Neisseria gonorrhoeae vaccines: a contemporary overview

SUMMARY

Neisseria gonorrhoeae infection is an important public health issue, with an annual global incidence of 87 million. N. gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health and may rarely cause life-threatening disease. Global rates of N. gonorrhoeae infection have increased over the past 20 years. Importantly, rates of antimicrobial resistance to key antimicrobials also continue to increase, with the United States Centers for Disease Control and Prevention identifying drug-resistant N. gonorrhoeae as an urgent threat to public health. This review summarizes the current evidence for N. gonorrhoeae vaccines, including historical clinical trials, key N. gonorrhoeae vaccine preclinical studies, and studies of the impact of Neisseria meningitidis vaccines on N. gonorrhoeae infection. A comprehensive survey of potential vaccine antigens, including those identified through traditional vaccine immunogenicity approaches, as well as those identified using more contemporary reverse vaccinology approaches, are also described. Finally, the potential epidemiological impacts of a N. gonorrhoeae vaccine and research priorities for further vaccine development are described.

INTRODUCTION

Epidemiology and clinical manifestations of Neisseria gonorrhoeae infection

Infection with Neisseria gonorrhoeae is an important public health issue, with an estimated annual global incidence of 87 million (1). Reported global rates of N. gonorrhoeae infection have significantly increased over the past 20 years (1, 2). In the United States (US), rates of N. gonorrhoeae infection increased by 111% between 2009 and 2020 (3); in Europe, rates increased by 218% between 2009 and 2018 (4); while in Australia, rates increased by 127% between 2012 and 2019 (5). N. gonorrhoeae infection disproportionately affects vulnerable populations, with over 90% of cases occurring in low- and middle-income (LMIC) settings (1). Within high-income countries, N. gonorrhoeae infection is more prevalent in certain populations, including men who have sex with men (MSM) (6, 7), transgender persons, sex workers, racial/ethnic minorities, and indigenous populations (8).

N. gonorrhoeae infection causes a wide range of diseases, including symptomatic urogenital disease, asymptomatic mucosal infection, and infrequently, disseminated gonococcal infection (9). Urogenital infection most commonly manifests as lower genital tract infection, usually presenting as purulent anterior urethritis in men, and as cervicitis in women (10). Up to 40% of cases of urogenital N. gonorrhoeae infections in women are asymptomatic (11, 12). If urogenital infection is not diagnosed and treated early, severe sequelae can ensue. In women, infection can ascend to the upper genital tract to cause salpingitis and pelvic inflammatory disease. Tubal infection can result in ectopic pregnancy and infertility, and infection during pregnancy is associated with preterm birth and low birthweight (9, 10, 13). Neonatal infection most commonly presents as ophthalmia neonatorum, purulent conjunctivitis that may result in blindness (14). In men, ascending infection can cause epididymitis, and untreated infection may result in male infertility and urethral strictures (15, 16). Extragenital mucosal infections in the oropharynx, rectum, and conjunctiva also occur. Oropharyngeal and rectal N. gonorrhoeae infections are more prevalent than urethral infections in certain high-risk populations, such as MSM in high-income settings, where regular asymptomatic screening with nucleic acid amplification testing (NAAT) at multiple anatomical sites is recommended (17). While infections of the oropharynx and rectum are often asymptomatic (18), they may represent a significant reservoir for N. gonorrhoeae transmission (19). Manifestations of disseminated gonococcal infection include purulent arthritis, tenosynovitis, dermatitis, polyarthritis, and osteomyelitis. Rare life-threatening complications of N. gonorrhoeae infection include meningitis and endocarditis (20). N. gonorrhoeae infection also promotes the transmission and susceptibility to human immunodeficiency virus (HIV) by causing local inflammation (21).

Importantly, resistance to all prior and currently recommended antimicrobials for the treatment of N. gonorrhoeae has been described (22). N. gonorrhoeae has the ability to develop antimicrobial resistance (AMR) through numerous mechanisms (22). Consequently, the World Health Organization (WHO) and the US Centers for Disease Control and Prevention (CDC) have identified antimicrobial-resistant N. gonorrhoeae as an urgent threat to public health (23, 24). N. gonorrhoeae has therefore been classified as a high-priority pathogen on the WHO Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery and Development of New Antibiotics (23). The first case of treatment failure due to an extensively drug-resistant (XDR) N. gonorrhoeae strain (resistant to both current first-line antimicrobials, ceftriaxone and azithromycin) was reported in the United Kingdom (UK) in 2016 (25); XDR N. gonorrhoeae with high-level resistance to both ceftriaxone and azithromycin has now been reported in the UK and Australia (26, 27). These cases demonstrate the growing global threat of untreatable N. gonorrhoeae infection. A number of novel gonococcal antimicrobial therapies have recently been tested in phase two and three trials, including solithromycin, zoliflodacin, and gepotidacin. These studies have demonstrated several limitations of these new anti-gonococcal antimicrobials (28); in brief, a randomized trial found solithromycin to be inferior to standard-of-care dual ceftriaxone and azithromycin therapy (29); the efficacy of zoliflodacin was suboptimal for pharyngeal infection (30); and current data on the performance of gepotidacin for extragenital infections are sparse (31). As the specter of untreatable N. gonorrhoeae infection looms, preventative strategies that overcome the extraordinary ability of N. gonorrhoeae to evade killing by antimicrobial therapy are therefore urgently required.

The need for a Neisseria gonorrhoeae vaccine

An effective and accessible N. gonorrhoeae vaccine could have a wide range of benefits, including (i) reduction of the individual and healthcare impact of urogenital infection; (ii) improvement in reproductive and neonatal health; (iii) reduction of individual and population antimicrobial usage and the unintended consequences arising from this, including the potential to drive further N. gonorrhoeae antimicrobial resistance; and (iv) reduction in the healthcare costs associated with frequent screening for N. gonorrhoeae infection in asymptomatic individuals. However, there are multiple significant barriers to the development of a N. gonorrhoeae vaccine, including (i) antigenic and phase variation of potential vaccine targets; (ii) the absence of protective immunity following natural infection; (iii) the lack of a known immune correlate of protection; and (iv) exclusive human host restriction, with limited appropriate animal models of infection (32). Encouragingly, the successes of vaccines for other sexually transmitted infections (STIs) such as human papillomavirus (HPV), hepatitis A virus (HAV), and hepatitis B virus (HBV) (33), as well as closely related pathogens, such as Neisseria meningitidis, have paved the way for further progress in N. gonorrhoeae vaccine development (34).

The development and implementation of safe and efficacious vaccines for HPV, HAV, and HBV have had a significant impact on the incidence and resulting complications of these diseases (33). These successes have provided additional motivation for the development of new STI vaccines. In 2014, the WHO and National Institutes of Health (NIH) announced a comprehensive roadmap to accelerate STI vaccine development (35). This roadmap comprised nine areas of focus, including obtaining improved epidemiological data, modeling vaccine impact, accelerating basic science research, outlining preferred product characteristics, and encouraging investment (35, 36). The WHO subsequently assembled a panel of international experts to define the potential public health value of and preferred product characteristics of a N. gonorrhoeae vaccine to inform vaccine development (37, 38). The WHO Global Health Sector Strategy on STIs has set a target of a 90% reduction in worldwide N. gonorrhoeae infection incidence by 2030. Given the rising incidence of N. gonorrhoeae infection worldwide and the limitations of current preventative interventions, this WHO strategy highlights N. gonorrhoeae vaccine development as a priority innovation to support this ambitious aim (39).

In this review, we examine the evidence for a N. gonorrhoeae vaccine, including (i) historical clinical trials; (ii) key N. gonorrhoeae vaccine preclinical studies; (iii) observational and randomized studies of the impact of N. meningitidis vaccines on N. gonorrhoeae infection; and (iv) clinical trials currently underway. In addition, we present a comprehensive survey of potential vaccine antigens, including those identified through traditional vaccine immunogenicity approaches, as well as those identified using more contemporary approaches, such as bioinformatics, transcriptomics, and proteomics. Finally, we review the potential epidemiological impacts of a N. gonorrhoeae vaccine and outline research priorities for N. gonorrhoeae vaccine development.

References for this review were identified on the basis of the topics described above, with the literature search conducted through PubMed and ClinicalTrials.gov. The websites of the WHO and US CDC were also reviewed, and an online search engine was used to access press releases, conference abstracts, and commercial information. Search terms included, “gonorrhoea,*” “gonococcal,” “Neisseria,” “vaccine,” “antigen,” “meningococcal,” “outer membrane vesicle,” “OMV,” “model,*” “impact,” “cost,” and “economic.” In addition, a search was undertaken for each vaccine antigen listed in column 1 of Table 2. Relevant articles published between 1 January 1900 and 1 March 2023 were included. Articles published in English resulting from these searches and their relevant references were reviewed.

NEISSERIA GONORRHOEAE VACCINE CHALLENGES

A number of obstacles have impeded progress toward the development of an effective vaccine against N. gonorrhoeae (32). First, N. gonorrhoeae demonstrates significant surface antigen variability, such that key surface antigens have variable genomic sequences and protein composition (antigenic variation) and/or change their protein expression (through phase variation). Second, there is no epidemiologic evidence that N. gonorrhoeae infection results in protective immunity against recurrent infections; indeed, repeated infections are relatively common in high-risk populations. Third, given the lack of protective immunity against reinfection, it has not been possible to define correlates of immunity that can be measured using immunologic methods (32).

As an exclusive human pathogen, the establishment of an appropriate animal infection model to study the pathogenesis and preclinical immune responses to N. gonorrhoeae infection and vaccines has been difficult. A 17-β-estradiol-treated mouse model (40, 41), using inbred mice and recently modified by the use of transgenic mice with additional human host-cell receptors such as human carcinoembryonic antigen cellular adhesion molecules (42, 43) and human transferrin (44) or supplementation of inbred mice with human transferrin (45, 46), has partially overcome this host-specific barrier. Although chimpanzees were also used in early infection models (47), they are no longer available or ethically appropriate for this work. A number of experimental systems have been used to assist drug development; however, these models are not appropriate for vaccine development. These include a hollow fiber infection model that is well suited to characterize the pharmacodynamic and pharmacokinetic responses of novel antimicrobials for the treatment of N. gonorrhoeae (48, 49), and an invertebrate Galleria mellonella greater wax moth of gonococcal infection (50). These latter models, however, lack the essential host immunity components required to test gonococcal vaccines.

A N. gonorrhoeae male urethritis-controlled human infection model (CHIM) was developed by investigators at Walter Reed Army Institute of Research and the University of North Carolina at Chapel Hill in the United States in the 1980s (51). Over 200 individuals have participated in N. gonorrhoeae urethritis CHIM studies. These studies have been reviewed for safety and compliance with modern ethical standards and have been undertaken without serious or unexpected adverse events (51). There are a number of advantages to a N. gonorrhoeae CHIM, compared to alternative study designs. In particular, compared to animal studies, the model not only assesses microbiological outcomes but also clinical disease and immune responses. In addition, CHIM studies provide a model that has the power to test for statistically significant vaccine efficacy in a much smaller study population (<100 participants) compared to an efficacy trial conducted in a population with a high risk for gonorrhoea infection (>1,000 participants) (52). Notably, the only N. gonorrhoeae CHIM that is currently available is a male urethritis model, which could limit the generalizability of vaccine efficacy findings to oropharyngeal, rectal, and cervical N. gonorrhoeae infections (52). N. gonorrhoeae male urethritis CHIM studies have already advanced the understanding of the complex pathogenesis and immune responses to N. gonorrhoeae infection (51). As promising vaccine candidates become available, N. gonorrhoeae CHIM studies may offer a safe and effective model for testing these novel vaccines, particularly if models of extragenital infection, such as an oropharyngeal N. gonorrhoeae CHIM become available (52).

