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. 2026 Mar 3;11:81. doi: 10.1038/s41541-026-01412-0

MtrE Loop2-specific multiple antigenic peptide vaccine and monoclonal antibody confer complement-dependent protection against Neisseria gonorrhoeae

Shuaijie Song 1,#, Haoyu Ge 1,#, Dailin Yuan 1, Xiaohua Gu 1, Wenyan Lu 1, Chen Ding 1, Yuling Qin 1, Shuai Gao 1, Xu’ai Lin 1, Hao Cheng 2, Stijn van der Veen 1,2,3,✉
PMCID: PMC13077049  PMID: 41776223

Abstract

Neisseria gonorrhoeae imposes a substantial global health burden due to its high incidence and escalating multidrug resistance. This study investigated the immunogenicity and efficacy of a peptide-based vaccine and a monoclonal antibody (mAb) targeting the conserved Loop2 epitope of the outer membrane protein MtrE. Two multiple antigenic peptide (MAP) vaccines, displaying four copies of MtrE Loop2 with or without a Cathepsin S cleavage site, were formulated with CpG1826 adjuvant. Immunization of mice elicited robust Loop2-specific IgM-dominant antibody responses with complement-dependent anti-gonococcal serum bactericidal activity. In a murine vaginal tract infection model, both vaccines demonstrated significant prophylactic and single-dose therapeutic efficacy. Furthermore, a human-mouse chimeric mAb (M01), consisting of mouse variable domains and human IgG1 constant domains, was generated from a dominant B-cell clonotype obtained from MAP vaccine-immunized mice. M01 exhibited high-affinity binding to MtrE and potent complement-dependent bactericidal activity. In a murine infection model, intravaginal administration of M01 significantly enhanced gonococcal clearance. Furthermore, Fc-engineered M01 variants confirmed that this efficacy was critically dependent on complement activity. These findings identify MtrE Loop2 as a promising target for both active and passive immunization strategies against N. gonorrhoeae, and underscore the critical role of complement-mediated activity as a mechanistic correlate of protection.

Subject terms: Biotechnology, Drug discovery, Immunology, Microbiology

Introduction

Multidrug-resistant gonorrhoea is an important global health threat, with approximately 87 million new cases annually1. The emergence and global dissemination of multidrug-resistant Neisseria gonorrhoeae, particularly high-level ceftriaxone-resistant strains associated with the FC428 clone or containing penA alleles related to penA 60.001, have severely compromised ceftriaxone-based first-line therapies2–5. Vaccination is the best long-term strategy for controlling infectious diseases, and vaccine development may be the only long-term, sustainable means of controlling gonorrhoea. Vaccination is the optimal strategy to curb transmission of multidrug-resistant gonorrhoea, however, antigenic variability of surface-expressed proteins has impeded vaccine development6,7. Conventional vaccines often incorporate multiple proteins or complex antigens, yet protective immunity typically hinges on a limited subset of critical epitopes. Non-protective components may dilute immune responses, increase reactogenicity, or impede manufacturing scalability8. This challenge has accelerated interest in precision-engineered peptide vaccines, which focus immune recognition on conserved and/or functionally critical epitopes while eliminating antigenic “noise“9. Such designs offer advantages in safety, reproducibility, thermal stability, and production efficiency, properties vital for global deployment8. For instance, previously it was shown that the multiple antigenic peptides (MAP) strategy, in which peptides are branched artificially through a scaffolding core with lysine residues, could be very suitable for the deployment of peptide multimers in vaccines10–14, thereby overcoming the lack of immunogenicity generally observed for peptide monomers. For N. gonorrhoeae, this strategy has previously been used to generate a peptide-mimetic vaccine for the 2C7 epitope of gonococcal lipooligosaccharide (LOS), which elicited bactericidal antibody responses in mice and enhanced clearance of N. gonorrhoeae in a murine vaginal tract infection model11,15,16.

Our previous work identified surface Loop2 of the MtrE outer membrane protein as a highly conserved and immunogenic region17. MtrE is the outer membrane channel of the MtrCDE multidrug efflux pump, which exports host-derived antimicrobial compounds such as antimicrobial peptides, fatty acids, and bile salts, and is furthermore a major determinant for multidrug resistance18–20. Importantly, gonococcal mtrCDE deletion mutants display fitness defects in a mouse vaginal tract infection model17,21–23. In contrast, a recent study demonstrated that fitness of an mtrD deletion mutant was not impaired in a human experimental genital tract infection model24. However, this study used the gonococcal FA1090 strain, which is non-inducible for mtrCDE expression and therefore not representative for establishing the in vivo contributions MtrCDE. Besides its role in the MtrCDE efflux system, MtrE also forms a functional efflux system with FarAB, which displays specificity for long-chain fatty acids25,26. MtrE forms a stable trimer, with Loop2 representing an extracellular, surface-accessible epitope17. Subunit vaccines displaying Loop2 as linear (Nloop2) or structural epitope (Intraloop2) on the IMX315 carrier and adjuvanted with CpG1826, elicited potent Th1-biased antibody responses with significant serum bactericidal activity against both reference and multidrug-resistant N. gonorrhoeae strains, including FC428-derivative isolates27. Critically, these vaccines demonstrated prophylactic and single-dose therapeutic efficacy in a murine vaginal tract infection model27. However, carrier proteins, such as full-length MtrE or IMX313, introduce extraneous epitopes that may compromise immune focusing, particularly when target epitopes are not immuno-dominant, as is likely the case for MtrE Loop2. Therefore, in the current study we used the four-branch approach to generate an MtrE Loop2 MAP. Furthermore, in a secondary strategy that putatively enhances peptide processing in the endosomes of dendritic cells and subsequent surface display, we introduced a minimal Cathepsin S (CatS) cleavage side between MtrE Loop2 and the lysine scaffolding core. This strategy has previously been used successfully to enhance processing of recombinant overlapping peptides provided as a single protein28.

