The fourth visual is a dot plot of area under the curve values in logarithm of colony forming units. The horizontal axis lists OMV-PorB wild type, K117Q / K171Q mutant, and Mock groups. The vertical axis ranges from 0 to 25. Each group has individual dots spread vertically with horizontal bars indicating group means, with the Mock group highest, OMV-PorB wild type intermediate, and OMV-PorBK117Q / K171Q lowest. P values of 0.04, 0.004, and 0.00005 are printed above brackets comparing the groups. All data are approximate.

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Vaccine efficacy of gonococcal OMVs in a mouse vaginal tract infection model. (A) Schematic diagram of the vaccine efficacy study. Mice received 3 vaccine doses of OMV-PorBWT, OMV-PorBK117Q/K171Q or a Mock control and on day 1 the mice were challenged with N. gonorrhoeae ATCC 49226. (B) Curves displaying the daily percentage of colonized mice. Significant differences were identified with the Mantel-Cox log-rank test (GraphPad Prism). (C) Curves displaying the daily CFU counts (mean ± SEM). (D) Individual area under the curve (AUC) for the daily CFU counts. Significant differences were identified by one-way ANOVA (GraphPad Prism).

Discussion

The urgent threat of ceftriaxone-resistant Neisseria gonorrhoeae [2,7] necessitates the accelerated development of an effective vaccine. The cross-protective effect of meningococcal OMV vaccines has revitalized interest in OMV-based platforms for gonorrhea [26–29,39]. However, the inherent immunomodulatory properties of gonococcal outer membrane components, particularly PorB, pose a significant challenge [32–34,37,38]. In this study, we identified PorB-dependent mitophagy in DCs as a novel mechanism that dampens adaptive immunity and reduces the efficacy of a gonococcal OMV vaccine. Importantly, we demonstrated that an OMV vaccine engineered to express a mitophagy-deficient PorB mutant (K117Q/K171Q) enhances DC activation, promotes a Th1-skewed immune response, elicits superior bactericidal antibody titres, and confers significantly improved protection in a mouse infection model.

One important finding of our work is that gonococcal OMVs induce mitophagy in DCs, a process distinct from the PorB-mediated apoptosis previously described for macrophages [37,38]. This cell-type-specific outcome highlights the complex interplay between N. gonorrhoeae and the host immune system. The OMV-induced degradation of mitochondrial markers TOM20 and TIM23, alongside the robust co-localization of mitochondria with autophagosomes and lysosomes, provides compelling evidence for autophagic degradation of mitochondria in DCs. The absolute dependence on PorB, and specifically on lysine residues 117 and 171, aligns with our prior mechanistic work in epithelial cells [40,41]. We demonstrated that OMVs deliver PorB to mitochondria to induce MMP dissipation, which is abolished for a PorB K117Q mutant that no longer is able to retain ATP in the barrel lumen to keep the channel in a prolonged open state [44]. A second mechanism is the result of direct K63-linked polyubiquitination of PorB lysine residue 171 by the E3 ubiquitin ligase RNF213, resulting in p62-dependent recruitment of the autophagy machinery.

The functional consequence of mitophagy on DC activity was evident. The prevention of mitophagy in DCs stimulated with OMV-PorBK117Q/K171Q led to significantly enhanced upregulation of the co-stimulatory receptor CD86 and MHC-II, suggesting that PorB-induced mitophagy modulates DC activation, a process important for initiating robust adaptive immunity. This observation is consistent with previous work showing that mitochondrial integrity and metabolism are integral to DC activation and immunogenicity [45], since chemical mitochondrial disruption in DCs through CCCP or carbon monoxide has been shown to impair antigen-loaded endosome trafficking, antigen presentation and T cell activation [46]. Our T cell polarization assays further revealed that enhanced DC activation by OMV-PorBK117Q/K171Q preferentially boosted the secretion of the Th1 cytokines TNF-α and IFN-γ, without affecting the Th2 cytokine IL-4. This skewing towards a Th1 response is highly relevant for anti-gonococcal immunity, as it has been suggested that Th1-polarized immune responses are critical for a successful gonococcal vaccine [43,47]. It has previously been demonstrated that specific enhancement of Th1-polarized immune responses after intravaginal or intranasal administration of an OMV vaccine resulted accelerated gonococcal clearance of the infection [48,49]. Similarly, immunization with MtrE Loop2-based antigens formulated with the Th1-polarizing adjuvant CpG1826 enhanced gonococcal clearance in a mouse infection model [43]. The significantly elevated IgG2a titres and IgG2a/IgG1 ratio observed in our study in mice immunized with OMV-PorBK117Q/K171Q are a classical serological signature of a Th1-driven response in mice [50]. This qualitative improvement in the antibody response translated into enhanced functional activity, because antibodies belonging to subclass IgG2a show superior complement activation and SBA than IgG1 subclass antibodies [51,52]. Therefore, sera from OMV-PorK117Q/K171Q-immunized mice exhibited significantly higher SBA titres against both the homologous strain and a heterologous ceftriaxone-resistant FC428-associated strain. Importantly, this enhanced bactericidal activity was maintained when using human complement, which strengthens the potential translational relevance of our findings [53,54].