Although these obstacles may have slowed the progress of N. gonorrhoeae vaccines, evidence suggesting partial effectiveness of the N. meningitidis serogroup B outer membrane vesicle (OMV) vaccines against N. gonorrhoeae infection (34, 53–62) has reinvigorated the field, with an increased international focus on the development of an effective N. gonorrhoeae vaccine.

HISTORICAL NEISSERIA GONORRHOEAE VACCINE STUDIES

The aim of developing a gonococcal vaccine has been pursued since the turn of the 20th century. Initially, these vaccines were designed as a therapeutic strategy for persistent N. gonorrhoeae infection, rather than as a preventative measure. At this time, there were numerous attempts made by different groups to immunize patients with symptomatic gonorrhoea with various whole-cell vaccines to promote opsonophagocytosis (63). With the development of effective antimicrobial therapy, therapeutic vaccine discovery stalled. Further efforts were made in the 1970s and 1980s when three different preventative N. gonorrhoeae vaccines were developed and trialed in humans, all of which were unsuccessful (64–66). These vaccine studies are described in Table 1. The first vaccine, a partially inactivated whole-cell vaccine prepared from two pooled N. gonorrhoeae strains, elicited specific antibody responses among the majority of the 54 participants included in the initial phase I study (67). A subsequent placebo-controlled double-blind field trial of this vaccine was undertaken in 1972–1973, this time using whole-cell preparations from three pooled N. gonorrhoeae strains. This study involved 62 participants from an Aboriginal Inuit population in the northern Canadian village of Inuvik, with the immunization schedule comprising three 1 mL intramuscular injections of vaccine or placebo at weekly intervals. No significant difference between the groups in the cumulative incidence of laboratory-confirmed N. gonorrhoeae infection was observed over the 12-month period following vaccination (30% incidence in the vaccinated group vs 24% in the placebo group; P = 0.78) (64).

TABLE 1.

Historical Neisseria gonorrhoeae vaccine trials in humans^a

Clinical trial design	Vaccine	Immunization schedule	Study population	Result	Reference
Randomized double-blind placebo-controlled trial	Inactivated whole-cell vaccine prepared from three strains of N. gonorrhoeae	1 mL dose of intramuscular immunization 3 times at 1-week interval	62 participants were recruited from an indigenous population of Inuit in northern Canada (background yearly N. gonorrhoeae infection incidence of 25%)	Cumulative infection rate of 30% in immunized participants compared to 24% in placebo in the 12-month follow-up period following immunization (ns)	(64)
Randomized double-blind placebo-controlled trial	Single-antigen pilus protein vaccine prepared from a single strain of N. gonorrhoeae	0.1 mL dose of intradermal immunization 2 times at 2-week interval	3,250 US military personnel stationed in Korea (96% men; 39% with the self-reported history of prior N. gonorrhoeae infection)	The cumulative infection rate of 6.9% in immunized participants compared to 6.5% in placebo in an 8-week follow-up period following immunization (ns)	(65)
Placebo-controlled human challenge trial	Outer membranes vaccine prepared from a single strain of N. gonorrhoeae	Participants were vaccinated (dosing schedule not available) and then inoculated with homologous N. gonorrhoeae strain per urethra 2–4 weeks later	63 male participants	Post-challenge infection rate of 54% in immunized participants compared to 64% in placebo (ns)	(66)

^a ns, not significant; US, United States.

The second vaccine, a N. gonorrhoeae pilus vaccine, elicited serum and genital anti-pilus antibody responses against heterologous strains and demonstrated efficacy in an initial N. gonorrhoeae urethral CHIM study after challenge with a homologous strain (68, 69). This was followed by a placebo-controlled, double-blind trial in Korea in the 1980s, involving 3,250 high-risk US military personnel, using an immunization schedule comprising two 0.1 mL intradermal injections of vaccine or placebo, 2 weeks apart. There was no significant difference in cumulative incidence of laboratory-confirmed N. gonorrhoeae infection in the 8-week period following vaccination between the two groups, with a cumulative incidence of 6.9% observed in the vaccinated group, compared with 6.5% in the placebo group (65). In a subsequent N. gonorrhoeae urethral CHIM study, no protection was observed against a heterologous N. gonorrhoeae strain expressing antigenically different pili (70), suggesting that despite the production of anti-pilus antibody responses against heterologous pili, these responses were insufficient to prevent infection with N. gonorrhoeae strains expressing antigenically different pili. Pilus antigen heterogeneity, a characteristic of circulating strains of N. gonorrhoeae, was the most likely explanation for the unsuccessful field trial.

The most recent N. gonorrhoeae vaccine to be trialed in humans was a N. gonorrhoeae outer membrane vaccine prepared from a single strain. In a randomized placebo-controlled N. gonorrhoeae urethral CHIM undertaken in 1985, 63 male participants received a single dose of intramuscular vaccine or placebo and underwent intraurethral challenge with a homologous N. gonorrhoeae strain 2–4 weeks later. No significant difference in infection was observed between the two groups. Infection rates were unexpectedly low in this study, with 46% of those vaccinated and 36% of placebo recipients remaining uninfected (66). Although designed to enrich the Porin (Por) outer membrane protein, this vaccine was contaminated with other membrane antigens, including lipooligosaccharide (LOS) and reduction modifiable protein (Rmp). Later studies demonstrated that anti-Rmp antibodies downregulate the bactericidal activity of antibodies against other antigens (71). The contamination of this vaccine by Rmp therefore likely resulted in anti-Rmp antibodies that may have antagonized the bactericidal effect of anti-Por and anti-LOS antibodies. This hypothesis was supported by a retrospective analysis of the vaccine trial data which incorporated data on risk for pre-existing immunity. This analysis demonstrated that the ratio of Por and LOS antibody concentration to Rmp antibody concentration correlated with protection from N. gonorrhoeae infection in both vaccine and placebo recipients (66).

These early studies demonstrate three key considerations for future gonococcal vaccine trials. First, CHIM trials that are appropriately designed to test investigational vaccines may serve as a go-no-go measure using a relatively small number of participants before more resource-intensive, larger-scale efficacy trials are undertaken. Second, pre-existing immunity should be incorporated into the design and analysis of future gonococcal vaccine trials by documenting baseline antibody levels and previous exposure. Finally, the heterogeneity of circulating N. gonorrhoeae strains must be considered both in the selection of potential vaccine antigens and the selection of challenge strains for future gonococcal CHIM vaccine trials.

POTENTIAL VACCINE TARGETS FOR NEISSERIA GONORRHOEAE VACCINES

Neisseria gonorrhoeae vaccine antigens

A number of potential N. gonorrhoeae vaccine candidates have been evaluated in preclinical testing including in vitro, in animal models, and occasionally, early phase human studies. Key features of an ideal N. gonorrhoeae vaccine antigen include (i) surface exposure; (ii) conservation (lack of phase or antigenic variation); (iii) high prevalence among globally circulating strains; (iv) immunogenicity; and (v) evidence that the antigen plays an important role in virulence or survival. In the absence of known immune correlates of protection against N. gonorrhoeae infection, a widely used approach has been to identify surface antigens that elicit an antibody response that confers complement-dependent bactericidal activity, and/or mediates opsonophagocytosis (72), hypothesizing that these may be surrogate predictors of prevention. However, antibody responses to natural uncomplicated N. gonorrhoeae infection are typically described as weak and short-lived (32). In addition, in early vaccine studies where the pilus, Por, and LOS antigens (described above) were evaluated, no correlates of protective immunity were apparent. The bactericidal and opsonophagocytic activity of antibodies induced by natural reinfection is influenced by a number of factors, including downregulation by blocking antibodies (e.g., anti-Rmp antibodies) (71) and soluble complement regulators (e.g., Factor H and C4b-binding protein) (66), as well as poor cross-protection of antibodies to polymorphic antigens (e.g., pilus and Por) (65, 71). Given the complex humoral immune responses to N. gonorrhoeae infection and the lack of protective immunity induced by natural gonococcal infection (32), an optimal N. gonorrhoeae vaccine will need to induce immune responses that are qualitatively and quantitatively different from that induced by natural immunity.

A number of novel strategies have informed the contemporary approach to gonococcal vaccine antigen discovery. Reverse vaccinology is a process of vaccine antigen discovery that harnesses genomics, proteomics, immunoproteomics, transcriptomics, and bioinformatics to identify highly conserved, widely distributed, and surface-exposed antigens that may represent promising vaccine antigens. A reverse vaccinology approach has been used to develop highly successful vaccines for other pathogens, such as N. meningitidis serogroup B (4CMenB; GlaxoSmithKline) (73). Identification of novel surface-exposed antigens of N. gonorrhoeae has used proteomic techniques to characterize N. gonorrhoeae membrane vesicle and cell envelope proteins (74). Such an approach can be coupled with a range of bioinformatic tools to predict function, subcellular localization, post-translational modification, and immunogenicity (74).

Using a proteomic-based approach, Zielke et al. identified 305 cell envelope and 46 membrane vesicle proteins that were uniformly present among four well-characterized N. gonorrhoeae strains, many of which were newly discovered proteins or proteins that had not previously been characterized in N. gonorrhoeae (75). Using such proteomic approaches, it has been possible to identify candidate vaccine antigens with a range of attractive characteristics, such as expression in physiologically relevant environmental conditions, including both aerobic and anaerobic, iron deprivation, exposure to normal human serum, and exposure to extended-spectrum cephalosporins (76–78). Analysis of the genes expressed during natural human mucosal infection, coupled with immune characterization, has also led to the discovery of a number of novel putative vaccine antigen targets (79). The availability of public genomic databases, such as Neisseria PubMLST, has also enabled the assessment of the presence and conservation of putative vaccine antigens across globally diverse strains (80).

Another strategy to improve vaccine discovery efforts for N. gonorrhoeae has been to target antigens that not only elicit an antibody response with bactericidal and opsonophagocytic activity but also those that elicit a functional antibody response that inhibits important physiological functions in the pathogenesis of gonococcal infection (81–83). These physiological functions include (i) adherence to and invasion of mucosal epithelial cells; (ii) nutrient acquisition and metabolism; (iii) immune evasion; (iv) intracellular survival; and (iv) protection from oxidative stress or antimicrobial substances. Promising vaccine antigens from each of these categories will be briefly highlighted below, and a comprehensive summary of potential N. gonorrhoeae vaccine antigens is presented in Table 2.

TABLE 2.