Besides vaccines, monoclonal antibodies (mAbs) could offer an alternative approach for treatment of multidrug-resistant N. gonorrhoeae. MAbs offer immediate, high-titer protection and can be manufactured at scale, standardized, and humanized for direct clinical use29. For instance, a human-mouse chimeric (human IgG1 constant region with mouse variable region) mAb developed against the LOS 2C7 epitope was able to clear N. gonorrhoeae from the murine vaginal tract infection model16. Importantly, introduction of the E430G mutation in the human IgG1 backbone, which increases complement activation30,31, enhanced gonococcal clearance, while introduction of the D270A/K322A mutation, which abolishes complement activation32,33, did not enhance clearance, indicating that the mAb anti-gonococcal activity was fully based on its complement-dependent bactericidal activity32. Advances in single-cell sequencing now enable rapid recovery of native heavy/light-chain pairs from antigen-specific B cells, accelerating mAb discovery while preserving natural affinity34. For instance, PorB-specific human mAbs derived from memory B cells of volunteers vaccinated with the meningococcal 4CMenB showed complement-dependent bactericidal activity against both Neisseria meningitidis and N. gonorrhoeae and enhanced gonococcal clearance of the murine vaginal tract infection model35,36. Here, we generated a human-mouse chimeric mAb targeting MtrE Loop2 and explored its in vitro and in vivo antimicrobial activity against N. gonorrhoeae.

Results

Immunogenicity of MtrE Loop2 MAP vaccines

Two MtrE Loop2 MAPs were generated consisting of a reverse MAP lysine core with four branches of MtrE Loop2 with or without an additional CatS cleavage site between the peptide and the lysine core (Fig. 1A). MAP-Loop2 and MAP-CatS-Loop2 were formulated with CpG1826 adjuvant and used to immunize groups of eight mice with three doses in a biweekly schedule. Immune sera were analyzed by ELISA for Loop2-specific IgG, IgM, and IgA antibody titers. Both MAP-Loop2 and MAP-CatS-Loop2 elicited significantly elevated MtrE Loop2-specific IgG titers compared with the adjuvant-only Mock control (Fig. 1B, C). Interestingly, the peptides raised very high MtrE Loop2-specific IgM titers (Fig. 1D, E), which were approximately fourfold higher compared with the IgG titers. As control, IgM titers elicited by the CpG1826 adjuvant only Mock control was poor, highlighting that the IgM response was specifically elicited by the MAP-Loop2 and MAP-CatS-Loop2 antigens. No MtrE Loop2-specific IgA titers were detected (Fig. 1F, G). To further investigate functional activity of the antibody responses, serum bactericidal activity assays were performed. MAP-Loop2 and MAP-CatS-Loop2 immune sera displayed strong complement-dependent bactericidal activity against both the ATCC 49226 reference strain (Fig. 2A) and against strain SRRSH240, a strain associated with the high-level ceftriaxone-resistant FC428 clone, using baby rabbit serum as complement source. (Fig. 2B). Importantly, enhanced bactericidal activity for MAP-Loop2- and MAP-CatS-Loop2-elicited sera compared with the Mock control was still observed using normal human serum as a more stringent complement source (Fig. 2C, D). Similarly, opsonophagocytosis assays with neutrophil-like HL-60 cells demonstrated that MAP-Loop2- and MAP-CatS-Loop2 elicited enhanced titers compared with the Mock control against both ATCC 49226 (Fig. 2E) and FC428-associated SRRSH240 (Fig. 2F). Therefore, these results indicate that CpG1826-formulated MAP-Loop2 and MAP-CatS-Loop2 vaccines elicited strong MtrE Loop2-specific responses with complement-dependent bactericidal and opsonophagocytosis-stimulating activity. Interestingly, since the MtrCDE efflux pump is an important factor in host colonization, the ability of MtrE Loop2-specifc immune sera to interfere in efflux pump activity was investigated using a Triton X-100 killing assay. Triton X-100-dependent killing activity was indeed enhanced by the MAP-Loop2- and MAP-CatS-Loop2-elicited immune sera compared with the Mock control (Fig. 2G, H), highlighting that the MtrE Loop2-specific antibodies functionally interfere with the MtrCDE efflux pump-mediated detergent resistance.

Fig. 1. Immunogenicity of MtrE Loop2-based multiple antigenic peptide (MAP) vaccines.

Fig. 1

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A Schematic representation of the MtrE Loop2 MAP designs, with and without inclusion of a Cathepsin S (CatS) proteolytic cleavage site. The reverse lysine core is depicted in magenta, and the CatS cleavage site sequence is shown in green. B Mice were immunized with three biweekly doses of CpG1826-formulated MAP-Loop2, MAP-CatS-Loop2 or a Mock control (8 mice/group) and one week following the final dose sera were collected and analyzed by ELISA for antibody responses against MtrE Loop2 fused with the IMX315 carrier protein. The graph presents total IgG reciprocal geometric mean titers (rGMTs) of individual mice against Loop2. Symbols represent the reciprocal geometric mean of three independent repeats for each mouse. C Graph displaying Loop2-specific IgG area under the curve (AUC) analysis on the full dilution series for individual mouse serum samples. Symbols represent the mean of three independent repeats for each mouse. D Total IgM rGMTs of individual mice against Loop2. E Loop2-specific IgM AUC analysis for individual mice. F Total IgA rGMTs of individual mice against Loop2. G Loop2-specific IgA AUC analysis for individual mice. Significant differences were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. MAP-Loop2 and MAP-CatS-Loop2 elicit bactericidal antibodies against Neisseria gonorrhoeae.

