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Murine models of Neisseria gonorrhoeae lower reproductive tract infection are valuable systems for studying N. gonorrhoeae adaptation to the female host and immune responses to infection. These models have also accelerated preclinical testing of candidate therapeutic and prophylactic products against gonorrhea. However, because N. gonorrhoeae infection is restricted to the murine cervicovaginal region, there is a need for an in vivo system for translational work on N. gonorrhoeae pelvic inflammatory disease (PID). Here we discuss the need for well-characterized preclinical upper reproductive tract infection models for developing candidate products against N. gonorrhoeae PID, and report a refinement of the gonorrhea mouse model that supports sustained upper reproductive tract infection. To establish this new model for vaccine testing, we also tested the licensed meningococcal 4CMenB vaccine, which cross-protects against murine N. gonorrhoeae lower reproductive tract infection, for efficacy against N. gonorrhoeae in the endometrium and oviducts following transcervical or vaginal challenge.

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances.

The increasing prevalence of Neisseria gonorrhoeae strains exhibiting decreased susceptibility to extended-spectrum cephalosporins (ESCs) presents a challenge for the successful treatment of gonorrhea infections. To address this challenge, we evaluated a panel of 23 cephalosporins against penicillin-binding protein 2 (PBP2) from the ESC-resistant (ESCR) N. gonorrhoeae strain H041 to determine which molecular features are important for antimicrobial activity. Structure-activity relationships (SARs) developed from acylation rate constants against PBP2 and antimicrobial susceptibilities against the H041 strain of N. gonorrhoeae, and interpreted against docking models, reveal that cephalosporins possessing large, lipophilic R1 side chains and electronegative R2 side chains with planar groups are associated with higher acylation rates against PBP2, but also that these same amphipathic features can lower antimicrobial activity. Based on these studies, we tested cefoperazone, one of the most effective ESCs for targeting PBP2, in the female mouse model infected with H041 and showed that it was equally or more effective than ceftriaxone or gentamicin for clearing infections. Taken together, our results reveal that two U.S. Food and Drug Administration (FDA)-approved agents (cefoperazone, ceftaroline) and one FDA-qualified infectious disease product (ceftobiprole) have potential as first-line treatments for gonorrhea and provide a framework for the future design of cephalosporins with improved activity against ESC-resistant N. gonorrhoeae.

The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside.

Background Increasing multidrug resistance rates in Neisseria gonorrhoeae have raised concerns and an urgent call for new antibiotics for treatment of gonorrhoea. Several decades of subdued drug development in this field and the recent failures of two new antibiotics to show non-inferiority compared with the current first-line antibiotics ceftriaxone plus azithromycin highlight the need for improved preclinical tools to predict clinical outcome of new drugs in the development process. Objectives To summarize current pharmacokinetic/pharmacodynamic (PK/PD) knowledge and dose-finding strategies for antibiotics against gonorrhoea. Sources Literature review of published papers and discussions by global experts at a special workshop on this topic. Content We review current knowledge of gonococcal specific PK/PD principles and provide an update on new in vitro and in vivo models to correlate drug exposure with clinical outcome, and identify challenges and gaps in gonococcal therapeutic research. Implications Identifying the ideal antimicrobial agent and dose for treating uncomplicated urogenital and pharyngeal gonococcal disease requires appropriate validated non-clinical PK/PD models. Recent advances in adapting in vitro and in vivo models for use in gonorrhoea are an important step for enabling the development of new drugs with reduced risk of failure in Phase 3 clinical development and diminish the risk of emergence of resistance.

Background Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. Methods We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. Results Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. Conclusions Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Gonorrhea remains a major public health challenge, and current recommendations for gonorrhea treatment are threatened by evolving antimicrobial resistance and a diminished pipeline for new antibiotics. Evaluations of potential new treatments for gonorrhea currently make limited use of new understanding of the pharmacokinetic and pharmacodynamic contributors to effective therapy, the prevention of antimicrobial resistance, and newer designs for clinical trials. They are hampered by the requirement to utilize combination ceftriaxone/azithromycin therapy as the comparator regimen in noninferiority trials designed to seek an indication for gonorrhea therapy. Evolving gonococcal epidemiology and clinical trial design constraints hinder the enrollment of those populations at the greatest risk for gonorrhea (adolescents, women, and persons infected with antibiotic-resistant Neisseria gonorrhoeae). This article summarizes a recent meeting on the evaluation process for antimicrobials for urogenital gonorrhea treatment and encourages the consideration of new designs for the evaluation of gonorrhea therapy.

