Neisseria gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted infection which is rising in incidence, with increasing drug-resistant strains posing a significant public health threat. To address the urgent need for novel therapies, we developed an antibody-drug conjugate (ADC) that targets this important human pathogen. We utilized Tridecaptin A1, a potent antimicrobial peptide (AMP) against Gram-negative bacteria that exhibits significant toxicity against human cells, limiting its development for clinical use. By conjugating the Tridecaptin A1 analogue, Oct-TriA1 to a monoclonal antibody (mAb) that specifically targets gonococcal MtrE, the outer membrane component of a drug efflux pump that is upregulated in resistant strains, we aim to selectively deliver the AMP to the gonococcus. However, Oct-TriA1 was not bactericidal when directly conjugated to mAb. To circumvent this, we exploited an immune evasion mechanism employed by the gonococcus by introducing a linker between Oct-TriA1 and the mAb which is specifically cleaved by the IgA protease (IgAP) secreted by the gonococcus; the IgAP inactivates human IgA. This ADC has no detectable toxicity for relevant human cells, kills the gonococcus in an MtrE- and IgAP-dependent manner, and is active against a strain which is resistant to first line agents. This modular ADC platform could be extended to other bacterial pathogens which employ proteases to evade immune killing, offering a strategy in the fight against antimicrobial resistance.

Sexually transmitted infections (STIs) and unintended pregnancy remain critical global public health concerns affecting millions of women worldwide. To support the development of new multipurpose products offering non-hormonal contraception and activity against sexually transmitted pathogens, we evaluated copper sulfate, zinc lactate, L-(+)-lactic acid and their combinations as candidate pharmaceutical actives. We determined virucidal (HIV-1, HSV-2) and bactericidal (Neisseria gonorrhoeae, Chlamydia trachomatis) activity in vitro followed by in vivo efficacy testing against HSV-2, N. gonorrhoeae, and C. trachomatis in mice. In vitro, zinc lactate alone showed no inhibitory activity. In contrast, copper sulfate and lactic acid alone and in combination demonstrated activity against all tested pathogens. Furthermore, these combinations remained potent in the presence of vaginal and seminal fluids. Importantly, the actives – alone or combined – at concentrations that inhibited STI pathogens did not inhibit the growth of Lactobacillus crispatus, bacteria associated with an optimal vaginal microbiota. Further, copper sulfate and lactic acid alone and in combination inhibited vaginal HSV-2 and N. gonorrhoeae infection in mice. The combination conferred limited protection against vaginal C. trachomatis infection. These data demonstrate the advantages of combining copper sulfate and lactic acid and support further evaluation and development of this combination as a multipurpose prevention product.

Gonorrhoea is a global health concern exacerbated by rising antimicrobial resistance. Retrospective analyses indicate that outer membrane vesicle (OMV) vaccines derived from Neisseria meningitidis (MeNZB, 4CMenB) may offer partial cross-protection against gonococcal infection. This review outlines the influence of gonococcal population biology on coverage, immune responses elicited by 4CMenB, and emerging strategies that offer the prospect of rationally designed vaccines dedicated to the prevention of gonococcal disease.

There is an unmet need for developing drugs for the treatment of gonorrhea due to rapidly evolving resistance of Neisseria gonorrhoeae against antimicrobial drugs used for empiric therapy, an increase in globally reported multidrug-resistant cases, and the limited available therapeutic options. Furthermore, few drugs are under development. Development of antimicrobials is hampered by challenges in clinical trial design, limitations of available diagnostics, changes in and varying standards of care, lack of robust animal models, and clinically relevant pharmacodynamic targets. On 23 April 2021, the US Food and Drug Administration, Centers for Disease Control and Prevention, and National Institute of Allergy and Infectious Diseases of the National Institutes of Health co-sponsored a workshop with stakeholders from academia, industry, and regulatory agencies to discuss the challenges and strategies, including potential collaborations and incentives, to facilitate the development of drugs for the treatment of gonorrhea. This article provides a summary of that workshop.