Potential Neisseria gonorrhoeae vaccine antigens discovered by traditional or reverse vaccinology approaches

Gene	Protein/ antigen name	Function	Location	Conservationa	Immunogenicity	Data	Reference
Adherence and invasion of epithelial cells
Phospholipase
pldA	Outer membrane phospho-lipase A (OMPLA)	Phospholipid hydrolysis of endogenous phospholipids. Autolysin	Outer membrane	Highly conserved	Murine antibodies elicited by N. meningitidis homolog are not bactericidal or protective against infection	Preclinical	(84–86)
PLD	Neisseria gonorrhoeae phospho-lipase D (NgPLD)	Regulator of gonococcal invasion of and survival within cervical epithelia	Outer membrane	Highly conserved	Antibodies decrease adherence to and invasion of primary cervical cells	Preclinical	(87–89)
Pilin
pilE	Major subunit of the type 4 pilus	Type 4 pilus fiber. Channel for pilus extrusion. Mediates adherence to epithelial cells	Outer membrane	Antigenically variable. Conserved at the C terminus	Antibodies to pili block cell attachment but are directed at variable epitopes	Historical vaccine trial	(65, 70, 90–95)
pilC	PilC	Type 4 pilus tip-associated adhesin. Plays a key role in pilus biogenesis and adhesion	Outer membrane	Antigenically variable. Phase variable	No data	Preclinical	(96, 97)
pilQ	PilQ	Outer membrane channel for pilus extrusion. The essential role in pilus biogenesis	Outer membrane	Antigenically variable. Conserved at the C terminus	Antibodies elicited by N. meningitidis homologs are bactericidal	Preclinical	(98–100)
Porin
porB	Porin	Major outer membrane protein. Nutrient channel. Binds complement factors C4bp and Factor H to down-regulate complement activation at the gonococcal surface. Suppresses neutrophil oxidative burst and neutrophil apoptosis	Outer membrane	Antigenically variable surface loops and conserved membrane-spanning regions	Antibodies are bactericidal, opsonophagocytotic, and block gonococcal entry into epithelial cells	Preclinical	(101–114)
Other outer membrane proteins
opa	Opacity proteins	Adherence and invasion of host cells Influence innate and adaptive immune responses by binding CEACAM receptors on T and B lymphocytes	Outer membrane	Antigenically variable. Phase variable	Antibodies are bactericidal	Preclinical and controlled human challenge studies	(115–121)
opcA	OpcA	Adhesion and invasion of host epithelial and endothelial cells	Outer membrane	Antigenically variable	Antibodies elicited by N. meningitidis homologs are bactericidal	Preclinical	(122–124)
ompA	Outer membrane protein A (OmpA)	Adhesion and invasion of host epithelial and endothelial cells	Outer membrane	Highly conserved	No data	Preclinical	(125, 126)
nhba	Neisseria heparin binding antigen (NHBA)	Involved in adherence to epithelial cells and serum survival	Outer membrane	Highly conserved	Antibodies are bactericidal, opsonophagocytotic, and block gonococcal adherence to epithelial cells	Preclinica	(127–130)
Nutrient acquisition and metabolism
Iron metabolism
tbpA	Transferrin-binding protein A (TbpA)	Essential receptor for iron uptake from transferrin	Outer membrane	Highly conserved	Antibodies are bactericidal	Preclinical and controlled human challenge studies	(51, 81, 131–135)
tbpB	Transferrin-binding protein B (TbpB)	Increases efficiency of iron uptake from transferrin	Outer membrane	Antigenically variable with conserved segments	Antibodies are bactericidal	Preclinical and controlled human challenge studies	(51, 81, 131, 133–137)
lbpA	Lactoferrin-binding protein A (LbpA)	Essential receptor for iron uptake from lactoferrin	Outer membrane	Highly conserved. Present in approximately half of the isolates.	Antibodies elicited by N. meningitidis homologs are bactericidal but cross-reactivity (in N. meningitidis) is limited	Preclinical and controlled human challenge studies	(138–143)
lbpB	Lactoferrin-binding protein B (LbpB)	Increases the efficiency of iron transport from lactoferrin	Outer membrane	Antigenically variable with conserved segments. Phase variable. Present in approximately half of the isolates	Antibodies elicited by N. meningitidis homologs are bactericidal but cross-reactivity (in N. meningitidis) is limited	Preclinical and controlled human challenge studies	(138–143)
fetA	Ferric entero-bactin transporter A (FetA)	Involved in iron uptake through scavenging siderophores from other bacteria via binding and transport of ferric enterobactin	Outer membrane	Antigenically variable. Phase variable	Antibodies elicited by N. meningitidis homologs are bactericidal but cross-reactivity (in N. meningitidis) is limited	Preclinical	(144–149)
fetB	Ferric entero-bactin transporter B (FetB)	Involved in iron uptake through scavenging siderophores from other bacteria via binding and transport of ferric enterobactin	Outer membrane	Antigenically variable	No data	Preclinical	(80)
Zinc metabolism
tdfJ	TonB-dependent family J (TdfJ)	Facilitates uptake of zinc via human protein S100A7	Outer membrane	Highly conserved	Antibodies elicited by N. meningitidis homolog are bactericidal	Preclinical	(150–152)
tdfH	TonB-dependent family H (TdfH)	Facilitates uptake of zinc via human calprotectin	Outer membrane	Highly conserved	No data	Preclinical	(153–155)
Anaerobic metabolism
aniA	Anaerobically induced protein A (AniA)	Inducible nitrite reductase, required for anaerobic growth and biofilm formation	Outer membrane	Highly conserved	Antibodies block nitrite reductase activity	Preclinical	(156–162)
Immune evasion
lst	Alpha-2,3-sialy-transferase (Lst)	Sialylates the surface lipooligo-saccharide to protect gonococci from complement-mediated killing and phagocytic killing by neutrophils. Incorporates keto-deoxyoctanoate (KDO) as the terminal glycan on the LOS	Cytoplasm (previously thought to be outer membrane)	Highly conserved	Antibodies partially inhibit sialyltransferase activity of N. gonorrhoeae; however, this is inhibited in the presence of exogenous 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) present in N. gonorrhoeae strains. KDO-specific monoclonal antibody 6E4 is opsonophagocytic.	Preclinical	(163–168)
nspA	Neisserial surface protein A (NspA)	Subverts complement pathway activation by binding to complement inhibitor factor H	Outer membrane	Highly conserved	Antibodies are bactericidal and opsonophagocytic	Preclinical	(169–171)
Intracellular survival
lgtG	Lipooligo-saccharide (LOS) epitope 2C7	Inner glycose core of LOS. Promotes colonization and survival	Outer membrane	High antigenic conservation. Phase variable.	Antibodies are bactericidal and opsonophagocytotic	Preclinical	(172–180)
iga	IgA1-specific protease (IgA1)	Promotes intracellular survival and release of inflammatory cytokines	Outer membrane	Highly conserved. Present in approximately 50% of isolates	No data	Preclinical	(181–186)
mip	Macro-phage Infectivity Potentiator (MIP) lipoprotein	Bacterial persistence within macrophages and protects Neisseria gonorrhoeae from bactericidal activity of immune effector cells	Outer membrane	Highly conserved	Antibodies are bactericidal	Preclinical	(187–189)
Oxidative stress and antimicrobial substance protection
msrA/B	Methionine sulfoxide reductase (MsrA/B)	Protects from oxidative stress by reducing methionine sulfoxide to methionine	Outer membrane	Highly conserved	Antibodies are bactericidal, opsonophagocytic, and functionally block the activity of MsrA/B by binding to its substrate, methionine sulfoxide	Preclinical	(83)
mtrE	Multiple transferable resistance protein E (MtrE)	Surface-exposed channel of the MtrCDE and FarAB-MtrE efflux pumps that export antimicrobial substances	Outer membrane	Highly conserved. Expression upregulated in multi-drug-resistant strains	Antibodies are bactericidal	Preclinical	(82, 190–195)
Other
NgoΦ6	Filamentous bacteriophage proteins	Encodes proteins needed for progeny phage production	Outer membrane	Highly conserved	Antibodies are bactericidal and block adherence to cervical epithelial cells	Preclinical	(196, 197)
Proteomic and bioinformatic vaccine antigen discovery
acp	Adhesin complex protein (ACP)	Inhibition of host lysozyme activity, promotes host cell colonization	Outer membrane	Highly conserved	Antibodies are bactericidal and inhibit human lysozyme	Preclinical	(198, 199)
iga2	IgA2 protease (AidA)	Putative adhesion and penetration protein	Cell envelope	Antigenically variable	No data	Preclinical	(77, 80, 200)
bamA	Beta-barrel assembly machinery protein A (BamA)	Folds and inserts beta-barrel proteins into the outer membrane	Outer membrane	Highly conserved	Antibodies are bactericidal	Preclinical	(76, 195)
bamE	Beta-barrel assembly machinery protein E (BamE)	Contributes to outer membrane assembly and integrity	Outer membrane	Highly conserved	No data	Preclinical	(77, 80)
csgG	Curl-specific gene G (CsgG)	Membrane protein	Outer membrane	Moderately conserved	No data	Preclinical	(77, 80)
lolB	Lipoprotein outer membrane localization lipoprotein B (LolB)	Putative role in lipoprotein trafficking to the outer membrane	Outer membrane	Moderately conserved	No data	Preclinical	(77, 80)
lprI	Lipoprotein I (LprI)	Putative lysozyme resistance protein	Cell envelope	Moderately conserved	No data	Preclinical	(77, 80)
lptD	Lipopolysaccharide assembly protein D (LptD)	Lipopolysaccharide assembly	Outer membrane	Moderately conserved	Antibodies are bactericidal	Preclinical	(75, 76)
lptE	Lipopolysaccharide assembly protein E (LptE)	Putative role in lipopolysaccharide assembly	Outer membrane	No data	No data	Preclinical	(77)
mafA	Multiple adhesin family A (MafA)	Adhesin	Cell envelope	Antigenically variable	No data	Preclinical	(77, 80)
metQ	Methionine binding lipoprotein Q (MetQ)	Methionine transport adhesin involved in epithelial cell adherence and survival	Outer membrane	Highly conserved	Antibodies are bactericidal and block gonococcal adherence to human cervical epithelial cells	Preclinical	(76, 77, 201, 202)
NGO0416	-	Hypothetical protein with conserved domain similarity to N-terminal domain of LamB carbohydrate-specific outer membrane porin	Periplasm	Moderately conserved	Limited bactericidal antibodies	Preclinical	(79)
NGO0425	-	Hypothetical protein	Cell envelope	Moderately conserved	No data	Preclinical	(77, 80)
NGO0690	-	Putative lipoprotein possibly involved in threonine biosynthesis and pilin antigenicity	Periplasm/ outer membrane	Moderately conserved	Antibodies are bactericidal	Preclinical	(79)
NGO0778	-	Membrane protein	Cell envelope	Highly conserved	No data	Preclinical	(77, 80)
NGO0948	-	Lipoprotein member of NlpB/DapX family	Periplasm/ outer membrane	Moderately conserved	Antibodies are bactericidal	Preclinical	(79)
NGO1043	-	Putative lipoprotein, possibly glycosylated and a substrate for phosphoethanolamine addition	Periplasm/ outer membrane	Moderately conserved	Antibodies are bactericidal	Preclinical	(79)
NGO1215	-	Putative protein with homology to a copper chaperone superfamily	Periplasm	Moderately conserved	Antibodies are bactericidal	Preclinical	(79)
NGO1251	-	Lipoprotein	Cell envelope	Highly conserved	No data	Preclinical	(77, 80)
NGO1701	-	Putative with homology to copper-binding protein of the DUF326 superfamily	Periplasm	Moderately conserved	Antibodies are bactericidal	Preclinical	(79)
NGO2054	-	Unknown	Outer membrane	Highly conserved	Antibodies are bactericidal	Preclinical	(76)
ompU	Outer membrane porin protein U (OmpU)	Putative iron uptake protein	Outer membrane	Moderately conserved	No data	Preclinical	(77, 80)
sliC	Surface-exposed lysozyme inhibitor of c-type lysozyme (SliC)	Inhibition of host lysozyme activity promotes host colonization	Outer membrane	Highly conserved	No data	Preclinical	(80, 203)
tamA	Trans-location and assembly module A (TamA)	Translocation assembly	Outer membrane	Moderately conserved	Antibodies are bactericidal	Preclinical	(76)

^a Amino acid sequence conservation between N. gonorrhoeae strains: highly conserved 80%, moderately conserved 50%, and antigenically variable <50%.