The bacterium Neisseria gonorrhoeae (Ngo) is the main cause of the sexually transmitted infection gonorrhea. The global incidence of 87 million new Ngo infections each year, rising infection rates, and the emergence of Ngo strains that are resistant to all clinically recommended antibiotics have raised the specter of untreatable infections (M. Unemo, H. S. Seifert, E. W. Hook, III, S. Hawkes, et al., Nat Rev Dis Primers 5:79, 2019, https://doi.org/10.1038/s41572-019-0128-6). Given their abundance in symptomatic disease, neutrophils are central to both Ngo infection and consequent damage to host tissues. This article highlights present knowledge and the main open questions about Ngo-neutrophil interactions in immunity versus disease pathogenesis.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways., Antibiotic-resistant bacterial pathogens pose a substantial threat to human health. Here, aided by structural analyses, the authors describe the molecular mechanism behind the activity of a series of compounds that inhibit trans-translation and are effective in eradicating N. gonorrhoeae infection in mice.

Declining gonococcal susceptibility to ceftriaxone and azithromycin has raised the possibility of untreatable gonorrhea in the future and reignited interest in gonococcal vaccine development. Despite decades of research, previous gonococcal vaccine candidates have been ineffective. A growing body of data suggests that meningococcal group B outer-membrane vaccines may be cross-protective against Neisseria gonorrhoeae. Clinical trials of a licensed vaccine against Neisseria meningitidis serogroup B containing an outer-membrane vaccine component are underway to determine its efficacy against N. gonorrhoeae. Other experimental gonococcal vaccine candidates are in the preclinical phases. Population impact of future gonococcal vaccines with different levels of efficacy and duration of protection in various populations is being evaluated using modeling studies. Despite recent progress, gaps in gonococcal vaccine research remain. Research is needed to evaluate vaccine efficacy in preventing gonococcal infections acquired via various anatomic routes and among patients coinfected with other sexually transmitted infections. Studies that model the impact of a future vaccine on high-burden populations such as men who have sex with men and estimate both vaccine cost-effectiveness and the incremental cost-effectiveness ratio of vaccination to antimicrobial resistance and treatment costs are warranted. This narrative review examines the current state of gonococcal vaccine research, the possible impact of a gonococcal vaccine on gonorrhea rates based on modeling studies, gaps in the gonococcal vaccine literature, and public health implications of a future gonococcal vaccine on reducing the gonorrhea burden in the United States.

Bacterial surface lipoproteins are emerging as attractive vaccine candidates due to their biological importance and the feasibility of their large-scale production for vaccine manufacturing. The global prevalence of gonorrhea, resistance to antibiotics, and serious consequences to reproductive and neonatal health necessitate development of effective vaccines. Reverse vaccinology identified the surface-displayed L-methionine binding lipoprotein MetQ (NGO2139) and its homolog GNA1946 (NMB1946) as gonococcal and meningococcal vaccine candidates, respectively. Here, we assessed the suitability of MetQ for inclusion in a gonorrhea vaccine by examining MetQ conservation, its function in Neisseria gonorrhoeae (Ng) pathogenesis, and its ability to induce protective immune responses using a female murine model of lower genital tract infection. In-depth bioinformatics, phylogenetics and mapping the most prevalent Ng polymorphic amino acids to the GNA1946 crystal structure revealed remarkable MetQ conservation: ~97% Ng isolates worldwide possess a single MetQ variant. Mice immunized with rMetQ-CpG (n=40), a vaccine containing a tag-free version of MetQ formulated with CpG, exhibited robust, antigen-specific antibody responses in serum and at the vaginal mucosae including secretory IgA. Consistent with the activity of CpG as a Th1-stimulating adjuvant, the serum IgG1/IgG2a ratio of 0.38 indicated a Th1 bias. Combined data from two independent challenge experiments demonstrated that rMetQ-CpG immunized mice cleared infection faster than control animals (vehicle, pp=0.002) and had lower Ng burden (vehicle, p=0.03; CpG, p