Emerging multidrug-resistant Neisseria gonorrhoeae strains possessing altered penA alleles (encoding penicillin-binding protein 2, PBP2) threaten the utility of ceftriaxone, the last remaining outpatient antibiotic for gonorrhoea treatment, posing a global health emergency. Here we report a benzoxaborinine-based penicillin-binding protein inhibitor series (boro-PBPi) developed to address penA-mediated ceftriaxone resistance. Optimization of boro-PBPi led to the identification of compound 21 (VNRX-14079), which exhibited potent antibacterial activity against multidrug-resistant N. gonorrhoeae through high-affinity binding to the PBP2 target. Boro-PBPi-PBP2 complex structures confirmed the covalent interaction of the boron atom with the catalytic residue Ser310 and the importance of the β3-β4 loop mobility for improved affinity. Boro-PBPi 21 elicits bactericidal activity, a low frequency of resistance, a good safety profile, suitable pharmacokinetic properties and in vivo efficacy in a murine infection model against ceftriaxone-resistant N. gonorrhoeae. Boro-PBPi 21 is therefore a promising antigonorrhoea agent poised for further advancement.

The dynamics of antimicrobial resistance in bacterial populations are influenced by the fitness impact of genetic determinants of resistance and antibiotic pressure. However, estimates of real-world fitness impact have been lacking. To address this gap, we developed a hierarchical Bayesian phylodynamic model to quantify contributions of resistance determinants to strain success in a 20-year collection of Neisseria gonorrhoeae isolates. Fitness contributions varied with antibiotic use, which over this period included ciprofloxacin, cefixime, ceftriaxone and azithromycin, and genetic pathways to phenotypically identical resistance conferred distinct fitness effects. These findings were supported by competition experiments both in vitro and in the mouse model of gonococcal infection. Quantifying these fitness contributions to lineage dynamics reveals opportunities for investigation into other genetic and environmental drivers of fitness. This work thus establishes a method for linking pathogen genomics and antibiotic use to define factors shaping ecological trends.

The 24th International Pathogenic Neisseria Conference (IPNC) marked a shift toward a balanced forum addressing both meningococcal and gonococcal disease. This drove discussion on how multivalent meningococcal vaccines are enabling the WHO roadmap to defeat meningitis by 2030, while highlighting growing evidence that effective gonococcal vaccines are achievable. Major challenges remain, including antimicrobial resistance, limited genomic surveillance and incomplete understanding of pathogenesis and immune evasion. These reflections shaped this perspective piece.

BACKGROUND:Neisseria gonorrhoeae is a major sexually transmitted pathogen with rising antimicrobial resistance. Epidemiological studies suggest meningococcal outer membrane vesicle (OMV) vaccines, eg 4CMenB (Bexsero), offer partial cross-protection, but immune correlates remain undefined. METHODS:Using a murine genital tract infection model, we compared systemic and mucosal responses after immunization with Bexsero, gonococcal OMVs (Ng OMVs) with alum, or alum alone. Vaccinated mice were challenged intravaginally with N. gonorrhoeae. Serum bactericidal activity (SBA) was measured, and antigen-specific IgG responses were profiled using gonococcal protein microarrays. Murine responses were compared with high-risk Bexsero-immunized humans and patients with gonococcal infection. RESULTS:Bexsero significantly accelerated bacterial clearance compared with Ng OMV or alum. Serum and vaginal IgG profiles were highly correlated, revealing distinct antigenic signatures between groups. Bexsero elicited responses to gonococcal orthologs of its recombinant antigens (GNA1030, GNA2091, NHBA), whereas Ng OMVs induced Opa, PilQ, and MtrE responses. SBA did not correlate with clearance; however, early PMN influx was associated with reduced bacterial load. Antigen-specific IgG patterns were concordant between Bexsero-immunized mice and humans. CONCLUSIONS:Protein microarrays with multivariate analysis identified antibody signatures associated with protection, supporting the translational value of this model for defining correlates of protection and guiding gonococcal vaccine development.

IntroductionLimited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear.MethodsTo study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae, 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 ΔABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model.ResultsMice immunized with 4CMenB or MC58 ΔABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58ΔABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae. Further, splenocytes in 4CMenB and MC58 ΔABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine.DiscussionOur findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