Adherence and invasion of mucosal epithelial cells

The potential of targeting with a vaccine a number of key mediators of attachment and invasion, such as type IV pili, LOS, and the opacity-associated outer membrane proteins (Opa), has been confounded by the high levels of antigenic variation and/or phase variation in these antigens. For example, although the gonococcal porin protein, PorB is the most highly abundant outer membrane protein and constitutively expressed, targeting it with a vaccine is confounded by a high level of antigenic variation within the eight surface-exposed loops in different gonococcal strains (32, 113). However, PorB is an essential protein that plays a key role in host cellular attachment, invasion, nutrient acquisition, apoptosis, and serum complement resistance (204) and has immune-enhancing activity, making it a promising vaccine adjuvant (114). Preclinical studies of putative PorB vaccines are described below. Alternative targets include mediators of host cell adherence such as the type IV pilus-associated outer membrane proteins PilC (96, 97), involved in pilus biogenesis and attachment; and PilQ, the secretin through which pili are extruded (98–100). Phospholipase D, which participates in host cell invasion and survival, is another potential outer membrane protein vaccine target (87, 88). In addition, the Neisseria heparin binding antigen (NHBA), which is also involved in host cell adherence and survival, has recently been demonstrated to be a promising vaccine antigen candidate, as it is widely distributed, highly conserved and induces bactericidal and opsonophagocytic antibodies (127, 129, 205).

Nutrient acquisition and metabolism

A number of antigens involved in nutrient acquisition through iron and zinc uptake have shown promise as potential vaccine antigen targets. The transferrin receptor proteins transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB) facilitate iron acquisition and are essential for experimental urethral infection of male volunteers when alternative iron acquisition mechanisms are not available (131). The transferrin receptor proteins are immunogenic, with the intranasal immunization of mice with TbpA and TbpB proteins fused to cholera toxin subunit B inducing serum and vaginal mucosal anti-TbpA and anti-TbpB bactericidal antibodies (134). However, preliminary evidence suggests that antibodies to gonococcal TbpA have only a modest inhibitory effect on ligand binding (81). Nitrate reductase (AniA) is required for anaerobic growth and biofilm formation of N. gonorrhoeae (159, 206). Antibodies against an AniA protein inhibit nitrite reductase activity (161, 162), suggesting this may be another promising function-blocking vaccine target.

Immune evasion and intracellular survival

Potential vaccine antigens involved in immune evasion and intracellular survival of N. gonorrhoeae include alpha-2,3-sialyltransferase (Lst) and Neisserial surface protein A (NspA). Lst expressed by gonococci scavenge sialic acid from the host and sialylate the gonococcal LOS, thereby inhibiting complement-mediated and polymorphonuclear leukocyte-mediated killing (166, 167). However, recent evidence suggests that Lst is a cytoplasmic rather than surface-exposed protein (168). NspA plays an important role in complement evasion by binding to complement regulator human factor H and factor H-like protein 1 (171). Immunization of mice with plasmid DNA containing the NspA gene followed by boosting with recombinant NspA protein-induced serum and mucosal antibodies with bactericidal and opsonophagocytic activities (170).

The conserved LOS epitope 2C7, defined by lactose substitutions at HepI and HepII in the LOS core, promotes gonococcal colonization and survival and is another important N. gonorrhoeae vaccine target. Although this epitope is a phase variable, the lgtG glycosyltransferase gene that controls this phase variation is expressed in 95% of gonococci in human infection (172, 179). Monoclonal antibodies against this epitope are bactericidal and opsonophagocytic (172). In an intraperitoneal mouse immunization study of a multi-antigenic 27C peptide mimic (MAP1) with a T helper type 1 (Th1)-inducing adjuvant (Monophosphoryl lipid A; MPL), immunization induced Th-1 biased anti-LOS antibodies that were also bactericidal. Immunization also reduced the length of gonococcal carriage and bacterial burden in experimentally infected mice (177). Further studies of a LOS 2C7 vaccine candidate with greater potential for scalability and economic production comprising a stable, homogeneous tetrapeptide 2C7 mimitope (TMCP2), administered with a glucopyranosyl lipid A adjuvant in a stable oil-in-water nanoemulsion replicated these findings (180). Anti-Rmp antibodies have been demonstrated to inhibit the efficacy of 2C7 monoclonal antibodies in mice in a dose-dependent fashion. Therefore, an effective LOS 2C7 vaccine would likely need to produce concentrations of protective antibodies sufficient to overcome this inhibitory effect in individuals with pre-existing anti-Rmp antibodies (207).

Protection from oxidative stress and antimicrobial substances

Proteins that protect N. gonorrhoeae from the threats of oxidative stress and antimicrobial substances play an important role in the pathogenesis of N. gonorrhoeae. A number of these proteins have recently been identified as promising vaccine targets. Gonococcal methionine sulfoxide reductase protein (MsrA/B) reduces methionine sulfoxide to methionine to protect the organism from oxidative stress (83). MsrA/B is surface-exposed and the gene encoding MsrA/B is highly conserved. Immunization of mice with an adjuvanted recombinant MsrA/B vaccine results in the production of function-blocking antibodies with bactericidal and opsonophagocytic activities (83). Another promising vaccine antigen is multiple transferable resistance protein E (MtrE), an outer membrane channel of a multidrug transporter system (MtrCDE) which mediates the export of hydrophobic antimicrobial substances (fatty acids, long-chain lipids, bile salts, and antimicrobials) from the cell and survival after neutrophil exposure (194). It also plays a key role in the FarA-FarB-MtrE active efflux pump, an additional efflux pump system that mediates resistance to hydrophobic agents (193). Mice immunized with an adjuvanted recombinant MtrE vaccine produce anti-MtrE antibodies that are bactericidal and reduce the activity of the MtrCDE efflux pump in the presence of hydrophobic compounds (82).

Key reverse vaccinology antigen discoveries

Finally, a number of promising vaccine antigens have also been discovered using reverse vaccinology approaches described above. These include several antigens involved in cell envelope homeostasis and translocation, including beta-barrel assembly machinery protein A (BamA), lipopolysaccharide assembly protein D (LptD), and translocation and assembly module A (TamA) as well as two human lysozyme inhibitors, adhesin complex protein (ACP), and surface-exposed lysozyme inhibitor of c-type lysozyme (SliC). BamA, LptD, and TamA are surface-exposed, highly conserved, and stably expressed, and immunization with them elicits antibodies with bactericidal activity (76). Both lysozyme inhibitor antigens ACP and SliC are highly conserved and stably expressed (198, 199, 203); antibodies to ACP are both bactericidal and inhibit binding to human lysozyme (199).

Another promising vaccine candidate discovered by reverse vaccinology is MetQ, the methionine-binding component of an ATP-binding cassette transporter system (201). MetQ plays a role in epithelial cell adherence and survival. It is a highly conserved surface-exposed protein that is constitutively expressed (76, 201). Anti-MetQ antibodies are bactericidal and reduce adherence of N. gonorrhoeae to cervical epithelial cells (201). Mice immunized with a recombinant MetQ lipoprotein formulated with a Th1-stimulating adjuvant (cytosine phosphoguanine; CpG) developed robust Th1-biased serum and vaginal antibodies. After vaginal challenge, the immunized mice demonstrated accelerated clearance of gonococcal infection and a lower bacterial burden (202).

Novel vaccine delivery systems

In addition to novel vaccine targets, there has been significant development in vaccine adjuvants that augment vaccine antigen immune responses. These include nanoparticle technologies such as liposome-based adjuvants which contain immunogens such as toll-like receptor ligands; and oil-in-water emulsions which activate myeloid cells to stimulate innate and adaptive immune responses (208). Novel adjuvants that have been assessed in preclinical N. gonorrhoeae vaccine studies include the Th1-stimulating adjuvants, microencapsulated interleukin-12 (IL-12) (209), and CpG oligodextronucleotides (202, 210). In recent mouse model studies, N. gonorrhoeae is able to suppress the development of Th1 and T helper type 2 (Th2) T-cell response and to induce a T helper type 17- (Th17)-driven immune response that facilitates immune evasion (211–213). Elevated levels of the Th17 cytokine, interleukin-17 (IL-17), have also been demonstrated in serum and genital secretions of patients with N. gonorrhoeae infection compared to healthy subjects or those with non-bacterial STIs, suggesting that the experimental observations in mice of a Th17-driven immune response may also apply in human N. gonorrhoeae infection (214, 215). Rational vaccine design using Th-1 stimulating adjuvants harnesses this key discovery. Th1-stimulating adjuvants have been shown to induce a Th1-driven response, generate anti-gonococcal antibodies and gamma-interferon secreting CD4+ T cells, and accelerated clearance of N. gonorrhoeae infection in preclinical mouse model studies (202, 209).

The past decade has seen a number of newly licensed vaccines for important infectious diseases that use novel vaccine delivery systems, including nucleic acid vaccines [such as those used in messenger ribonucleic acid (mRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines], virus-like particles (as used in HPV vaccines), and OMV vaccines (used in serogroup B meningococcal vaccines) (216). A number of these novel vaccine delivery systems have been studied in preclinical mouse models of N. gonorrhoeae vaccines. Nucleic acid vaccines, viral replicon particles, and recombinant vaccines are particularly attractive for putative PorB gonococcal vaccines as these techniques avoid potential problems of contamination by Rmp and inadvertent stimulation of anti-Rmp blocking antibodies by the putative vaccine. Zhu et al. have undertaken a number of studies investigating various vaccine delivery techniques and prime-boost schedules for putative PorB vaccines, including PorB deoxyribonucleic acid (DNA), renatured recombinant PorB (rrPorB), PorB expressed from Venezuelan equine encephalitis virus replicon particles (PorB VRPs), and OMV vaccines (in which the major constituent antigen is PorB) (108, 109). These studies have demonstrated that different immune responses are triggered by various vaccine antigen delivery systems and sites of inoculation. For example, mice immunized subcutaneously with a rrPorB vaccine developed high levels of PorB-specific IgG antibodies, with immunization administered in the hind footpad inducing a Th1 response and immunization administered in the dorsal area inducing a Th2 response. In this study, immunization with PorB VRPs induced a Th1 response while an intranasal OMV vaccine was the only vaccine that generated serum bactericidal antibodies (109). Antibodies induced by a PorB DNA vaccine alone were modest; however, boosting by either rrPorB or PorB VRPs significantly increased PorB-specific serum antibody levels (108).

Other novel vaccine delivery technologies such as bacterial ghosts have also been explored in preclinical N. gonorrhoeae vaccines. Bacterial ghosts are empty Gram-negative bacterial cell envelopes that retain the cellular morphology and antigenic determinants of the cell envelope and provide a promising system for the delivery of nucleic acid (DNA or RNA) vaccines. This delivery system provides intrinsic adjuvant activity due to the enhanced immune responses produced against cell envelope antigens, including T-cell activation and mucosal immunity (217). Jiao et al. have demonstrated that N. gonorrhoeae PorB and NspA DNA vaccines delivered using Salmonella enteritidis ghosts induce serum IgG antibodies that are bactericidal in an experimental mouse model (218, 219).