Renewed interest in developing vaccines against Neisseria gonorrhoeae has been sparked by the increasing threat of gonococcal antimicrobial resistance (AMR) and growing optimism that gonococcal vaccines are biologically feasible. Evidence suggests serogroup B Neisseria meningitidis vaccines might provide some cross-protection against N. gonorrhoeae, and new gonococcal vaccine candidates based on several approaches are currently in preclinical development. To further stimulate investment and accelerate development of gonococcal vaccines, greater understanding is needed regarding the overall value that gonococcal vaccines might have in addressing public health and societal goals in low-, middle-, and high-income country contexts and how future gonococcal vaccines might be accepted and used, if available. In January 2019, the World Health Organization (WHO) convened a multidisciplinary international group of experts to lay the groundwork for understanding the potential health, economic, and societal value of gonococcal vaccines and their likely acceptance and use, and for developing gonococcal vaccine preferred product characteristics (PPCs). WHO PPCs describe preferences for vaccine attributes that would help optimize vaccine value and use in meeting the global public health need. This paper describes the main discussion points and conclusions from the January 2019 meeting of experts. Participants emphasized the need for vaccines to control N. gonorrhoeae infections with the ultimate goals of preventing adverse sexual and reproductive health outcomes (e.g., infertility) and reducing the impact of gonococcal AMR. Meeting participants also discussed important PPC considerations (e.g., vaccine indications, target populations, and potential immunization strategies) and highlighted crucial research and data needs for guiding the value assessment and PPCs for gonococcal vaccines and advancing gonococcal vaccine development.

The trans-translation pathway for rescuing stalled ribosomes is conserved and essential in bacterial pathogens but has no mammalian homolog, making it an ideal target for new antibiotics. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, resulting in broad-spectrum antibiotic activity. Optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles produced MBX-4132, which cleared multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Cryo-EM studies of non-stop ribosomes showed that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein L27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules.One Sentence SummaryRibosome rescue inhibitors reveal a new conformation of the ribosome and kill drug-resistant Neisseria gonorrhoeae in vivo.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero®) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. High titers of serum IgG1 and IgG2a and vaginal IgG1 that cross-reacted with Ng OMVs were induced by vaccination via either the subcutaneous or intraperitoneal routes, and a 4-fold increase in the serum bactericidal50 titers was detected against the challenge strain. Antibodies from vaccinated mice recognized several surface proteins in a diverse collection of Ng strains, including PilQ, BamA, MtrE, PorB, and Opa, and 4CMenB-induced antibodies bound PilQ and MtrE in native form on the surface of viable bacteria. In contrast, the antibodies were only cross-reactive against lipooligosaccharide species from a few Ng strains. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against Ng and implicate several Ng surface antigens as potentially protective targets. This work also validates the murine infection model as a relevant experimental system for investigating mechanisms of vaccine-mediated protection against gonorrhea.Author summaryOver 78 million Neisseria gonorrhoeae (Ng) infections occur globally each year and control of gonorrhea through vaccination is challenged by a lack of strong evidence that immunity to gonorrhea is possible. This contention was recently challenged by epidemiological evidence suggesting that an outer membrane vesicle (OMV) vaccine from the related species Neisseria meningitidis (Nm) protected humans against gonorrhea. Here we provide experimental evidence in support of this hypothesis by demonstrating that a licensed, modified version of this Nm OMV-based vaccine accelerates clearance of Ng in a mouse infection model. These results confirm the possibility cross-species protection and are important in that they support the biological feasibility of vaccine-induced immunity against gonorrhea. We also showed that several Ng outer membrane proteins are recognized by antisera from vaccinated mice that may be protective targets of the vaccine. Additionally, our demonstration that a vaccine that may reduce the risk of gonorrhea in humans protects mice against Ng, a highly host-restricted pathogen, validates the mouse model as a potentially useful tool for examining mechanisms of protection, which could be exploited in the development of other candidate gonorrhea vaccines.