We investigated the trends and antimicrobial resistance (AMR) of Neisseria gonorrhoeae (NG) in Kenya with whole-genome sequencing (WGS) of isolates collected in 2002-2009 (n = 108) and 2020-2022 (n = 110). Phenotypic AMR was confirmed by agar dilution. Predicted minimum inhibitory concentrations (MICs), multi-locus sequence typing (MLST), multi-antigen ST (NG-MAST), NG-STAR, and AMR genetic determinants were determined using WGS and detection of molecular markers. The WGS cgMLST typing used LIN codes. Resistance to penicillin, ciprofloxacin, and tetracycline was common throughout. In 2020-2022, azithromycin resistance (n = 2) and cephalosporin alert values (n = 5) were observed. Phylogenetic clusters were congruent with the LIN code lineage, though other typing schemes (MLST, NG-STAR, and NG-MAST) were not as consistent. There were major shifts over time in the lineages and genetic determinants. Circumcision and HIV status were associated with several AMR, housekeeping, metabolism, and iron acquisition genetic determinants. These findings highlight dynamic NG genomic trends, emerging macrolide resistance, and the value of WGS for surveillance. Behavioral and biological factors may contribute to AMR emergence and warrant further investigation. IMPORTANCE This work highlights the significant value of using whole-genome sequencing to track the evolution and epidemiology of gonorrhea over 20 years: (i) we documented the emergence of azithromycin resistance and cephalosporin reduced susceptibility and relationship to genetics of gonorrhea; (ii) by combining epidemiological and genetic data, we found that circumcision and HIV status were linked to specific genetic features of gonorrhea, including those tied to antibiotic resistance; and (iii) we used novel and traditional genetic typing methods to expand and refine the understanding of lineage shifts and genetic determinants, enhancing surveillance and intervention efforts. Some isolates had potential decreasing susceptibility for cephalosporins, highlighting the critical importance of ongoing surveillance and the opportunity for novel resistance gene identification. Studying how gonorrhea strains relate to a person's immune system, other bacteria (microbiome), and sexual networks could help us understand how certain strains spread and what the potential factors amplifying antimicrobial resistance are.

The dynamics of antimicrobial resistance in bacterial populations are informed by the fitness impact of genetic determinants of resistance and antibiotic pressure. However, estimates of real-world fitness impact have been lacking. To address this gap, we developed a hierarchical Bayesian phylodynamic model to quantify contributions of resistance determinants to strain success in a 20-year collection of Neisseria gonorrhoeae isolates. Fitness contributions varied with antibiotic use, and genetic pathways to phenotypically identical resistance conferred distinct fitness effects. These findings were supported by in vitro and experimental infection competition. Quantifying these fitness contributions to lineage dynamics reveals opportunities for investigation into other genetic and environmental drivers of fitness. This work thus establishes a method for linking pathogen genomics and antibiotic use to define factors shaping ecological trends.

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to first-line antibiotics has focused efforts on understanding how alternative therapies, such as the expanded use of gentamicin (Gen), might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for the treatment of gonorrheal infections, we used RNA-seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level. We found that sub-lethal Gen levels altered the expression of 23 genes in Ng strain FA19. Many of the differentially regulated genes were associated with known stress responses elaborated by Ng under different harmful conditions. We found that the transcripts of the hicAB operon, which encodes a putative HicA-HicB toxin-antitoxin system that is encoded by tandem genes with the prophage Ngo φ3, were increased in response to Gen. Although the loss of hicAB did not impact gonococcal susceptibility to a variety of antimicrobial agents or harmful environmental conditions, it did reduce biofilm formation in Ng strains F62, FA1090, WHO X, and CDC200 but not that of strain FA19. Furthermore, in strain F62, but not FA19, loss of hicAB reduced the in vivo fitness of Ng during experimental lower genital tract infection of female mice. Furthermore, we found that expression of hicAB can influence levels of the norB transcript, which encodes the nitrate reductase shown previously to be upregulated in gonococcal biofilms. We propose that sub-lethal Gen has the capacity to influence gonococcal pathogenesis through the action of the HicAB toxin-antitoxin system. IMPORTANCE:During antibiotic treatment, bacteria can be exposed to sub-lethal levels that could serve as a stress signal, resulting in changes in gene expression. The continued emergence of multi-drug-resistant strains of Ng has rekindled interest in expanded use of gentamicin (Gen) for the treatment of gonorrheal infections. We report that sub-lethal levels of Gen can influence levels of Ng transcripts, including that of the gonococcal hicAB-encoded toxin-antitoxin (TA) locus, which is embedded within an integrated prophage. Although the loss of this TA locus did not impact Ng susceptibility to Gen, it reduced the biofilm-forming ability of four of five Ng strains. Furthermore, in an examined strain in this group, we found that Ng fitness during experimental infection was negatively impacted. We propose that levels of the hicA-hicB transcripts can be increased by sub-lethal levels of an antibiotic used in the treatment of gonorrhea and that this could influence pathogenicity.