A useful pre-clinical gonococcal vaccine test is the in vivo efficacy study in a mouse vaginal tract infection model [20,55,56]. Consistent with our in vitro findings, immunization with OMV-PorBK117Q/K171Q led to significantly accelerated clearance of N. gonorrhoeae and a reduction in bacterial burden compared to the OMV-PorBWT vaccine. These results support the hypothesis that PorB-induced mitophagy may compromise vaccine-induced immunity. Our findings offer a potential mechanistic explanation for previous observations that the immunomodulatory properties of gonococcal PorB can hinder vaccine efficacy [31,34,39], thereby identifying a potential target for rational vaccine optimization.

The conservation of the critical PorB lysine residues supports the broad relevance of this mechanism. Although gonococcal PorB is highly polymorphic and divided into two major variants, PorB1a and PorB1b, our previous study demonstrated that OMVs expressing PorB1a or PorB1b are both able to induce mitophagy through an identical mechanism, with lysine 170 being the ubiquitination target in PorB1b [40,41]. Lysine residues 117 for both PorB1a and PorB1b, 171 for PorB1a and 170 for PorB1b are highly conserved, because PorB sequence analysis of 785 clinical isolates from Hangzhou, China, covering the years 2011–2012 [57], 2015–2017 [58], and 2019–2022 [12,59] demonstrated that all strains carry PorB alleles containing K117/K171 (PorB1a) or K117/K170 (PorB1b). Furthermore, analysis of 13,889 NG-MAST PorB alleles (739 PorB1a alleles and 13,150 PorB1b alleles) currently available in the PubMLST database (up to porB allele 15401; https://pubmlst.org/neisseria) demonstrated that only seven PorB alleles did not contain lysine 117, while 31 PorB1a alleles did not contain lysine 171 and 9 PorB1b alleles did not contain lysine 170. Therefore, gonococcal OMV-induced mitophagy is likely a highly conserved process. Future studies should explore the efficacy of this approach using OMVs derived from clinically prevalent strains, including the FC428 clone, to further validate the translational potential.

In conclusion, this study provides evidence for a previously uncharacterized role of PorB-induced mitophagy in DCs as a modulator of adaptive immunity. Our findings indicate that mitigating this process through engineered OMVs can enhance both immunogenicity and protective efficacy. Further work is needed to elucidate the molecular cascade connecting mitophagy to antigen presentation in DCs. Nevertheless, this study established the principle that targeting bacterial immune modulation mechanisms, such as PorB-driven mitophagy, represents a viable and rational avenue to explore in the design of more effective gonococcal OMV vaccines.

Acknowledgements

We thank Lin Zhaoxiaonan and Xiao Guifeng from the Core Facilities of Zhejiang University School of Medicine for assistance.

Funding Statement

This work was supported by the National Natural Science Foundation of China [grant numbers 82572622, 82272382, 82072320]; and the Zhejiang Province Natural Science Foundation [grant number LZ24H190001]. The funder had no role in study design, data collection and interpretation, writing of the manuscript, or the decision to submit the manuscript for publication.

Disclosure statement

No potential conflict of interest was reported by the author(s).

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