Antimicrobial resistance in Neisseria gonorrhoeae is a global health problem. Enhanced international collaborative surveillance and disease control are needed to reduce the global burden of this important pathogen. Currently the antimicrobial resistance properties and molecular mechanisms of multidrug-resistant N. gonorrhoeae in the Republic of Georgia represent a significant knowledge gap. Here we report the isolation of a strain of N. gonorrhoeae exhibiting resistance to cefixime and ciprofloxacin with reduced susceptibility to penicillin and tetracycline from a patient being treated at a Georgian medical centre. Notably, this isolate was found to contain a mosaic penA allele and to harbour mutations in genes conferring susceptibility to the β-lactam, cephalosporin, fluoroquinolone, macrolide and penicillin classes of antibiotic. To our knowledge, this is the first report to describe the key mutations conferring the antimicrobial resistance properties of an isolate of N. gonorrhoeae from Georgia. Keywords: Antimicrobial, Georgia, gonorrhoea, mutation, Neisseria, resistance

Gonorrhea, an obligate human infection, is on the rise worldwide and gonococcal strains resistant to many antibiotics are emerging. Appropriate antimicrobial treatment and prevention, including effective vaccines, are urgently needed. To guide investigation, an experimental model of genital tract infection has been developed in female mice to study mechanisms by which Neisseria gonorrhoeae evades host-derived antimicrobial factors and to identify protective and immunosuppressive pathways. Refinements of the animal model have also improved its use as a surrogate host of human infection and accelerated the testing of novel therapeutic and prophylactic compounds against gonococcal infection. Reviewed herein are the (a) history of antibiotic usage and resistance against gonorrhea and the consequences of resistance mechanisms that may increase gonococcal fitness and therefore the potential for spread, (b) use of gonococcal infection in the animal model system to study mechanisms of pathogenesis and host defenses, and (c) current status of vaccine development.

Effective vaccines are urgently needed to combat gonorrhea, a common sexually transmitted bacterial infection, for which treatment options are diminishing due to rapid emergence of antibiotic resistance. We have used a rational approach to the development of gonorrhea vaccines, and genetically engineered nanoparticles to present antigenic peptides of Neisseria gonorrhoeae, the causative agent of gonorrhea. We hypothesized that the ferritin nanocage could be used as a platform to display an ordered array of N. gonorrhoeae antigenic peptides on its surface. MtrE, the outer membrane channel of the highly conserved gonococcal MtrCDE active efflux pump, is an attractive vaccine target due to its importance in protecting N. gonorrhoeae from host innate effectors and antibiotic resistance. Using computational approaches, we designed constructs that expressed chimeric proteins of the Helicobacter pylori ferritin and antigenic peptides that correspond to the two surface-exposed loops of N. gonorrhoeae MtrE. The peptides were inserted at the N terminus or in a surface-exposed ferritin loop between helices αA and αB. Crystal structures of the chimeric proteins revealed that the proteins assembled correctly into a 24-mer nanocage structure. Although the inserted N. gonorrhoeae peptides were disordered, it was clear that they were displayed on the nanocage surface, but with multiple conformations. Our results confirmed that the ferritin nanoparticle is a robust platform to present antigenic peptides and therefore an ideal system for rational design of immunogens.

Background Antimicrobial resistant (AMR) Neisseria gonorrhoeae has emerged as a global public health concern. Widespread resistance to all known antibiotics prescribed for treatment greatly hinders prevention, control, and management of gonorrhea. Misuse of antibiotics and substandard antibiotic quality and dosing have contributed to the threat of untreatable gonorrhea. As a globally deployed force, the U.S. military is vulnerable to diminished operational capability and readiness as this threat looms overhead. Methods The need for rapid, timely, and actionable information for force health protection precipitated the establishment of the Department of Defense (DoD) Gonococcal (GC) Reference Laboratory and Repository in 2011. The aims of the Reference Laboratory are to monitor trends in AMR GC through confirmatory identification, antimicrobial susceptibility testing (AST) and advanced molecular characterization, and to improve N. gonorrhoeae culture capability at international DoD surveillance sites. To our knowledge, this is the only global AMR GC surveillance program led by a military organization. Results Gonococcal surveillance at international DoD sites is conducted at partner military medical centers, private medical clinics, and U.S. government-sponsored care clinics. Laboratories currently submitting isolates include: AFRIMS (Bangkok, Thailand); NAMRU-3 (Accra, Ghana); NAMRU-6 (Lima, Peru); USAMRD-G (Tbilisi, Republic of Georgia); and USAMRD-A (Nairobi, Kenya). To date, 436 presumptive isolates have been shipped, from which 247 isolates were confirmed as N. gonorrhoeae. Resistance is commonly observed in these isolates, particularly with respect to tetracycline, penicillin, and ciprofloxacin. Of greater concern, reduced susceptibility to azithromycin (MIC ≥ 0.125 µg/mL) and cefixime (MIC ≥ 0.06 µg/mL) have been observed. Conclusion The risk of AMR GC and the associated loss of convenient outpatient therapy is of great concern to the military medical community. Comparable data across geographically distinct regions is essential for monitoring AMR GC and implementing appropriate countermeasures in locations where service members are or could be deployed. Disclosure No significant relationships.