Ng is acquiring increased resistance to antibiotics that is outpacing new antibiotic development. Treatment of gonorrhea remains problematic given that new annual cases approximate 82 million worldwide and 1.6 million in the US. Natural immunity to gonorrhea is ineffective. Thus, an efficacious gonococcal vaccine is much needed. The vaccine against Group B Neisseria meningitidis, 4CMenB, can confer partial cross-protection against gonorrhea, suggesting that a gonococcal vaccine is feasible. In light of this finding, we hypothesize that Snodgrassella alvi, which branches within the family Neisseriaceae, can be deployed as a naturally attenuated, nonpathogenic live vector vaccine for Ng. S. alvi is not expected to colonize humans due to its niche-restriction to bees. In silico analysis found that S. alvi lacks virtually all major virulence factors associated with pathogenic Neisseriaceae, including a dedicated machinery to retrieve iron from the host. To test our hypothesis, groups of mice were vaccinated by the intraperitoneal (IP) route three times, and elicited high serum and vaginal IgG antibody (Ab) titers that cross-react to Ng. Serum IgG Abs were robustly bactericidal. When groups of IP-vaccinated BALB/c females were subjected to vaginal Ng challenge, only 13% of S. alvi-vaccinated mice showed Ng colonization post-1 wk challenge compared to 76% unvaccinated controls. Thus, S. alvi confers exquisite protection against vaginal Ng challenge. Work supported by NIH AI151424. Mucosal and Regional Immunology (MUC)

BACKGROUND:Identification of immune correlates in murine gonorrhea models has been hampered by study-dependent differences in vaccine antigens and administration routes. We previously showed that detergent-detoxified outer membrane vesicles (dOMVs) isolated from a PorA-, PorB-, and RmpM-deficient meningococcal strain (ΔABR) elicit antibodies that cross-react with Neisseria gonorrhoeae and enhance gonococcal clearance in a mouse model of lower reproductive tract infection. In this study, we investigated whether (1) ΔABR dOMVs can protect mice from ascending gonococcal infection and (2) vaccination route influences immune responses. METHODS:Mice were vaccinated subcutaneously (SC) or intraperitoneally (IP) and then vaginally inoculated with gonococci. Bioburden of mice was measured and assessed relative to ΔABR dOMV-induced cellular and humoral immune responses. RESULTS:Subcutaneous and intraperitoneal vaccination accelerated gonococcal clearance from the lower and upper reproductive tract at similar rates. Probing of gonococcal protein microarrays with immune sera from the 2 groups identified multiple vaccine targets that were commonly immunogenic. Despite comparable clearance patterns in vaccinated mice, differences in immune induction were observed that were dependent on administration route. SC immunized mice demonstrated a neutrophil influx that correlated with decreased vaginal bioburden; higher serum bactericidal activity against nonsialylated gonococci was also noted. In contrast, IP immunization induced higher serum and vaginal IgA levels, serum bactericidal activity against sialylated gonococci, and antigonococcal opsonophagocytic killing activity of neutrophils. CONCLUSIONS:This work demonstrates that ΔABR dOMVs protect against ascending gonococcal infection and that cellular and functional antibody responses to the same candidate vaccine may vary depending on immunization route.

ponA L421P encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MIC PEN ) above the clinical breakpoint in Neisseria gonorrhoeae . Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s, ponA L421P is present in nearly 50% of current N. gonorrhoeae isolates in the PubMLST database. Bioinformatic analysis indicates that ponA L421P is exclusive to N. gonorrhoeae isolates, whereas Leu-421 is 100% conserved in other Neisseria species. To understand the involvement of ponA L421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors ponA L421P . Proline-421 was the only mutation that increased the MIC PEN to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant ponA alleles over multiple serial passages, both with and without sub-MIC levels of penicillin. There was no fitness defect attributed to ponA L421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponA L421P is driven by its capacity to increase the MIC pen above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponA L421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the ponA L421P allele emerged in gonococcal isolates, increasing the MIC PEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines. Importance:The emergence of antibiotic-resistant Neisseria gonorrhoeae threatens effective treatment of gonorrhea, one of the most common sexually transmitted infections worldwide. Understanding the genetic changes that drive and maintain resistance is crucial for anticipating future resistance trends. Here, we investigated the impact of a key resistance mutation in PBP1 (encoded by ponA L421P ). Although penicillin has not been used to treat gonorrhea for decades, this mutation remains widespread even in recent N. gonorrhoeae isolates. ponA L421P confers clinically relevant penicillin resistance without imposing an in vitro fitness cost. ponA L421P also increases resistance to ceftriaxone in strains with penA alleles that are associated with ceftriaxone resistance. This work highlights the role of the ponA L421P allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population.