Fig. 2

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Sera of mice immunized with CpG1826-formulated MAP-Loop2, MAP-CatS-Loop2 or a Mock control (8 mice/group) were analyzed for serum bactericidal activity (SBA) using baby rabbit complement- and human complement-mediated killing, opsonophagocytosis-mediated killing, and Triton-X-100-mediated killing by antibodies interfering with the MtrCDE efflux pump activity. A SBA reciprocal geometric mean titers (rGMTs) against N. gonorrhoeae strain ATCC 49226 using baby rabbit complement. Symbols represent the reciprocal geometric mean of three independent repeats for each mouse. B SBA rGMTs against FC428-associated N. gonorrhoeae strain SRRSH240 using baby rabbit complement. C SBA rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain ATCC 49226 using human complement. Symbols represent the independent repeats. D SBA rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain SRRSH240 using human complement. E Opsonophagocytosis rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain ATCC49226 using differentiated HL-60 cells. Symbols represent the independent repeats. F Opsonophagocytosis rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain SRRSH240 using differentiated HL-60 cells. G Triton X-100 killing rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain ATCC 49226. Symbols represent the independent repeats. H Triton X-100 killing rGMTs of pooled sera from each immunization group against N. gonorrhoeae strain SRRSH240. Significant differences were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). **P < 0.01, ***P < 0.001.

MtrE Loop2 MAP vaccines provide protection in a mouse infection model

The prophylactic vaccine efficacy of MAP-Loop2 and MAP-CatS-Loop2 formulated with CpG1826 adjuvant was investigated in a mouse vaginal tract infection model. Groups of mice (9–10 mice/group) were immunized with three biweekly doses followed one week later by inoculation with the N. gonorrhoeae reference strain ATCC 49226 (Fig. 3A). The number of colonized mice in the adjuvant-only Mock control group showed a gradual decline between day 7 and day 12 after challenge (Fig. 3B). In contrast, the number of colonized mice reduced more rapidly in the MAP-Loop2 and MAP-CatS-Loop2 groups, where mice were cleared between days 3 and 6 for the MAP-Loop2 group and between days 3 and 5 for the MAP-CatS-Loop2 group. Similarly, bacterial loads in the mouse vaginal tracts declined more rapidly for the MAP-Loop2 and MAP-CatS-Loop2 groups compared with the Mock control group (Fig. 3C), resulting in significantly reduced area under the curves (Fig. 3D). Overall, these results demonstrate that MAP-Loop2 and MAP-CatS-Loop2 are effective antigens that are able to elicit MtrE Loop2-specific protective immune responses against a gonococcal challenge.

Fig. 3. Prophylactic vaccine efficacy of MAP-Loop2 and MAP-CatS-Loop2.

Fig. 3

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A Schematic diagram of the prophylactic vaccine efficacy study using the mouse vaginal tract infection model for N. gonorrhoeae. Mice were immunized with 3 biweekly doses of CpG1826-formulated MAP-Loop2, MAP-CatS-Loop2 or a Mock control (9–10 mice/group) on days −35, −21, and −7, and subsequently challenged with N. gonorrhoeae ATCC 49226 on day 0. B Kaplan–Meier curves representing daily percentage of colonized mice. C Curves displaying the average and SEM of daily CFU counts from the colonized mice. D Graph displaying the individual area under the curve (AUC) for the daily CFU counts from the colonized mice. Significant differences between the Kaplan–Meier curves were identified with the Mantel-Cox log-rank test (GraphPad Prism). Significant differences between the AUCs were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). ***P < 0.001.

Therapeutic efficacy of MtrE Loop2 MAP vaccine in a mouse infection model

To further investigate the therapeutic vaccine efficacy of MAP-Loop2 and MAP-CatS-Loop2, groups of mice (10–11 mice/group) were challenged with N. gonorrhoeae and 6 h after the challenge they were vaccinated with a single dose of the CpG1826-formulated MAP-Loop2 and MAP-CatS-Loop2 vaccines or an adjuvant-only Mock control (Fig. 4A). The number of colonized mice showed a gradual decline between day 5 and day 11 for the Mock control group (Fig. 4B). In contrast, the number of colonized mice in the MAP-Loop2 group started to decline by day 3 and all mice were cleared by day 7. For the MAP-CatS-Loop2 group, mice were cleared between day 2 and day 7. Bacterial loads in the vaginal tracts reflected the decline in colonized mice (Fig. 4C), by day 4 most were already mostly cleared, resulting in significantly reduced area under the curves (Fig. 4D). To understand the rapid clearance observed after therapeutic immunization, early MtrE Loop2-specific IgM responses were analyzed in mice immunized with MAP-Loop2 or an adjuvant-only Mock control (3 mice/group/time-point). A significant increase in MtrE Loop2-specific IgM in the MAP-Loop2 group was detected from day 2 onwards, which peaked by day 4 post immunization (Fig. 4E). This rapid MtrE Loop2-specific IgM response is in accordance with the observed bacterial clearance. Overall, these results show that MAP-Loop2 and MAP-CatS-Loop2 are highly effective therapeutic vaccine antigens, eliciting rapid bactericidal responses.

Fig. 4. Therapeutic vaccine efficacy of MAP-Loop2 and MAP-CatS-Loop2.