Background New antibiotics for gonorrhea are needed due to the emergence of resistance to extended-spectrum cephalosporins in Neisseria gonorrhoeae (Ng). We recently established the 17β-estradiol mouse model of gonococcal lower genital tract infection for testing antibiotics that utilize free time above MIC as the pharmacokinetic (PK) driver to predict efficacy. We further established the mouse model for antibiotic testing by defining the in vivo efficacy of ciprofloxacin (CIP), an antibiotic that uses the free area under the curve over MIC (fAUC/MIC). Methods Lower genital tract infection with Ng strain FA1090 was established in female mice using published methods for two days, after which increasing oral doses of CIP (or controls) were administered (n = 10–20 mice/group) and infection was quantified for 8 days. Plasma drug levels from uninfected mice were measured after administration of similar doses of CIP, and PK parameters (modeled using WinNonlin software) were correlated with observed efficacy. Results Single oral doses ranging from 5 to 60 mg/kg CIP showed significant activity against strain FA1090, with the highest doses (15, 30, and 60 mg/kg) clearing 100% of infections within 8 days; these correspond to predicted fAUC/MICs of 66–264. The 60 mg/kg dose cleared infection in all mice within 48 h, which we defined previously as the endpoint in the model that best correlates with in vivo exposures required for successful CRO/CFX treatment regimens. Conclusion The gonorrhea mouse model shows a dose-dependent response for CIP against a CIPS strain with a dose of 60 mg/kg required to clear infection in 48 hrs. PK modeling suggests that achieving exposures necessary for effective treatment of CIPR strains (mic ≥1 µg/ml) would be challenging. These data that establish PK/PD correlations for CIP - with a fAUC/MIC driver- further strengthens the usefulness of this mouse model to test novel antimicrobial compounds against gonorrhea. Disclosure No significant relationships.

Background The Global Emerging Infectious Surveillance Program of the U.S. Department of Defense, Armed Forces Health Surveillance Branch supports a repository for Neisseria gonorrhoeae (GC) clinical isolates recovered from routine care at U.S. Military Treatment Facilities in the continental US (CONUS) and at several overseas (OCONUS) labs though collaborative surveillance projects. Here we report the use of phenotypic data in conjunction with molecular typing and whole genome sequencing (WGS) of GC to describe the antimicrobial resistance trends from isolates collected from three geographically different clinics in Lima, Callao and Iquitos, Peru. Methods Putative GC collected from patients between 2012 and 2015 were confirmed as GC using standard biochemical and serological methods. Susceptibility to eight different antibiotics was determined by Etest. β-lactamase (sL) activity was determined by nitrocefin hydrolysis. NG-MAST types were determined by standard methods and WGS analysis. Results Sixty eight out of 90 isolates examined were confirmed as GC. Antimicrobial susceptibility testing showed a high level of resistance to ciprofloxacin (70%) and lower percentages of resistant strains to other common antibiotics. Although 63% percent of isolates were β-lactamase positive by the nitrocefin test, only 70% of these isolates were PenR. The other 30% had reduced susceptibility to Pen (PenRS). Whole Genome Sequencing (WGS) revealed mutations in the blaTEM-1B gene for these PenRS isolates. These isolates were collected from different clinics, but showed genetic relatedness based on nucleotide polymorphism (SNP)-based analysis. Several novel NG-MAST types were detected among the isolates. Conclusion These findings highlight the high prevalence of multidrug resistant GC in Peru. The identification of NG-MAST types not identified in surveillance reports from Europe or the United States is important. Further, WGS allowed us to discern false positive β-lactamase isolates by detecting mutations in the blaTEM genes observed in PenRS isolates and showed the clonally relatedness of these isolates. Disclosure No significant relationships.