[This corrects the article DOI: 10.3389/fimmu.2025.1539795.].

The rapid emergence of antimicrobial-resistant strains of Neisseria gonorrhoeae threatens treatment options and control efforts. The Uniformed Services University Gonococcal Reference Laboratory and Repository of the Global Emerging Infections Surveillance Program receives isolates from several geographically distinct regions worldwide. We analyzed 962 isolates collected during 2014-2022 for genomic and phenotypic antimicrobial resistance. Resistance to antimicrobial drugs previously used for gonococcal infections was high, but of most concern were increases of resistance to currently used antibiotic drugs, such as extended-spectrum cephalosporins and the alternative antibiotic treatment gentamicin. The percentage of isolates with reduced susceptibility to ceftriaxone was 3.6%, to cefixime was 2.5%, and to gentamicin was 15.0%. Although isolates were collected from populations of limited diversity, 706 (73.4%) of isolates demonstrated novel multiantigen sequence types, and 225 (23.4%) had novel multilocus sequence types. Continued surveillance of N. gonorrhoeaeis essential to monitoring the prevalence and spread of resistant organisms worldwide

Background: We characterized the antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae (NG) isolated from symptomatic men at a sexually transmitted infection clinic in Kisumu, Kenya. Methods: Two urethral swabs were obtained from symptomatic men between 2020 and 2022, one for Gram's stain and the other inoculated directly onto modified Thayer-Martin media containing 1% VCNT and 1% IsoVitaleX enrichment. Culture results were confirmed by colony morphology, Gram's stain and oxidase test. Duplicate isolates were shipped to Uniformed Services University for confirmation and characterization. Susceptibility to eight drugs was assessed by E-test. Agar dilution confirmed resistance to ceftriaxone, cefixime, and azithromycin. Susceptibility, intermediate resistance (IR), and resistance (R) were determined according to published criteria. Results: Of 154 enrolled participants, 112 were culture-positive for NG. Agar dilution results in 110 (98.2%) showed the following: azithromycin-R (1.8%), and 4.5% R or IR to ceftriaxone or cefixime: ceftriaxone-R (0.9%), ceftriaxone-IR (2.7%), and cefixime-IR (2.7%). By E-test, most isolates were IR or R to tetracycline (97.2%), penicillin (90.9%), and ciprofloxacin (95.4%). Conclusions: We detected NG with resistance to azithromycin and ceftriaxone, indicating a growing threat to the current Kenyan dual syndromic treatment of urethritis with cephalosporin plus macrolides. Ongoing AMR surveillance is essential for effective drug choices.

Chlamydia trachomatis infections are the most common bacterial STIs globally and can lead to serious morbidity if untreated. Development of a killed, whole-cell vaccine has been stymied by coincident epitope destruction during inactivation. Here, we present a prototype Chlamydia vaccine composed of elementary bodies (EBs) from the related mouse pathogen, Chlamydia muridarum (Cm). EBs inactivated by gamma rays (Ir-Cm) in the presence of the antioxidant Mn2+-Decapeptide (DEHGTAVMLK) Phosphate (MDP) are protected from epitope damage but not DNA damage. Cm EBs gamma-inactivated with MDP retain their structure and provide significant protection in a murine genital tract infection model. Mice vaccinated with Ir-Cm (+MDP) exhibited elevated levels of Cm-specific IgG and IgA antibodies, reduced bacterial burdens, accelerated clearance, and distinctive cytokine responses compared to unvaccinated controls and animals vaccinated with EBs irradiated without MDP. Preserving EB epitopes with MDP during gamma inactivation offers the potential for a polyvalent, whole-cell vaccine against C. trachomatis.