Meningococcal outer membrane vesicle vaccines

Meningococcal OMV vaccines have been a key focus of both observational and preclinical N. gonorrhoeae vaccine studies. OMVs are spherical lipid bi-layer membrane structures that are released spontaneously from the outer membrane of Gram-negative bacteria and contain surface-exposed phospholipids, lipopolysaccharide/LOS, and membrane proteins as well as RNA, DNA, proteins, and peptidoglycans within the lumen of the vesicle (220–222). The role of OMVs in bacterial pathogenesis includes modulation of host immune response, nutrient acquisition, and biofilm formation (220–222). OMVs present a number of advantages as a novel vaccine platform, including the ability to enter lymphatic vessels for uptake by antigen-presenting cells and the presentation of membrane surface antigens in their native configuration, thereby evoking humoral and cell-mediated responses (220–222). The association between immunization with currently available meningococcal B vaccines and N. gonorrhoeae infection and preclinical studies of these vaccines will be discussed in further detail below. The focus in preclinical studies of novel OMV vaccines has recently shifted to optimizing meningococcal OMV design based on known features of gonococcal pathogenesis, such as the use of meningococcal isolates lacking Rmp proteins and avoiding detergent-based preparation of outer membranes (223, 224). Although detergent-based preparation of outer membrane vesicles extracts LOS and decreases endotoxin activity, it also removes key meningococcal antigens, including factor H binding protein (fHbp) (223). A detoxified meningococcal OMV vaccine (lacking PorA, PorB, and Rmp) has been shown to improve gonococcal clearance in a murine model (223). In addition, a meningococcal native OMV vaccine with attenuated endotoxin and overexpressed fHbp has been shown to induce high levels of serum immunoglobulin G (IgG) anti-FHbp as well as serum bactericidal antibodies against heterologous gonococcal strains (224). Furthermore, the next generation of OMV vaccines developed from N. gonorrhoeae strains and designed specifically to induce protection against N. gonorrhoeae infection is under study, with a number of preclinical studies demonstrating promising results, including production of serum and vaginal antibodies and accelerated clearance of gonococcal infection in the estradiol-treated female mouse model (225–227). These include the dmGC_0817560 (226) and NGoXIM (227) native OMV vaccines described in further detail below.

Route of immunization

The route of immunization may also play a significant role in determining the immunogenicity of a N. gonorrhoeae vaccine. It has been observed that the ability of parenteral immunization to induce mucosal immunoglobulin A (IgA) antibodies for other sexually transmitted pathogens is limited (228). By contrast, mucosal administration of vaccines via intranasal immunization has demonstrated relatively higher mucosal IgA and IgG antibodies compared with parenteral vaccines (134, 228). This has been shown in mouse model studies of a number of different experimental N. gonorrhoeae vaccines (109, 118, 134, 202, 225, 227). For a number of these vaccines, accelerated clearance of gonococcal infection has been observed in intranasally immunized mice, including a gonococcal OMV preparation (225) and a recombinant MetQ-CpG adjuvant vaccine (202). In addition, intravaginal and intranasal immunization using a native gonococcal OMV plus microencapsulated IL-12 vaccine (NGoXIM) in a female mouse model induced serum and vaginal IgG and IgA antibodies and accelerated clearance of gonococcal infection (209, 227). Other novel routes of N. gonorrhoeae vaccine delivery studied in preclinical settings include a transdermal microneedle skin patch that enables slow release of antigens using a formalin-inactivated whole-cell gonococcal microparticle vaccine formulation. Mouse model studies of this vaccine demonstrated that the transdermal skin patch vaccine induced increased IgG antibody titers compared with the comparator subcutaneously administered vaccine (229).

Table 3 provides a summary of N. gonorrhoeae vaccines that have proceeded to contemporary preclinical studies in the experimental mouse model, many of which have included novel vaccine antigens, vaccine delivery systems, or routes of immunization.

TABLE 3.

Contemporary Neisseria gonorrhoeae vaccines that have proceeded to preclinical studies in the experimental mouse model^a

Trial design	Vaccine	Immunization schedule	Immunogenicity	Attenuation of gonococcal infection	Reference
Estradiol-treated BALB/c mouse model inoculated vaginally with N. gonorrhoeae MS11 approximately 3 weeks after immunization	Gonococcal outer membrane preparation from N. gonorrhoeae strain MS11	IN or SC administration 3 times at 3-week interval	Serum and vaginal antibodies were induced by both IN and SC immunization. SBA to heterologous gonococcal strain induced by IN immunization.	Clearance of gonococcal colonization was significantly faster in IN immunized compared to control mice.	(225)
BALB/c mouse model immunized with a putative vaccine	Gonococcal recombinant plasmid encoding PorB DNA (PorB DNA) from N. gonorrhoeae strain FA1090 prime vaccine followed by PorB DNA, renatured recombinant PorB protein (rrPorB) plus Ribi R-700 adjuvant or PorB expressed from Venezuelan equine encephalitis virus replicon particles (PorB-VRPs)	IM or epidermal gene gun bombardment administration with prime PorB DNA followed by boost with PorB DNA, rrPorB, or PorB-VRPs 4 weeks later	Serum antibodies induced by both IM and epidermal gene gun bombardment, with Th1 response induced by IM administration and Th2 response induced by gene gun bombardment. Boosting with rrPorB and PorB VRPs significantly increased PorB IgG and IgA antibodies. Serum OPA to homologous gonococcal strain, SBA not produced.	No data	(108)
BALB/c mouse model immunized with various combinations of putative vaccines	N. gonorrhoeae vaccines produced from strain FA1090, including renatured recombinant PorB protein (rrPorB) plus Ribi R-700 adjuvant or PorB expressed from Venezuelan equine encephalitis virus replicon particles (PorB-VRPs) or outer membrane vesicle (OMV) vaccine	SC administration into dorsal area or hind footpad (rrPorB), SC administration into hind footpad (PorB VRP) or IN (OMV) 3–4 times, 3 weeks apart.	Serum anti-PorB antibodies were induced by all vaccines tested, with Th1 bias for PorB-VRP and rrPorB in the footpad and Th2 bias when rrPorB was given in dorsal area. IN OMV induced SBA while other vaccines did not.	No data.	(109)
BALB/c mouse model immunized with various combinations of vaccine components	Various combinations of N. gonorrhoeae strain FA19 1) recombinant transferrin binding protein A (rTbpA) plus Ribi R-700 adjuvant, 2) recombinant transferrin binding protein B (rTbpB) and 3) cholera toxin B subunit (Ctb), either as conjugates or admixed.	IN or SC administration 3 times at 10-day intervals	Serum and vaginal antibodies induced by IN immunization for each Tbp antigen combined with Ctb. SBA induced by IN immunization.	No data	(134)
BALB/c mouse model immunized with a putative vaccine	N. gonorrhoeae vaccines produced from strain FA1090, including renatured recombinant TbpB (rrTbpB) and TbpB expressed from Venezuelan equine encephalitis virus replicon particles (TbpB-VRPs)	SC immunization at 0, 4, 7, and 10 weeks	Serum antibodies induced by both TbpB vaccines, with the highest titers in mice immunized with rrTbpB. TbpB-VRP responses Th1-biased. Mucosal antibodies produced by both vaccines with the highest titers in mice immunized or boosted with rrTbpB. Bactericidal antibodies not produced	No data	(230)
Estradiol-treated BALB/c female mouse model inoculated with N. gonorrhoeae strain FA1090 approximately 2 weeks after immunization	OMV preparation from N. gonorrhoeae strain FA1090 combined with IL-12 microspheres	Intravaginal immunization 3 times at 1-week interval	Intravaginal OMV/IL-12 microsphere vaccination induced serum and vaginal IgG and IgA antibodies against homologous and heterologous strains.	Clearance of N. gonorrhoeae colonization significantly faster in mice immunized with OMV/IL-12 microsphere vaccine candidate compared to those immunized with OMV or IL-12 microspheres alone	(209)
Smith Webster (CFW) mouse model immunized with a putative vaccine	Whole-cell formalin-inactivated microparticle vaccine from N. gonorrhoeae strain CDC-F62 loaded in dissolvable microneedles	Transdermal immunization was applied for 20 minutes, three times, 2 weeks apart	Transdermal microparticle vaccination induced greater serum IgG antibodies than SC vaccination and induced elevated CD4+ and CD8+ responses comparable to SC vaccination.	No data	(229)
BALB/c mouse model immunized with a putative vaccine	porB gene DNA from N. gonorrhoeae strain WHO-A inserted into the eukaryotic expression vector pVAX1 (pVAX1-porB) loaded in S. enteritidis ghosts (SE ghosts (pVAX1-porB))	PO immunization 3 times at 2-week interval	SE bacterial ghosts (pVAX1-porB) vaccination induced greater serum IgG antibodies, CD4+ and CD8+ T-cell responses than pVAX1-porB DNA vaccine alone. SBA induced.	No data	(218)
BALB/cAnNCr mouse model inoculated with N. gonorrhoeae F62 3 weeks after immunization	Meningococcal detoxified outer membrane vesicle (dOMV) vaccine prepared from meningococcal strains deleted for major outer membrane proteins (including PorA, PorB, and RmpM) plus Alhydrogel adjuvant	IP immunization 3 times at 3-week interval	dOMV vaccine produced induced serum and vaginal antibodies. SBA not detected.	A significantly higher proportion of mice immunized with meningococcal dOMV vaccines prepared from strains deleted for major outer membrane proteins were cleared of Ng compared to control.	(224)
BALB/c mouse model immunized with a putative vaccine	nspA gene DNA from N. gonorrhoeae strain WHO-A inserted into the eukaryotic expression vector pVAX1 (pVAX1-nspA) either alone (SE ghosts pVAX1-nspA) or in combination with SE ghosts (pVAX1-porB) vaccine described above	PO immunization 3 times at 2-week interval	Co-administered SE ghosts (pVAX1-nspA) and SE ghosts (pVAX1-porB) vaccination induced the highest level of anti-nspA and anti-porB serum IgG and the highest SBA titers	No data	(219)
BALB/c mouse model immunized inoculated with N. gonorrhoeae FA1090 approximately 3 weeks after immunization	Gonococcal recombinant MetQ protein combined with Titermax gold oil-in-water immersion adjuvant subcutaneous vaccine, then subsequently combined CpG 1826 adjuvant intranasal vaccine (rMetQ-CpG)	SC immunization, followed by 3 IN boosts on days 14, 24, and 35	Immunization with rMetQ-CpG induced the highest level of anti-MetQ IgG and IgA serum and vaginal antibodies, with a serum IgG1/IgG2a ratio suggestive of a Th1 response.	Clearance of N. gonorrhoeae colonization was significantly faster and with a lower burden of infection in mice immunized with the rMetQ-CpG vaccine candidate compared to those immunized with PBS or adjuvant alone.	(202)
CD-1 mouse model immunized with a putative vaccine	Meningococcal native outer membrane vesicle (NOMV) vaccine prepared from meningococcal strain with genetically attenuated endotoxin and overexpressed factor H binding protein (FHbp) or inactivated gene encoding FHbp (NOMV-KO) or recombinant FHbp	IP immunization 2 times at 3-week interval	Immunization with NOMV-FHbp and NOMV-KO-induced gonococcal SBA	No data.	(223)
Estradiol-treated BALB/c mouse model inoculated with N. gonorrhoeae strain FA1090, FA19, or WHO strain F, L, or W approximately 2 weeks after immunization; plus BALB/c male mouse model.	Gonococcal native outer membrane vesicle vaccine (NOMV) from strains FA1090; Gonococcal detergent-extracted OMV (dMV) from strain FA19 and double deletion mutant OMV (dm OMV) prepared from mutant N. gonorrhoeae strain MS11 in which genes for Rmp and LpxL1 were deleted to eliminate induction of blocking antibodies against Rmp and to decrease LOS endotoxicity; all vaccines combined with IL-12 microspheres (ms)	IN or intravaginal immunization 2 times at 2-week interval.	IN and intravaginal immunization of female mice with NOMV plus IL-12 ms induced comparable serum IgG, salivary IgA and vaginal IgG, and IgA anti-gonococcal antibodies. IN immunization of male mice with NOMV plus IL-12 ms induced comparable serum IgG and saliva IgA antigonococcal antibodies to female mice. IFN-gamma production by CD4+ T cells from iliac lymph nodes was elevated after IN or intravaginal immunization with NOMV plus IL-12 ms.	Female mice immunized with IN or intravaginal NOMV plus IL-12 ms cleared gonococcal infection faster than mice immunized with control immunization. In addition, female mice cleared gonococcal infection with heterologous strains faster than mice immunized with control immunization. Gonococcal clearance was also accelerated in mice immunized with deOMVs comparable to that seen for NOMV immunized mice; Gonococcal clearance was accelerated in mice immunized with OMV plus IL-12 ms vaccine produced from mutant N. gonorrhoeae in which genes for Rmp and LpxL1 were deleted to eliminate induction of blocking antibodies against Rmp and to decrease LOS endotoxicity comparable to that seen for NOMV immunized mice	(227)
Estradiol-treated BALB/c mouse model inoculated with N. gonorrhoeae strain FA1090	Gonococcal native outer membrane vesicle vaccine (NOMV) from Chilean gonococcal strain GC_08175680 (dmGC0817560 NOMV) and FA1090 (dmFA1090 NOMV) with lpxL1 and rmp genes deleted to reduce reactogenicity, minimize the production of potentially unprotective antibodies and increase NOMV yield; both vaccines formulated with aluminum hydroxide	Parenteral administration.	Immunization with dmGC_08175680 NOMV and dmFA1090 NOMV- induced gonococcal specific serum and vaginal mucosal IgG and IgA antibodies and gonococcal-specific Th1/Th17 CD4+ T-cell responses	Immunization of mice with dmGC_08175680 OMV and dmFA1090 NOMV accelerated clearance of FA1090 from mice significantly faster than 4CMenB	(226)