Background With an incidence rate of 106 million infections a year, Neisseria gonorrhoeae has a significant effect on global morbidity. Rapid development of gonococcal antibiotic resistance, and reports of treatment failures with last-line cephalosporins, has caused the Centers for Disease Control and Prevention to label N. gonorrhoeae as an urgent threat and has sparked renewed interest in development of a gonococcal vaccine. Methods In this study, we immunized mice with detoxified outer membrane vesicles (dOMVs) isolated from the closely-related pathogen Neisseria meningitidis and examined the effect on gonococcal clearance in a murine vaginal colonization model. dOMV vaccines were derived from (1) wild type (WT) bacteria, (2) an isogenic strain (ΔABR) deleted for expression of the major outer membrane proteins PorA, PorB, and RmpM, or (3) an isogenic strain (OCh) deleted for PorA and expressing a varying PorB sequence type relative to the parental strain. ELISAs were used to evaluate anti-dOMV IgG and IgA antibody titers present in sera and vaginal washes. Sera were also used to identify potential gonococcal vaccine antigens using immunoblot and immunoprecipitation experiments. Results Although vaccination with WT dOMVs significantly enhanced gonococcal clearance relative to adjuvant-only controls, vaccination with ΔABR dOMVs resulted in clearance of a higher percentage of mice relative to WT dOMV-vaccinated mice one week post-immunization. Higher levels of clearance in ΔABR dOMV-immunized mice correlated with significantly increased vaginal IgA titers and enhanced immunogenicity of unique meningococcal protein antigens. Conclusion Immunization with meningococcal dOMVs deleted for PorA, PorB, and RmpM promotes gonococcal clearance in a murine model. Deletion of the major porins likely enhances immunogenicity of proteins that are less abundant on the meningococcal surface but exhibit a high degree of homology with corresponding gonococcal proteins, suggesting the potential utility of these dOMVs as a broadly cross-protective Neisseria vaccine. Disclosure No significant relationships.

Mouse models of infection are important tools in the study of infectious disease or host the development of products to prevent or treat infections. The estradiol-treated mouse model of Neisseria gonorrhoeae genital tract infection has proved to be a valuable system for determining the importance of gonococcal factors that mediate evasion of host innate effectors in vivo or host gonococcal adaptation to hormonally driven host factors in females. Examination of mechanisms that Neisseria gonorrhoeae uses to subvert the host immune response also has been greatly aided by this whole model system, as have studies on the consequence of antibiotic resistance mutations on gonococcal fitness in vivo and the search for new antibiotics to treat antibiotic-resistant infections. The strict human specificity of N. gonorrhoeae limits the ability of experimental murine infection to mimic human infection. However, in recent years, the development of transgenic mice and protocols for supplementing mice with human factors has improved animal modeling of gonorrhea. To date, however, because the mouse estrous cycle is much shorter than the human reproductive cycle, all reported gonorrhea mouse models require treatment with estradiol and antibiotics to maintain an estrus-like state and suppress the overgrowth of inhibitory commensal flora that occurs under the influence of estrogen to allow sustained N. gonorrhoeae infection. In this chapter, we detail the methods used to (1) prepare the mice for experimental infection with N. gonorrhoeae, (2) inoculate mice and quantitatively culture vaginal swabs for noncompetitive and competitive infection experiments, and (3) monitor the host innate immune response to infection.

There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESCs strain requiring doses that yielded an fTMIC in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCr strain and were useful in designing effective dosing regimens.

Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

There is an urgent need for the development of an antigonococcal vaccine due to the increasing drug resistance found in this pathogen. The U.S. Centers for Disease Control (CDC) have identified multidrug-resistant gonococci (GC) as among 3 “urgent” hazard-level threats to the U.S. population. In light of this, on 29 to 30 June 2015, the National Institute for Allergy and Infectious Diseases (NIAID) sponsored a workshop entitled “Gonorrhea Vaccines: the Way Forward.” The goal of the workshop was to gather leaders in the field to discuss several key questions on the current status of gonorrhea vaccine research and the path forward to a licensed gonorrhea vaccine. Representatives from academia, industry, U.S. Government agencies, and a state health department were in attendance. This review summarizes each of the 4 scientific sessions and a series of 4 breakout sessions that occurred during the one and a half days of the workshop. Topics raised as high priority for future development included (i) reinvigoration of basic research to understand gonococcal infection and immunity to allow intervention in processes essential for infection; (ii) clinical infection studies to establish parallels and distinctions between in vitro and animal infection models versus natural human genital and pharyngeal infection and to inform in silico modeling of vaccine impact; and (iii) development of an integrated pipeline for preclinical and early clinical evaluation and direct comparisons of potential vaccine antigens and adjuvants and routes of delivery.

Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform—isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry—to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.