Fig. 4

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A Schematic diagram of the therapeutic vaccine efficacy study using the mouse vaginal tract infection model for N. gonorrhoeae. Mice were colonized with N. gonorrhoeae ATCC 49226 on day 0 and subsequently injected with a single dose of CpG1826-formulated MAP-Loop2, MAP-CatS-Loop2 or a Mock control (10–11 mice/group) 6 h after challenge. B Kaplan–Meier curves representing daily percentage of colonized mice. C Curves displaying the average and SEM of daily CFU counts from the colonized mice. D Graph displaying the individual area under the curve (AUC) for the daily CFU counts from the colonized mice. E Early MtrE Loop2-specific IgM responses in mice immunized on Day 0 with MAP-Loop2 or an adjuvant-only Mock control (3 mice/group/time-point). The graph presents total IgM reciprocal geometric mean titers (rGMTs) of individual mice against Loop2. Symbols represent the reciprocal geometric mean of three independent repeats for each mouse. Significant differences between the Kaplan–Meier curves were identified with the Mantel-Cox log-rank test (GraphPad Prism). Significant differences between the AUCs and IgM titers were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). ***P < 0.001.

Generation of an MtrE Loop2-specific mAb (M01)

Since the therapeutic MtrE Loop2-based MAP vaccines worked remarkably well in clearing an already established N. gonorrhoeae infection, antibodies might also be a suitable alternative treatment. Therefore, two mice were immunized with five biweekly doses of the CpG1826-formulated MAP-Loop2 vaccine and splenocytes were harvested for subsequent single-cell sequencing. A total of 9068 single cells were isolated and sequenced (Fig. 5A), with an average number of 35,761 reads per cell and a 70% sequencing saturation. After filtering, 8431 single cells were classified as B cells (Fig. 5B), with 7344 cells identified containing a productive V-J spanning pair. The majority of the B cells expressed IgM-type antibodies (Fig. 5C) and the most abundant clonotype was identified in 22 cells (Fig. 5D). This clonotype belonged to an IgM type antibody with a kappa light chain. The variable domain of this antibody was used to generate a human-mouse chimeric mAb (M01) containing the mouse variable domains and human IgG1 heavy and IgK light constant domains (Fig. 6A). M01 showed strong binding to full-length MtrE by Western blotting analysis, since concentrations as low as 80 ng/mL still resulted in a positive Western blotting signal (Fig. 6B, Supplementary Fig. 1). Similarly, ELISA analysis demonstrated strong binding of M01 to full length MtrE and Loop2, with titers as low as 375 ng/mL for both MtrE and Loop2 (Fig. 6C, D).

Fig. 5. Single cell sequencing of splenocytes from mice immunized with CpG1826-formulated MAP-Loop2.

Fig. 5

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A Effective single cell identification. B Clustering of B cells and monocytes. C Quantification of antibody heavy chain types. D Quantification of B cell clonotypes.

Fig. 6. MtrE Loop2 specificity of human-mouse chimeric monoclonal antibody (mAb) M01.

Fig. 6

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A Graphic representation of mAb M01 consisting of mouse variable domains and human IgG1 constant domains. The sequence of the mouse variable heavy (VH) and light (VL) chain domains is shown with colored complementarity determining regions (CDRs). B Western blotting analysis of full-length recombinant MtrE with different concentrations of M01 used for detection. C ELISA analysis of M01, a control mAb and a Mock control (PBS) for binding specificity to full-length recombinant MtrE. D ELISA analysis of M01, a control mAb and a Mock control (PBS) for binding specificity to MtrE Loop2 fused with the IMX315 carrier protein. Graphs represent the mean and SEM of three biological repeats.

Efficacy of mAb M01 in a murine infection model

The local passive antibody treatment efficacy of M01 was investigated in a mouse vaginal tract infection model. Groups of eight mice were colonized with N. gonorrhoeae strain ATCC 49226, and from 1 day onwards mice were daily treated by direct intravaginal injection with M01 or a human IgG1 control antibody at a dose of 10 µg, or with a Mock control (Fig. 7A). The number of colonized mice in the Mock control group showed a gradual decline between day 7 and day 11 after challenge, while mice treated with the control antibody were cleared between day 5 and day 9 (Fig. 7B). In contrast, mice treated with Loop2-specific antibody M01 were fully cleared by day 5. Similarly, bacterial loads rapidly declined between day 3 and day 5 for the M01-treated group (Fig. 7C), resulting in significantly reduced area under the curves for the M01 group (Fig. 7D). These results demonstrate that MtrE Loop2-specific monoclonal antibody M01 is highly effective for treatment of N. gonorrhoeae infections.

Fig. 7. In vivo efficacy of MtrE Loop2-specific monoclonal antibody (mAb) M01.

Fig. 7

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A Schematic diagram of the efficacy study of mAb M01 using the mouse vaginal tract infection model for N. gonorrhoeae. Mice were challenged with N. gonorrhoeae ATCC 49226 on day 0 and from day 1 onwards daily injected intravaginally with 10 μg M01, a control mAb or a Mock control (8 mice/group). B Kaplan–Meier curves representing daily percentage of colonized mice. C Curves displaying the average and SEM of daily CFU counts from the colonized mice. D Graph displaying the individual area under the curve (AUC) for the daily CFU counts from the colonized mice. Significant differences between the Kaplan–Meier curves were identified with the Mantel-Cox log-rank test (GraphPad Prism). Significant differences between the AUCs were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). **P < 0.01, ***P < 0.001.