^a IM, intramuscular; IN, intranasal; IN, intraperitoneal; OPA: opsonophagocytic antibodies; PO, per oral; SBA: serum bactericidal antibodies; SC, subcutaneous.

THE IMPACT OF NEISSERIA MENINGITIDIS OUTER MEMBRANE VESICLE VACCINES ON GONORRHOEA INFECTION

The most significant step in N. gonorrhoeae vaccine progress in the past decade was a landmark study that demonstrated 31% vaccine efficacy of a N. meningitis serogroup B outer membrane vesicle (OMV) vaccine (MeNZB) against N. gonorrhoeae infection in a retrospective observational case-control study of 15- to 30-year-olds attending sexual health clinics in New Zealand (34). This finding demonstrated the biological plausibility of vaccine-mediated protective immunity against N. gonorrhoeae and provided a proof-of-concept that an effective N. gonorrhoeae vaccine may be possible (231). This observation was supported by evidence from ecological studies in Cuba, Norway, and Canada where an association between the introduction of N. meningitidis serogroup B OMV-containing vaccines and reduced rates of gonorrhoea infection were apparent (58–62). The impact of 4CMenB (Bexsero; GSK) has been assessed in further retrospective observational case-control and cohort studies. 4CMenB is a N. meningitidis serogroup B OMV-containing vaccine that incorporates the OMV included in MeNZB, as well as three recombinant antigens, Neisseria adhesin A (NadA), fHbp and NHBA, as well as two accessory proteins (GNA2091 fused with fHbp and GNA1030 fused with NHBA) that increase the immunogenicity of the target recombinant antigens (232). These studies have also demonstrated a protective effect of 4CMenB on N. gonorrhoeae infection, with estimated vaccine effectiveness for a two-dose schedule ranging between 33% and 46% in various settings across the world, including in the United States, Australia, and Italy (53–56). These studies are summarized in Table 4.

TABLE 4.

Completed observational trials assessing vaccine effectiveness of meningococcal vaccines on gonorrhoea infection^a

Clinical trial design	Vaccine	Immunization schedule	Study population	Result	Reference
Retrospective ecological study of N. gonorrhoeae infection incidence in Cuba before and after the introduction of VA-MENGOC-BC vaccine	VA-MENGOC-BC N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain CU385 plus serogroup C capsular polysaccharide from N. meningitidis serogroup C strain C11)	Intramuscular; single dose given in mass vaccination program; 2-dose schedule, 2 months apart given in routine schedule.	Cuban national health registry data comprising annual incidence rates of N. gonorrhoeae infection, meningococcal disease, and syphilis between 1970 and 2018, including the period of vaccine efficacy trial of VA-MENGOC-BC, mass vaccination campaign in age 3 months to 20 years from 1989 to 1990; and incorporation in routine vaccination schedule from 1991 to 2018.	Decreased incidence of N. gonorrhoeae infection compared to other STIs observed between 1990 and 1993 after mass vaccination campaign and between 2010 and 2018 compatible with possible impact of routine infant vaccination programs.	(58–60, 233)
Retrospective ecological study of N. gonorrhoeae infection incidence in Norway before and after the introduction of MenBvac vaccine.	MenBvac N. meningitidis serogroup B vaccine (OMV from N. meningitidis serogroup B strain H44/76)	Intramuscular 2-dose schedule.	Norwegian national health registry data comprising incidence rates of gonorrhoea from 1993 onward and data of vaccine efficacy trial in 13- to 15-year-old students enrolled in secondary schools between 1988 and 1992. 93,611 (63%) of the 148,589 children resident in Norway and born during 1973–1976 received MenBvac. A total of 2,601 cases of N. gonorrhoeae infection was reported during 1993–2008.	Incidence rate ratio (IRRs) analysis (defined as number of new diagnoses of N. gonorrhoeae infection per 100,000 population for vaccinated cohort compared to pre-vaccination and post-vaccination cohorts) demonstrated reduced crude IRR for women aged 20–24 years in the vaccinated cohort (IRR 0.58, 95% CI 0.42–0.8) and reduced adjusted IRR for men aged 20–24 years in the vaccinated cohort (0.68, 95% CI 0.51–0.93) and post-vaccination cohort (0.51, 95% CI 0.33–0.78) between 1993 and 2008.	(62)
Retrospective case-control study of 15- to 30-year-old sexual health clinic patients eligible to receive MeNZB vaccine in New Zealand. Cases are defined as confirmed laboratory detection of N. gonorrhoeae only from clinical specimen; and controls defined as confirmed laboratory detection of C. trachomatis only from clinical specimen	MeNZB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254)	Intramuscular, 3-dose schedule. Infants: age 6 weeks, 3 months and 5 months Children > 6 months: 3 doses, 6 weeks apart	Sexual health clinic patients aged 15–30 years and eligible to receive MeNZB (mass vaccination program 2004–2006 age 6 weeks to 20 years and available in schools and primary care until 2008) and diagnosed with N. gonorrhoeae and/or C. trachomatis infection between 1 January 2005 and 31 December 2016. 14,730 cases and controls for analysis: 1,241 incidences of N. gonorrhoeae infection; 12,487 incidences of C. trachomatis infection; and 1,002 incidences of co-infection.	Vaccinated individuals significantly were less likely to be cases than controls [511 (41%) vs 6,424 (51%)]; adjusted OR 0.69 (95% CI 0.61–0.79; P < 0.0001). Estimated vaccine effectiveness of MeNZB against N. gonorrhoeae infection adjusted for ethnicity, deprivation, geographical area, and sex 31% (95% CI 21–39; P < 0.0001)	(34)
Retrospective ecological study of N. gonorrhoeae infection incidence in Sanguenay-Lac-Saint-Jean region of Quebec, Canada before and after the introduction of 4CMenB vaccination program.	4CMenB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254 plus three recombinant protein antigens)	Intramuscular, 2-dose schedule.	Public Health Registry data comprising cases of N. gonorrhoeae infection notified between January 2006 and June 2017 and vaccination uptake data for the mass vaccination campaign (mass vaccination campaign of individuals aged 6 months to 20 years conducted from May to December 2014). Overall vaccine coverage was 82%. A total of 231 gonorrhoea cases were reported among persons aged 14 years and older between January 2006 and June 2017.	A decrease in the number of N. gonorrhoeae infections and incidence rate of among the vaccinated cohort (age 14–20 years) were observed post-vaccination period, whereas it increased in unvaccinated cohort (age 21 years and older). Estimated vaccine impact: N. gonorrhoeae infection risk reduction of 59% (95% CI 22–84; P = 0.1).	(61)
Retrospective cohort study of individuals born 1984–1999 eligible for MeNZB vaccination 2004–2008 in New Zealand with primary outcome hospitalization for primary diagnosis of N. gonorrhoeae infection.	MeNZB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254)	Intramuscular vaccine, 3-dose schedule. Infants: age 6 weeks, 3 months, and 5 months Children > 6 months: 3 doses, 6 weeks apart.	Individuals born 1984–1999 and residing in New Zealand from 2004 until 2015 (mass vaccination program 2004–2006 age 6 weeks to 20 years and available in schools and primary care until 2008) with data available through the National Registry on vaccination status, sex, ethnicity, and deprivation. 935,496 individuals were included in the analysis. Overall vaccination coverage was 59.2%. 261 cases of hospitalization attributable to N. gonorrhoeae.	Vaccinated individuals were significantly less likely to be hospitalized due to N. gonorrhoeae infection after adjusting for gender, ethnicity, and deprivation (HR 0.76, 95% CI 0.58–0.99) with estimated vaccine effectiveness of 24% (95% CI 1%–42%).	(57)
Retrospective case-control study of 16- to 23-year-old individuals with N. gonorrhoeae or Chlamydia trachoomatis infection in New York City and Philadelphia. Cases defined as confirmed laboratory detection of N. gonorrhoeae (NAAT or culture) but not C. trachomatis; and controls defined as confirmed laboratory detection of C. trachomatis only (NAAT or culture) but not N. gonorrhoeae	4CMenB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254)	Intramuscular vaccine, 2-dose schedule minimum 30 days and maximum 180 days apart (single dose categorized as partial vaccination).	Individuals aged 16–23 years old with C. trachomatis and/or N. gonorrhoeae reported to STI surveillance systems of the New York City Department of Health and Mental Hygiene and the Philadelphia Department of Public Health, with data matched to the vaccine registry data system to obtain number and dates of MenB-4C vaccine doses between January 1 2016 and December 31 2018. 109,737 individuals with 167,706 reported STIs for analysis. 124,876 C. trachomatis infections, 18,099 N. gonorrhoeae infections, and 24,731 were gonococcal and chlamydia co-infections. 3,058 STIs occurred after the complete vaccination series, 6,519 after the partial vaccination series, and 155,330 among vaccine-naïve individuals.	Vaccinated individuals were significantly less likely to be diagnosed with N. gonorrhoeae infection. Complete vaccination series unadjusted prevalence ratio (UPR) 0.64, 95% CI 0.51–0.79; P < 0.0001 in bivariate analyses and adjusted prevalence ratio (APR) 0.60, 95% CI 0.47–0.77; P < 0.0001 in multivariate analyses. Partial vaccination series UPR 0.83, 95% CI 0.72–0.96, P = 0.0204 in bivariate analyses and APR 0.74, 95% CI 0.63–0.88; 0 = 0.0012. Estimated vaccine effectiveness for complete vaccination series 40% (95% CI 23–53) and partial vaccination series 26% (95% CI 12–37%).	(53)
Retrospective case-control study of adolescents and young adults with gonorrhoea or chlamydia infection in the state of South Australia, Australia. Cases defined as all gonorrhoea-positive cases who did or did not have C. trachomoatis co-infection at the time of first episode N. gonorrhoeae infection. Controls defined as C. trachomatis-positive infections only.	4CMenB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254)	Intramuscular vaccine, 2-dose schedule, 8 weeks apart.	Individuals born between 1 February 1998 and 1 February 2005 that had N. gonorrhoea or C. trachomatis disease notification between 1 February 2019 and 31 January 2021 (in 2019, a 2-dose vaccination schedule for 15- to 17-year-old school-based immunization program was implemented and between 2019 and 2020, a catch-up program was available for those aged 17–20 years). 53,356 individuals received at least 1 dose of 4CMenB and 46,083 received 2 doses. 512 patients with total 575 episodes of gonorrhoea and 3,140 patients with 3847 episodes of chlamydia included in analysis.	Estimated vaccine effectiveness using C. trachomatis infection as control was 32.6% (95% CI 10.6–49.1) for individuals who received at least one dose; and 32.7% (95% CI 8.3–50.6) for people who received two doses compared to those who were unvaccinated.	(54)
Retrospective matched cohort study of 15–30 year-olds who received 4CMenB (plus/minus MenACWY) or MenACWY only in Southern California, United States. The exposed group comprising recipients of 4CMenB was matched in a ratio of 1:4 to the unexposed group comprising recipients of MenACWY only by age, sex, and year of index vaccination with study outcome positive gonorrhoea NAAT or culture or chlamydia NAAT (negative control).	4CMenB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254); MenACWY N. meningitidis vaccine (serogroup A, C, W, and Y polysaccharide conjugate vaccine)	4CMenB intramuscular vaccine, 2-dose schedule. Minimum 1 dose included in analysis. MenACWY intramuscular vaccine, 2-dose schedule. Minimum 1 dose included in analysis	Individuals aged 15–30 years old in Kaiser Permanente Southern California health records were noted to be vaccinated with 4CMenB, matched in a ratio of 1:4 to recipients of MenACWY only by age, sex, and year of index vaccination between 1 January 2016 and 12 December 2019. 6,641 4CMenB recipients; matched to 26,471 MenACWY-only recipients.	Incident gonorrhoea rates 2.0 (95% CI 1.3–2.8) per 1000-person years for 4CMenB recipients; 5.2 (95% CI 4.6–5.8) per 1000-person years for MenACWY-only recipients. Incident chlamydia rates 12.4 (95% CI 10.7–14.4) per 1000-person years for 4CMenB recipients; 15.2 (95% CI 14.2–16.2) per 1000-person years for MenACWY only recipients. Hazard ratio (HR) for incident gonorrhoea in 4CMenB recipients compared to MenACWY-only recipients 0.54 (95% CI 0.34–0.86) in multivariable analyses.	(55)
Retrospective case-control study of ≥18-year old MSM living with HIV with gonorrhoea or syphilis, chlamydia or anal HPV in Milan, Italy. Cases defined as all gonorrhoea-positive cases by NAAT or culture; controls were defined as chlamydia positive by NAAT, syphilis positive by serology, and HPV positive by anal NAAT to 28 HPV genotypes or following a diagnosis of condylomatosis.	4CMenB N. meningitidis vaccine (OMV from N. meningitidis serogroup B strain NZ98/254)	Intramuscular vaccine, 2-dose schedule, 8 weeks apart.	≥18-year-old MSM living with HIV diagnosed with gonorrhoea or syphilis, chlamydia or anal HPV included in the database of the Infectious Diseases Unit at the San Raffaele Scientific Institute, Milan, Italy between July 2016 and February 2021. 349/1,051 (33%) received 4CMenB vaccination. 103 cases and 948 controls analyzed. Median follow-up 3.8 years (2.1–4.3)	Estimated vaccine effectiveness was 42% (95% CI 6–64, P = 0.027) and remained significant at 44% (95% CI 9–65, P = 0.020) after adjustment in multivariable analysis.	(56)