In 2014, the World Health Organization, the US National Institutes of Health, and global technical partners published a comprehensive roadmap for development of new vaccines against sexually transmitted infections (STIs). Since its publication, progress has been made in several roadmap activities: obtaining better epidemiologic data to establish the public health rationale for STI vaccines, modeling the theoretical impact of future vaccines, advancing basic science research, defining preferred product characteristics for first-generation vaccines, and encouraging investment in STI vaccine development. This article reviews these overarching roadmap activities, provides updates on research and development of individual vaccines against herpes simplex virus, Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum, and discusses important next steps to advance the global roadmap for STI vaccine development.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development., Author summary Eighty-seven million Neisseria gonorrhoeae (Ng) infections occur globally each year and control of gonorrhea through vaccination is challenged by a lack of strong evidence that immunity to gonorrhea is possible. This contention was recently challenged by epidemiological evidence suggesting that an outer membrane vesicle (OMV) vaccine from the related species Neisseria meningitidis (Nm) protected humans against gonorrhea. Here we provide experimental evidence in support of this hypothesis by demonstrating that a licensed, modified version of this Nm OMV-based vaccine accelerates clearance of Ng in a mouse infection model. These results confirm the possibility cross-species protection and are important in that they support the biological feasibility of vaccine-induced immunity against gonorrhea. We also showed that several Ng outer membrane proteins that may be protective targets of the vaccine are recognized by antiserum from vaccinated mice, at least two of which (PilQ and NHBA) are also detected with sera from 4CMenB-immunized human subjects. Our demonstration that a vaccine that may reduce the risk of gonorrhea in humans protects mice against Ng, a highly host-restricted pathogen, also validates the mouse model as a useful tool for guiding the development of other candidate gonorrhea vaccines.

The mucosa is colonized with commensal Neisseria. Some of these niches are sites of infection for the STD pathogen Neisseria gonorrhoeae (Ngo). Given the antagonistic behavior of commensal bacteria toward their pathogenic relatives, we hypothesized that commensal Neisseria may negatively affect Ngo colonization. Here, we report that commensal species of Neisseria kill Ngo through a mechanism based on genetic competence and DNA methylation state. Specifically, commensal-triggered killing occurs when the pathogen takes up commensal DNA containing a methylation pattern that it does not recognize. Indeed, any DNA will kill Ngo if it can enter the cell, is differentially methylated, and has homology to the pathogen genome. Consistent with these findings, commensal Neisseria elongata accelerates Ngo clearance from the mouse in a DNA-uptake-dependent manner. Collectively, we propose that commensal Neisseria antagonizes Ngo infection through a DNA-mediated mechanism and that DNA is a potential microbicide against this highly drug-resistant pathogen.

Mouse models of infection are important tools in the study of infectious disease or host the development of products to prevent or treat infections. The estradiol-treated mouse model of Neisseria gonorrhoeae genital tract infection has proved to be a valuable system for determining the importance of gonococcal factors that mediate evasion of host innate effectors in vivo or host gonococcal adaptation to hormonally driven host factors in females. Examination of mechanisms that Neisseria gonorrhoeae uses to subvert the host immune response also has been greatly aided by this whole model system, as have studies on the consequence of antibiotic resistance mutations on gonococcal fitness in vivo and the search for new antibiotics to treat antibiotic-resistant infections. The strict human specificity of N. gonorrhoeae limits the ability of experimental murine infection to mimic human infection. However, in recent years, the development of transgenic mice and protocols for supplementing mice with human factors has improved animal modeling of gonorrhea. To date, however, because the mouse estrous cycle is much shorter than the human reproductive cycle, all reported gonorrhea mouse models require treatment with estradiol and antibiotics to maintain an estrus-like state and suppress the overgrowth of inhibitory commensal flora that occurs under the influence of estrogen to allow sustained N. gonorrhoeae infection. In this chapter, we detail the methods used to (1) prepare the mice for experimental infection with N. gonorrhoeae, (2) inoculate mice and quantitatively culture vaginal swabs for noncompetitive and competitive infection experiments, and (3) monitor the host innate immune response to infection.