Modulation of complement-dependent bactericidal activity of M01

Since M01 was very effective in clearing N. gonorrhoeae from the mouse infection model, the functional activity of M01 was further investigated. Therefore, two M01 derivatives were generated with mutations that enhanced or abolished complement activation. A single E430G in the Fc region enhances Fc:Fc interactions and hexamerization, resulting in enhanced complement activation30. In contrast, a double D270A/R322A mutation in the Fc region abolishes complement interactions32. Both M01 derivatives were compared with M01 for complement-dependent bactericidal activity against both N. gonorrhoeae ATCC 49226 and FC428-associated strain SRRSH240. Importantly, M01 displayed excellent bactericidal activity against both strains, with a titer of 2 µg/mL for ATCC 49226 and 0.4 µg/mL for SRRSH240 (Fig. 8). In contrast, M01-E430G showed enhanced bactericidal activity, with titers of 0.016 µg/mL and 0.08 µg/mL against ATCC 49226 and SRRSH240, respectively, while M01-D270A/R322A completely lost complement-dependent bactericidal activity. Subsequently, the monoclonal antibodies were investigated for in vivo activity in the mouse infection model. Groups of eight mice were colonized with N. gonorrhoeae strain ATCC 49226, and from 1 day onwards mice were daily treated by direct intravaginal injection with M01, M01-E430G, M01-D270A/R322A or a Mock control (Fig. 9A). M01-D270A/R322A or Mock control mice gradually cleared N. gonorrhoeae between day 7 and day 9–10 (Fig. 9B). In contrast, M01 cleared N. gonorrhoeae between day 4 and day 6, while M01-E430G cleared N. gonorrhoeae significantly quicker between day 2 and day 5. Similarly, bacterial loads in the mouse vaginal track were lower for M01 and M01-E430G (Fig. 9C), resulting in significantly reduced area under the curves (Fig. 9D). Therefore, these results demonstrate that the efficacy of MtrE Loop2-specific monoclonal antibody M01 was fully based on complement-dependent activity.

Fig. 8. Complement-dependent bactericidal activity of MtrE Loop2-specific monoclonal antibodies (mAbs).

Fig. 8

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The bactericidal activity of mAbs M01, M01-E430G (enhanced complement activation) and M01-D270A/R322A (abolished complement activity) was investigated using baby rabbit complement. A Reciprocal bactericidal geometric mean titers against N. gonorrhoeae strain ATCC 49226. Symbols represent the independent repeats. B Reciprocal bactericidal geometric mean titers against FC428-associated N. gonorrhoeae strain SRRSH240.

Fig. 9. In vivo efficacy of MtrE Loop2-specific monoclonal antibodies (mAbs) modulated for complement activity.

Fig. 9

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A Schematic diagram of the efficacy study of mAbs M01, M01-E430G and M01-D270A/R322A using the mouse vaginal tract infection model for N. gonorrhoeae. Mice were challenged with N. gonorrhoeae ATCC 49226 on day 0 and from day 1 onwards daily injected intravaginally with 10 μg M01, M01-E430G or M01-D270A/R322A or a Mock control (8 mice/group). B Kaplan–Meier curves representing daily percentage of colonized mice. C Curves displaying the average and SEM of daily CFU counts from the colonized mice. D Graph displaying the individual area under the curve (AUC) for the daily CFU counts from the colonized mice. Significant differences between the Kaplan–Meier curves were identified with the Mantel-Cox log-rank test (GraphPad Prism). Significant differences between the AUCs were identified by one-way ANOVA with posthoc Tukey test (GraphPad Prism). *P < 0.05, ***P < 0.001.

Discussion

Developing vaccines against N. gonorrhoeae is challenging due to the absence of clear correlates of protection, high rates of phase and antigenic variation, and effective immune evasion mechanisms, resulting in the current lack of an approved gonococcal vaccine7. Although several gonococcal vaccine candidates have made notable progress, they remain at the stages of antigen discovery and the identification of protective immune responses, with no product having advanced beyond early development in recent years16,27,37–40. Critically, the emergence of ceftriaxone-resistant strains, notably FC428 derivatives, and rising treatment failures threaten our last-line therapy3, underscoring an urgent need for novel prophylactic and therapeutic strategies.

Peptide-based vaccines offer a compelling alternative to conventional approaches, enabling precise targeting of conserved epitopes while overcoming limitations of traditional platforms: synthetic production ensures scalability, cost-effectiveness, and thermal stability41. Our epitope-focused design employed a tetravalent MAP scaffold displaying MtrE Loop2 on a reverse lysine core. This architecture may favor recognition as a T cell-independent antigen, characterized by repetitive epitopes that directly activate B cells without classical T-cell support42. T cell-independent antigens typically elicit rapid T cell-independent IgM responses by engaging innate-like B cells or marginal zone B cells, which are primed to respond to highly organized multivalent epitopes43. Importantly, T cell-independent antigen responses typically lack germinal center formation, limiting affinity maturation and class switching to IgG. However, they can robustly induce IgM, which mediates potent complement-dependent effector functions44. This aligns with our observation that MAP-Loop2 elicited dominant IgM over IgG responses, yet achieved strong complement-dependent bactericidal activity. The efficacy of IgM, despite its lower affinity compared to IgG, may be attributed to its pentameric structure, which enables high-avidity binding and efficient C1q engagement, leading to complement cascade activation even at low antigen densities44.

Our results are in line with the dominant IgM response elicited by the 2C7 LOS epitope peptide mimetic MAP, although bactericidal activity of antibodies elicited by this MAP appeared to be mostly contributed to the 2C7-specific IgG responses11,15. This discrepancy between the two MAP vaccines may reflect differences in epitope accessibility, BCR clustering efficiency, or the role of T cell help. Notably, while IgG responses generally require T cell help for class switching, certain multivalent peptide scaffolds may still induce low-level IgG through alternative pathways, such as bystander T cell activation or TLR-mediated B cell stimulation45. Strikingly, the IgM-dominant response induced by our MAP-Loop2 vaccines proved functionally protective. Despite limited class switching to IgG, pentameric IgM efficiently activated complement, driving potent serum bactericidal activity against both reference and a ceftriaxone-resistant FC428-associated strains. These results are similar to observations with pneumococcal polysaccharide vaccines, where IgM mediates rapid bacterial clearance, despite limited longevity46. Thus, while IgG remains desirable for long-term immunity, IgM induction represents an important and underappreciated protective mechanism for peptide vaccines against bacterial pathogens.