^a HIV, human immunodeficiency virus; HPV, human papillomavirus; MSM, men who have sex with men; NAAT, nucleic acid amplification test; OMV, outer membrane vesicle.

Several studies are currently recruiting participants into randomized placebo-controlled trials of the 4CMenB vaccine to assess efficacy against N. gonorrhoeae infection (https://clinicaltrials.gov/study/NCT04415424; https://clinicaltrials.gov/study/NCT04350138; https://clinicaltrials.gov/study/NCT05766904; https://clinicaltrials.gov/study/NCT05766904; https://clinicaltrials.gov/study/NCT05294588; 234). Furthermore, in recently reported interim analysis of a randomized, open-label factorial study of the 4CMenB vaccine coupled with doxycycline post-exposure prophylaxis in MSM on HIV pre-exposure prophylaxis (PrEP) (DOXYVAC), a reduced incidence of first-episode N. gonorrhoeae infection in the 4CMenB group was observed compared to the no vaccine group (adjusted hazard ratio 0.49; 95% CI 0.27–0.88) (235). However, the final study report is awaited, as the review of the study data indicates that a number of N. gonorrhoeae infections were not included in the interim analysis (236). The randomized studies of 4CMenB will provide further high-level evidence of the protective efficacy of N. meningitidis serogroup B OMV vaccines against N. gonorrhoeae infection. Here we describe the clinical and basic science studies of meningococcal serogroup B OMV vaccines in further detail.

Observational studies

The first studies to suggest an association between various meningococcal serogroup B OMV vaccines and N. gonorrhoeae incidence were ecological analyses of the impact of mass serogroup B meningococcal OMV vaccination programs on N. gonorrhoeae infection rates in Cuba, Norway, and Canada (58–62). In Cuba, a N. meningitidis serogroup B OMV-containing meningococcal vaccine, VA-MENGOC-BC, was used in a national mass vaccination program of individuals aged 3 months to 24 years between 1989 and 1990, and subsequently incorporated into the national infant immunization schedule (233). Reported vaccine coverage of the mass vaccination program in the target population was 95% (233). In the years immediately after the program (1989–1993), the incidence of gonorrhoea decreased from 381.9 to 190.3 cases per 100,000 (r = 0.9607, P = 0.001), despite an increase in other sexually transmitted infections such as syphilis (58–60).

In a second ecological study undertaken in Norway using trial registry data of a N. meningitidis serogroup B OMV-containing vaccine, MenBvac, delivered to 63% of 13- to 15-year-olds between 1988 and 1992, a reduced incidence rate ratio (IRR) of gonorrhoea was observed in the subsequent years 1993–2008 among 20- to 24-year-olds in the vaccinated cohort compared to the pre-vaccination cohort (IRR 0.58, 95% CI 0.42–0.8 for women and adjusted IRR 0.68, 95% CI 0.51–0.93 for men) (62). In a third ecological study, the incidence of N. gonorrhoeae infection was studied in the context of a mass vaccination campaign undertaken in Canada in 2014 among individuals aged 6 months to 20 years vaccinated with the N. meningitidis serogroup B OMV-containing 4CMenB vaccine in the Sanguenay-Lac-Saint-Jean region of Quebec. Although an association between vaccination and reduced gonorrhoea incidence of 59% (95% CI −22% to 84%; P = 0.1) was observed, this finding was not statistically significant (61).

A landmark retrospective observational case-control study of the MeNZB N. meningitidis serogroup B OMV vaccine was the first to describe vaccine efficacy against N. gonorrhoeae infection among 14,730 sexual health clinic patients aged 15–30 years who were eligible to receive MeNZB vaccination through a mass vaccination program of individuals aged 6 weeks to 20 years implemented in New Zealand between 2004 and 2006 (34). This study demonstrated that vaccinated individuals were significantly less likely to be cases (N. gonorrhoeae mono-infection) than controls (Chlamydia trachomatis mono-infection); 41% vs 51%; adjusted odds ratio (OR) 0.69 (95% CI 0.61–0.79; P < 0.0001). After adjustment for ethnicity, deprivation status, geographical area, and sex, the estimated vaccine effectiveness of MeNZB against N. gonorrhoeae infection was 31% (95% CI 21–39; P < 0.0001) (34). Further study of individuals vaccinated with MeNZB during New Zealand’s mass vaccination program demonstrated that vaccinated individuals were also significantly less likely to be hospitalized due to N. gonorrhoeae infection, with an estimated vaccine effectiveness against N. gonorrhoeae-related hospitalization of 24% (95% CI 1–42%) (57).

A similar association between the N. meningitidis serogroup B OMV-containing vaccine 4CMenB and reduced risk of N. gonorrhoeae infection has been reported in subsequent retrospective observational case-control studies using jurisdictional health registry and immunization data in various populations in the United States, Australia, and Italy, with vaccine effectiveness of a two-dose schedule ranging between 33% and 46% (53–56). In a retrospective matched cohort study of 15- to 30-year-old residents in Southern California, the incidence of N. gonorrhoeae infection among individuals who received 4CMenB (with or without a MenACWY N. meningitidis serogroup A, C, W, Y polysaccharide conjugate vaccine) was compared with the incidence of N. gonorrhoeae infection among individuals who received the MenACWY vaccine alone; the hazard ratio (HR) of incident N. gonorrhoeae infection was 0.54 (95% CI 0.34–0.86) (55).

By contrast, no association has been noted between receipt of an alternative N. meningitidis serogroup B vaccine, MenB-fHbp (Trumenba; Pfizer) and N. gonorrhoeae infection. The MenB-fHbp vaccine contains recombinant fHbp but does not contain OMVs. Importantly, the homolog of fHbp in N. gonorrhoeae is not surface exposed, does not bind factor H, and is therefore not predicted to be protective against N. gonorrhoeae infection (237). In a retrospective, observational case-control study of 96,235 persons aged 16–23 years of age with a diagnosis of N. gonorrhoeae or C. trachomatis infection between 2016 and 2018 in New York City and Philadelphia, no significant association between MenB-fHbp vaccination and N. gonorrhoeae mono-infection was observed after adjustment for ethnicity, gender, and jurisdiction (adjusted prevalence ratio 0.97, 95% CI = 0.79–1.19) (237). This suggests that healthy vaccinee bias (where persons who adopt preventive vaccinations may be more likely to adopt other protective behaviors and therefore have reduced risk of disease acquisition) has not played a significant role in the association between meningococcal serogroup B OMV vaccine and N. gonorrhoeae protection.