Introduction Sexually transmitted infections (STIs) continue to plague militaries and defense forces. While the historical recognition of the impact of STIs on operations is evident, contemporary surveillance and research activities are limited. As Neisseria gonorrhoeae and other sexually transmitted pathogens become increasingly resistant to antibiotics, the role of the Department of Defense (DoD) in disease surveillance and clinical research is essential to military Force Health Protection. Methods The Infectious Disease Clinical Research Program (IDCRP) of the Uniformed Services University of the Health Sciences partnered with the DoD Global Emerging Infections Surveillance (GEIS) program to monitor the distribution of gonorrhea antimicrobial resistance (AMR) both domestically and abroad. The DoD gonococcal reference laboratory and repository was established in 2011 as a resource for confirmatory testing and advanced characterization of isolates collected from sites across the continental United States (CONUS) and GEIS-funded sites outside the continental United States (OCONUS). The IDCRP is currently implementing surveillance efforts at CONUS military clinics, including Madigan Army Medical Center, Naval Medical Center Camp Lejeune, Naval Medical Center Portsmouth, Naval Medical Center San Diego, and San Antonio Military Medical Center (efforts were also previously at Womack Army Medical Center). The reference laboratory and repository receives specimens from OCONUS collaborators, including Armed Forces Research Institute of Medical Sciences (AFRIMS; Bangkok, Thailand), Naval Medical Research Unit No. 3 (NAMRU-3), Ghana Detachment (Accra, Ghana), Naval Medical Research Unit No. 6 (NAMRU-6; Lima, Peru), U.S. Army Medical Research Unit – Georgia (USAMRD-G; Tbilisi, Republic of Georgia), and U.S. Army Medical Research Directorate – Kenya (USAMRD-K; Nairobi, Kenya). The gonococcal surveillance program, to include findings, as well as associated clinical research efforts are described. Results Among N. gonorrhoeae isolates tested within the United States, 8% were resistant to tetracycline, 2% were resistant to penicillin, and 30% were resistant to ciprofloxacin. To date, only one of the 61 isolates has demonstrated some resistance (MIC=1 μg/ml) to azithromycin. No resistance to cephalosporins has been detected; however, reduced susceptibility (MIC=0.06–0.125 μg/ml) has been observed in 13% of isolates. Resistance is commonly observed in N. gonorrhoeae isolates submitted from OCONUS clinical sites, particularly with respect to tetracycline, penicillin, and ciprofloxacin. While no azithromycin-resistant isolates have been identified from OCONUS sites, reduced susceptibility (MIC=0.125–0.5 μg/ml) to azithromycin was observed in 23% of isolates. Conclusion Continued monitoring of circulating resistance patterns on a global scale is critical for ensuring appropriate treatments are prescribed for service members that may be infected in the U.S. or while deployed. Domestic surveillance for gonococcal AMR within the Military Health System has indicated that resistance patterns, while variable, are not dramatically different from what is seen in U.S. civilian data. Global patterns of gonococcal AMR have been described through the establishment of a central DoD gonococcal reference laboratory and repository. This repository of global isolates provides a platform for further research and development into biomedical countermeasures against gonococcal infections.

The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA and wild type bacteria revealed enrichment of TadA–which recodes proteins through mRNA editing–as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence., Author summary The Gram-negative outer membrane is a formidable barrier, primarily because of its asymmetric composition. A layer of lipopolysaccharide is exposed to the external environment and phospholipids are on the internal face of the outer membrane. MlaA is part of a bacterial system that prevents phospholipid accumulation within the lipopolysaccharide layer. If MlaA is removed, membrane asymmetry is disrupted and bacteria become more vulnerable to certain antimicrobials. Neisseria gonorrhoeae causes millions of infections worldwide annually. A growing number are resistant to available antibiotics. Improving our understanding of gonococcal pathogenicity and basic biological processes is required to facilitate the discovery of new weapons against gonorrhea. We investigated the role of MlaA in N. gonorrhoeae and found that when MlaA was absent, bacteria were more sensitive to antibiotics and human defensins. However, the mutant bacteria produced more membrane vesicles–packages of proteins wrapped in membrane material. Mutant vesicles and cell envelopes were enriched in proteins that contribute to disease. These alterations significantly increased mutant fitness during experimental infection of the female mouse genital tract. Our results provide new insights into the processes N. gonorrhoeae uses to fine-tune its ability to stay fit in the hostile environment of the genital tract.

There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESC(s)) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fT(MIC)]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESC(r)) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESC(s) strain requiring doses that yielded an fT(MIC) in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESC(r) strain and were useful in designing effective dosing regimens.