The strongly IgM-dominant profile of our MAP vaccines highlights both a potential strength and a limitation. The rapid, high-titer IgM response provides potent, immediate effector function via complement activation, as evidenced by the significant prophylactic and therapeutic protection observed. However, the apparent bias towards a T-independent response suggests limited affinity maturation and class switching, which are hallmarks of germinal center reactions driven by CD4+ T cell help47. Consequently, the long-term durability of protection and the establishment of immunological memory may be suboptimal compared to vaccines that elicit robust IgG and memory B cell responses48. Future iterations of a Loop2-based vaccine could aim to preserve the advantageous avidity and complement-activating features of the IgM response while improving immunogenicity towards a more traditional T-dependent profile. Strategies may include linear fusion or chemical conjugation of the MAP to a carrier protein containing strong T-helper epitopes, or co-formulation with adjuvants specifically designed to promote Th1/Th2 responses and germinal center formation49. Such modifications could enhance IgG switching, affinity maturation, and the generation of long-lived plasma cells and memory B cells, potentially leading to more sustained protection against gonococcal infections.

Complementing our active immunization approach, we developed a human-mouse chimeric antibody. This antibody, termed M01, employed the variable domain of the most abundant IgM clonotype elicited with our MAP-Loop2 vaccine and combined it with the human IgG1 isotype constant regions. M01 demonstrated good binding affinity to MtrE Loop2 and strong complement-dependent bactericidal activity both in vitro in bactericidal assays and in vivo in a mouse infection model. Therefore, our study demonstrates that besides mAbs targeting the most highly abundant surface antigens LOS32,35 and PorB35, mAbs targeting low-abundance surface antigens such as MtrE can be functional active. The in vivo functional activity of M01 was fully dependent on complement, since an engineered M01-derivative with enhanced hexamerization and C1q binding through the E430G mutation, commonly referred to as hexabody30, demonstrated enhanced in vivo anti-gonococcal activity. In contrast, an M01-derivative with abolished complement activity through the D270A/R322A mutation32,33 fully lost in vivo anti-gonococcal activity. The interaction between antibodies and C1q is a critical step in initiating the classical complement pathway. C1q binds to the Fc regions of antigen-bound IgG or IgM, triggering a cascade that results in the formation of the membrane attack complex31. Antigen avidity plays a key role in this process, as higher avidity enhances the clustering of antibody-Fc regions, promoting efficient C1q binding and complement activation31,50. Therefore, low-abundant or monovalent antigens may fail to induce sufficient Fc multimerization, impairing C1q engagement and downstream complement effector functions51,52. Although MtrE is a low-abundant gonococcal surface antigen, its trimeric nature likely benefits effective antibody multimerizarion. Besides, the complement dependence of in vivo functional activity of our MtrE Loop2 mAb and the LOS 2C7 epitope mAb32 also establishes complement-dependent activity as an important correlate of protection for a vaccine against N. gonorrhoeae. While our in vitro data highlights complement-mediated bactericidal activity as a critical effector function, our in vivo model does not rule out activity through complement-mediated opsonophagocytsosis by neutrophils. However, the LOS 2C7 epitope mAb lost in vivo activity when the complement pathway was inactive, such as in C1q−/− or C9−/− mice or when C5 was blocked, while the mAb retained activity in neutrophil-depleted mice or when the C5a receptor was inhibited32. Therefore, complement-dependent bactericidal activity is mechanistically most likely the critical mode of activity of our MtrE Loop2 mAb.

A limitation of our study is the lack of direct measurement of antibody titers at the mucosal site of infection. While systemic antibodies likely reach the genital mucosa via transudation, future studies should quantify vaginal wash antibodies and evaluate intramuscular or mucosal immunization strategies. Intraperitoneal dosing of the MAP-Loop2 and MAP-CatS-Loop2 vaccines and the daily intravaginal administration of mAb M01 establishes proof-of-concept for vaccine efficacy and local passive antibody treatment, respectively, but does not reflect a practical therapeutic modality. The translational potential of MtrE Loop2-targeting vaccines and antibodies will depend on demonstrating efficacy with systemic administration via routes used in human dosing regimens and favorable pharmacokinetics, as shown for other gonococcal mAbs11,16.

Several limitations of this study should be acknowledged. First, while the murine vaginal tract infection model provides a robust platform for preclinical evaluation of gonococcal vaccines and antibody therapies, it does not fully recapitulate human genital tract physiology, immune responses, or microbiota composition. Therefore, the observed protective and therapeutic effects may not directly translate to clinical efficacy in humans. Second, although we demonstrate strong complement-dependent bactericidal activity and in vivo clearance, we did not directly quantify antibody concentrations at the mucosal site of infection, nor did we formally dissect the relative contributions of complement-mediated lysis versus opsonophagocytic mechanisms in vivo. Finally, the strong IgM-dominant response elicited by the MAP vaccines, while highly effective in this short-term model, may limit the durability of protective immunity. Future studies should therefore focus on optimizing vaccine formulations to promote durable memory responses and on validating efficacy in additional translational models.

In conclusion, this study demonstrates that MtrE Loop2-based MAP vaccines elicit robust IgM-dominant responses with potent complement-dependent activity, providing both prophylactic and therapeutic protection against N. gonorrhoeae in murine vaginal tract infection models. Notably, a derived monoclonal antibody (M01) targeting MtrE Loop2 exhibited strong in vivo effects, with Fc-engineered variants confirming complement activation as the primary mechanism of action. These findings support the potential of MtrE Loop2 as candidate for gonorrhoea prevention and treatment, leveraging complement-mediated antimicrobial activity as treatment strategy for antibiotic-resistant strains.

Methods

Multiple antigenic peptide (MAP) synthesis

MAP-Loop2, containing a tetravalent 13-amino acid MtrE Loop2 peptide (Fig. 1A), and MAP-CatS-Loop2, containing a tetravalent MtrE Loop2 peptide with an additional minimum cathepsin S cleavage site (Fig. 1A), were synthesized by GenScript with a purity over 90%. Shortly, the tetravalent MAPs were synthesized on a reverse lysine core scaffold covalently attached to a solid-phase resin through a cleavable linker53. The MAPs were purified and characterized by high performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS).

Single cell sequencing of splenocytes

Splenocytes from immunized mice were digested (0.35% collagenase IV, 2 mg/mL papain, 120 U/mL DNase I; 37 °C, 20 min). CD19+ B cells were sorted (FITC anti-mouse CD19; BioLegend, #115505) and subjected to single-cell V(D)J sequencing (10x Genomics; Hangzhou Lianchuan Biotech). Shortly, BCR-enriched cDNA libraries were sequenced on an Illumina platform (Illumina Novaseq™ 6000). Data were processed using Cell Ranger (10x Genomics) and VDJ Tools software for repertoire construction, clustering, and differential analysis.

Generation of MtrE Loop2-specific monoclonal antibodies (mAbs)

The mAbs M01, M01-E430G and M01-D270A/R322A were generated by GenScript. Shortly, the variable domains of the most abundant mouse B cell clonotype identified by single B cell sequencing (IgM and IgK types heavy and light chains) were synthesized with human IgG1 and IgK constant domains and cloned into expression vector pcDNA3.4 for expression of human-mouse chimeric mAb M01 (Fig. 6A). Similarly, human-mouse chimeric antibody genes with complement-modulating SNPs were synthesized for expression of M01-E430G and M01-D270A/R322A. Vectors were transfected into CHO cells for expression of the mAbs. Subsequently, cell cultures were harvested by centrifugation, and cell pellets were disrupted by sonication on ice in a lysis buffer containing protease inhibitors. The soluble fraction was applied on a Protein A affinity column and mAbs were further purified by ion exchange chromatography and size exclusion chromatography. Purified mAbs were concentrated using Amicon Ultra centrifugal filters and stored in PBS buffer at −80 °C.

Animal ethics statement

Animal experiments were approved by the Zhejiang University Animal Care and Use Committee (license number 17324) and experiments complied with the guidelines of Administration of Affairs Concerning Experimental Animals of the People’s Republic of China and the principles of the Declaration of Helsinki.

Animal experiments

All animal experiments were performed on five- to six-week-old female BALB/c mice obtained from the Shanghai SLAC Laboratory Animal Company. Mice were kept in a 24-cage individually ventilated caging system (Suzhou Suhang Technology) in a biosafety level 2 laboratory. Mice were acclimatized to the facility for one week before commencement of experiments and they received water and feeding ad libitum. Mice were anesthetized using 4% isoflurane and upon completion of the experiments anesthetized mice were euthanized by cervical dislocation. Mice were assigned randomly to treatment groups. For immunization and prophylactic vaccine efficacy experiments, mice received three biweekly intraperitoneal injections of CpG1826 (20 µg)-formulated MAP-Loop2 (50 µg), MAP-CatS-Loop2 (50 µg) or a Mock control (PBS) on days −35, −21, and −7, and on day 0 (one week post final immunization) sera was collected for immunogenicity assays, or mice were challenged with N. gonorrhoeae. For early antibody response analysis, mice received a single intraperitoneal injection of CpG1826 (20 µg)-formulated MAP-Loop2 (50 µg) or a Mock control (PBS) and sera were collected on days 0, 2, 4, 6, 8 or 10 for immunogenicity assays. For single-cell sequencing of splenocytes, mice received five biweekly intraperitoneal injections of CpG1826-formulated MAP-Loop2 before dissection of the spleen. For the therapeutic vaccine efficacy experiments, mice received a single vaccine dose of CpG1826-formulated MAP-Loop2, MAP-CatS-Loop2 or a Mock control six hours after challenge, while for the therapeutic mAb efficacy experiments mice received daily intravaginal doses of 10 µg PBS-formulated mAbs M01, M01-E430G, M01-D270A/R322A, a control mAb (humanized IgG1 with IgK light chain targeting influenza H7N9 HA antigen; Sino Biological, #HG1K) or a Mock control on days 1 to 11 or until cleared. For the challenge experiments, mice received 1 mg β-estradiol (Aladdin) subcutaneously on days −2, 0 and 2, and 0.6 mg vancomycin (Meilunbio) and 1.2 mg streptomycin (BBI) on days −2, −1, 0, 1, and 2. Furthermore, from day-2 onwards the drinking water was lashed 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 and formulated in PBS containing 0.5 mM CaCl2 (Sigma), 1 mM MgCl2 (Sigma) and 1% (w/v) gelatin (Aladdin). From day 1 onwards, vaginal mucosa were daily swabbed for determination of bacterial loads. Mice that were not successfully colonized by day 1 (<1,000 CFU) and remained culture-negative by day 3, were excluded from the analysis of vaccine efficacy to ensure a synchronized infection baseline. Group sizes were estimated based on comparable gonococcal challenge experiments performed previously27. Bacterial samples were plated on GC agar (Oxoid) supplemented with 1% (v/v) Vitox (Oxoid), vancomycin, colistin (Meilunbio), nystatin (Meilunbio), trimethoprim and streptomycin.

Complement-dependent bactericidal activity assays

For complement-dependent serum bactericidal activity and mAb bactericidal activity assays, N. gonorrhoeae ATCC 49226 and FC428-associated strain SRRSH240 were suspend in PBS containing 1% (v/v) Vitox, 0.9 mM CaCl2, 0.5 mM MgCl2, 2.5% (v/v) baby rabbit serum (Cedarlane) or 25% (v/v) normal human serum from healthy volunteers and twofold dilution series of heat-inactivated serum from immunized mice or mAbs. Complement sera were confirmed to be free of intrinsic bactericidal activity and gonococcus-specific antibodies by testing heat-inactivated and non-heat-inactivated sera alone in bactericidal activity assays prior to use. For Triton X-100 killing assays, complement was replaced by 0.005% (v/v) Triton X-100 (VETEC), which was used to probe whether antibody binding to MtrE Loop2 functionally interferes with MtrCDE efflux pump activity, resulting in enhanced detergent susceptibility.

Reactions were incubated for one hour at 37 °C and 5% CO2 and aliquots of 10 µL were spot-plated onto GC agar containing 1% Vitox. The titer was determined as the highest dilution able to kill over 50% of the bacteria compared with the no sera/mAb control wells.

Opsonophagocytosis assays

Human promyelocytic HL-60 cells (ATCC CCL-240) were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Biological Industries) supplemented with 10% fetal bovine serum (FBS; Bovogen), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco) at 37 °C under 5% CO2. To induce differentiation into neutrophil-like cells, HL-60 cells were transferred to antibiotic-free IMDM medium containing 10% FBS and 0.8% (v/v) N,N-dimethylformamide (DMF; CST) for 5–6 days. Differentiated cells were washed and resuspended in IMDM with 1% (v/v) Vitox, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 8% (v/v) normal human serum and a serial twofold dilution of heat-inactivated immune serum. Cells (5 × 10⁴) were challenged with N. gonorrhoeae ATCC 49226 and SRRSH240 at a multiplicity of infection (MOI) of 1, incubated for 90 min at 37 °C with 5% CO₂ and spot-plated onto GC agar supplemented with 1% Vitox. The opsonophagocytic titer was defined as the highest serum dilution able to kill over 50% of the bacteria compared to control wells containing no immune serum.

Enzyme-linked immunosorbent assay (ELISA)

Total IgG, IgM and IgA titers of sera for MtrE Loop2 expressed as a linear peptide on the N-terminus of the IMX315 carrier protein (Nloop2)17,27, or mAb titers for Nloop2 and MtrE were determined by ELISA. Maxisorp microtiter plates (NUNC) were coated overnight at 4 °C with 150 ng/well recombinant Nloop2 or MtrE and subsequently blocked with 4% Bovine Serum Albumin (BSA; Biosharp). After washing, plates were incubated for one hour at 37 °C with twofold dilution series of individual or pooled mouse sera or with mAbs. After washing, plates were incubated with HRP-conjugated polyclonal goat anti-mouse IgG (Thermo Fisher, #31430), HRP-conjugated polyclonal goat anti-mouse IgM (Thermo Fisher, #31440), HRP-conjugated polyclonal goat anti-mouse IgA (Thermo Fisher, #62-6720), or HRP-conjugated polyclonal goat anti-human IgG (Thermo Fisher, #62-8420) and finally, HRP activity was determined with TMB substrate (Beyotime).

Western blotting analysis

Recombinant MtrE was boiled for 10 min in SDS PAGE loading buffer (Beyotime), run on SDS polyacrylamide gels and transferred onto PVDF membranes (Biorad). Membranes were blocked with 5% BSA (Biosharp) and subsequently incubated with mAb M01 (0.08–10 µg/mL) for 1 h at room temperature. After washing, membranes were incubated with HRP-conjugated polyclonal goat anti-human IgG (Thermo Fisher, #62-8420) for 1 h at room temperature. Finally, HRP activity was detected with Immobilon Western Substrate (Millipore, Cat #WBKLS) and a ChemiDoc Touch Imaging System (Bio-Rad).

Supplementary information

Supplementary Information (52.8KB, pdf)

Acknowledgements

This work was supported by the National Natural Science Foundation of China [grant numbers 82572622, 82272382, 82150610507, 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. We thank Lin Zhaoxiaonan and Xiao Guifeng from the Core Facilities of Zhejiang University School of Medicine for assistance.

Author contributions

S.S. and H.G. are joined-first author. S.V. conceived the project. S.S. and S.V. designed the experiments. S.S., H.G., D.Y., X.G., W.L., C.D., Y.Q., and S.G. performed the experiments. S.S., X.L., H.C,. and S.V. analyzed the data. S.S. and S.V. wrote the manuscript. X.L. and H.C. edited the manuscript. All authors approved the final manuscript.

Data availability

All data generated or analysed during this study are included in this published article. The original datasets generated and analysed during this study are available from the Zenodo repository at 10.5281/zenodo.18129123.

Competing interests

S.V., S.S., H.G., D.Y., W.L., C.D., and Y.Q. are named inventors on a pending Chinese patent application (Application number: 2025115678059) covering intellectual property for mAb M01. X.G., S.G., X.L., and H.C. declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Shuaijie Song, Haoyu Ge.

Supplementary information

The online version contains supplementary material available at 10.1038/s41541-026-01412-0.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information (52.8KB, pdf)

Data Availability Statement

All data generated or analysed during this study are included in this published article. The original datasets generated and analysed during this study are available from the Zenodo repository at 10.5281/zenodo.18129123.

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