Collectively, these retrospective, observational studies are limited by potential biases resulting from possible missing data associated with the use of health and immunization registry data. In addition, this non-randomized data may be confounded by differences in risk behavior between vaccinated and non-vaccinated persons, such that those who adopt a preventative meningococcal vaccine may also be more likely to adopt preventative behaviors that reduce the risk of N. gonorrhoeae infection. Further data are also required to determine vaccine effectiveness against N. gonorrhoeae infection in subpopulations at high risk of N. gonorrhoeae infection, such as people living with HIV (PLHIV) and men who have sex with men (MSM). Encouragingly, the first study investigating the impact of 4CMenB in PLHIV demonstrated promising results. This retrospective case-control study comprised 1,051 MSM living with HIV in Milan, Italy and demonstrated vaccine effectiveness of 4CMenB against N. gonorrhoeae infection of 42% (95% CI 6–64, P = 0.027), a figure that remained significant after adjustment in multivariable analysis (56). A further uncertainty remains regarding whether there are any differences in the protective efficacy of meningococcal serogroup B OMV-containing vaccines against N. gonorrhoeae infection at specific anatomical sites (i.e., genital, anorectal, or oropharyngeal infections) as well as the duration of vaccine-induced protection against N. gonorrhoeae infection.

Finally, the evidence regarding vaccine effectiveness against N. gonorrhoeae and C. trachomatis co-infection in published studies is mixed. In the initial New Zealand retrospective case-control study of MeNZB, vaccine effectiveness was observed against N. gonorrhoeae/C. trachomatis co-infection, albeit with a lower effect size. The estimated vaccine effectiveness against N. gonorrhoeae/C. trachomatis co-infection compared to C. trachomatis-only controls was 14% (95% CI, 1−26%) in this study, while estimated vaccine effectiveness against N. gonorrhoeae-only infection was 31% (95% CI, 21−39%) (34). By contrast, in the large retrospective case-control study described above of 109,737 individuals aged 16–23 years with C. trachomatis or N. gonorrhoeae infection in New York City and Philadelphia between 2016 and 2018, vaccination with 4CMenB was not protective against N. gonorrhoeae/C. trachomatis co-infection, despite an estimated two-dose vaccine effectiveness against N. gonorrhoeae-only infection of 40% (95% CI 23–53%) (53).

Randomized studies

The interim findings of the first randomized study of a meningococcal serogroup B OMV vaccine with 4CMenB were made available in February 2023. The French National Agency for AIDS Research (ANRS) DOXYVAC trial was a phase III randomized open-label factorial design trial of MSM on HIV PrEP with a history of STI in the previous 12 months (235). In this study, participants were randomized to two interventions: (i) two doses of 4CMenB or no vaccine (randomized 1:1) and (ii) doxycycline post-exposure prophylaxis (PEP) (200 mg within 72 hours of condomless sex) or no PEP (randomized 2:1). Participants underwent testing for N. gonorrhoeae infection at baseline, every 3 months and whenever they had symptoms of STIs. Testing for N. gonorrhoeae infection comprised NAAT (nucleic acid amplification test) of urine, oropharyngeal, and anorectal swabs every 3 months. The primary endpoint of the study was the incidence of a first episode of N. gonorrhoeae infection 1 month after the second dose using an intention-to-treat analysis. Of 546 MSM enrolled, 502 were included in the intention-to-treat analysis. The interim findings reported a significant reduction in incident N. gonorrhoeae infection between the two-dose 4CMenB recipients and unimmunized participants followed for 9 months, with the incidence of a first episode of N. gonorrhoeae infection 9.8 and 19.7 per 100 person-years in the 4CMenB arm and no vaccine arms, respectively (adjusted hazard ratio 0.49; 95% CI 0.27–0.88). There was no interaction for the primary endpoints between the doxycycline PEP and 4CMenB vaccination. No vaccine-related serious adverse events were reported. However, the results of this study are now under independent review due to a discrepancy between the results of the reported interim and final results, explained by the omission of a number of N. gonorrhoeae infections from the interim analysis (236). As the first reported randomized trial of a meningococcal B OMV vaccine, the final results of this trial and independent review are highly anticipated.

Further randomized studies of a two-dose schedule of 4CMenB are currently underway and are described in Table 5. Notably, four double-blind randomized-controlled trials are actively enrolling participants, including two large placebo-controlled multi-center clinical trials and a CHIM study (https://clinicaltrials.gov/study/NCT04415424; https://clinicaltrials.gov/study/NCT04350138; https://clinicaltrials.gov/study/NCT05766904; https://clinicaltrials.gov/study/NCT05294588). In addition, a randomized, open-label, single-site trial of 18- to 50-year-old gay and bisexual men on HIV pre-exposure prophylaxis or recent N. gonorrhoeae infection is planned (234). The three double-blind, randomized, placebo-controlled trials evaluating the impact of 4CMenB on natural infection will recruit from different populations, including (i) a multi-site Australian study of 18- to 50-year-old men (cis and trans), transexual women and non-binary people who have sex with men (https://clinicaltrials.gov/study/NCT04415424); (ii) a multi-site American study of 18- to 50-year-old healthy men and women (https://clinicaltrials.gov/study/NCT04350138); and (iii) a single-site Hong Kong study of MSM aged 18 or above with risk factors for gonorrhoea infection (https://clinicaltrials.gov/study/NCT05766904). The randomized-controlled CHIM study is a single-site, double-blind randomized controlled trial where two doses of 4CMenB are compared to quadrivalent influenza and tetanus/diphtheria vaccination. The aim is to recruit up to 140 male participants who will undergo urethral challenge with N. gonorrhoeae strain FA1090 after immunization with 4CMenB or the comparator vaccine. Participants will be randomized 1:1 to the 4CMenB or control vaccine arm and receive two immunizations prior to the anterior urethral bacterial challenge with 10⁶ colony-forming units of N. gonorrhoeae strain FA1090 in suspension. The primary outcome measured will be microbiological confirmation of urethral infection via detection of N. gonorrhoeae by culture or NAAT of urine or urethral swab (https://clinicaltrials.gov/study/NCT05294588). In addition, this CHIM will measure the proportion of participants that develop symptomatic disease and also present an opportunity for intensive biological sampling and immunological characterization of responses in those who have received 4CMenB compared to the control group. Furthermore, subgroup analysis of this data regarding anatomical site-specific risk of N. gonorrhoeae infection (e.g., genital, anorectal, and oropharyngeal) between the vaccinated and non-vaccinated groups in these randomized studies will be important to inform the potential impact of the 4CMenB vaccine on N. gonorrhoeae transmission at a population level. Increasing evidence suggests that oropharyngeal N. gonorrhoeae infection may play a significant role in N. gonorrhoeae transmission (19) and modeling studies suggest that the impact of a N. gonorrhoeae vaccine will be significantly reduced if the vaccine is not effective at the oropharynx (238).

TABLE 5.

Randomized trials of vaccine effectiveness of meningococcal vaccines on gonorrhoea infection currently in design, recruitment, or pre-publication phases^a

Clinical trial design	Vaccine and immunization schedule	Study population	Recruitment strategy	Primary outcome	Reference
Phase III, double-blinded, randomized, placebo-controlled, multi-centered trial evaluating the efficacy of 4CMenB in the prevention of gonorrhoea infection (GoGoVax).	4CMenB; Intramuscular administration, 2 doses, 3 months apart OR placebo.	18- to 50-year-old men (cis and trans), transexual women and non-binary people who have sex with men; either HIV negative and on PrEP or HIV positive with HIV viral load <200 copies/mL and CD4 count >350 cells/cmm.	730 participants enrolled and randomized 1:1. Recruitment for 12 months. After vaccination, all participants followed up 3 months for 24 months.	To measure whether 4CMenB changes the incidence of a first episode of N. gonorrhoeae infection. To compare the overall incidence of all episodes of N. gonorrhoeae infection diagnosed during the study period between vaccine and placebo arms.	Seib et al (https://clinicaltrials.gov/study/NCT04415424)
Phase II, randomized, observer-blind, placebo-controlled, multi-center trial evaluating the efficacy of 4CMenB in the prevention of urogenital/and or anorectal gonorrhoea infection	4CMenB; Intramuscular administration, 2 doses, 2 months apart OR placebo.	18- to 50-year-old healthy men and women	Approximately 2,200 participants and randomized 1:1. After vaccination, participants followed up 3 months for 16 months.	To measure the efficacy of 4CMenB in the prevention of urogenital and/or anorectal infection.	Marazzo et al (https://clinicaltrials.gov/study/NCT04350138)
Single-site double-randomized controlled trial evaluating the efficacy of 4CMenB in the prevention of gonorrhoea using a controlled human experimental infection with N. gonorrhoeae strain FA1090	Initial vaccination phase: 2 doses of intramuscular 4CMenB OR quadrivalent influenza and tetanus/diphtheria vaccine; post-challenge vaccination: crossover arm with receipt of either quadrivalent influenza and tetanus/diphtheria vaccines or 2 doses of 4Cmen B.	18- to 35-year-old men without a history of 4CMenB vaccination	Approximately 120–140 participants enrolled and randomized 1:1.	Infectivity of N. gonorrhoeae inoculum defined as the proportion of participants with microbiological evidence of N. gonorrhoeae by culture or NAAT in urine or urethral swab culture on the post-inoculation antibiotic treatment day in each study group.	Duncan et al (https://clinicaltrials.gov/study/NCT05294588)
Single-site, parallel, double-blind, randomized, placebo-controlled trial evaluating the efficacy of 4CMenB in the prevention of gonorrhoea infection	4CMenB; intramuscular administration, 2 doses, 1 month apart OR placebo	MSM aged 18 or above at risk of gonorrhoea infection (condomless sex with >1 man in last 6 months, the history of STI, inclination to have condomless sex, and other HIV PrEP-eligible criteria)	150 participants.	Incidence of N. gonorrhoeae infection between control and intervention groups	Kwan T et al (https://clinicaltrials.gov/study/NCT05766904)
Randomized, open-label, single-site trial evaluating the efficacy of 4CMenB in the prevention of gonorrhoea infection (MenGo)	4CMenB; intramuscular administration, 2 doses, 3 months apart	18- to 50-year-old gay and bisexual men that are currently taking HIV PrEP or have been diagnosed with gonorrhoea in the past 3 months	130 participants enrolled and randomized 1:1. Followed 3 months for 24 months.	Number of N. gonorrhoeae infections in participants over 2 years measured by NAAT	(234)

^a HIV, human immunodeficiency virus; MSM, men who have sex with men; NAAT, nucleic acid amplification test; PrEP, pre-exposure prophylaxis.

Biological plausibility

Biological plausibility for the association between meningococcal serogroup B OMV vaccines and protection against N. gonorrhoeae infection has been strengthened by basic science studies demonstrating a high level of genomic sequence identity between N. gonorrhoeae and the serogroup B N. meningitidis OMV protein antigens present in the MeNZB and 4CMenB vaccines (127, 128). Bioinformatic analysis of 22 proteins that comprise >90% of 4CMenB OMV content resulted in the identification of twenty orthologues of these proteins in N. gonorrhoeae strain FA1090, including sixteen with >90% identity and two with >80% identity (128). Of the OMV proteins that have an ortholog in N. gonorrhoeae, fourteen of these also have a high level of sequence identity with >400 N. gonorrhoeae strains available on GenBank (128). A further study comprising bioinformatic analysis of abundant 4CMenB OMV vaccine antigens among 940 N. gonorrhoeae strains from the United States, found that of all the predicted outer membrane proteins, OpcA (45%) and PorB (70%) had the lowest mean sequence similarity between the NZ98/254 N. meningitidis strain from which the 4CMenB OMV is derived and N. gonorrhoeae. In addition, although the porA gene was identified in 99.5% of N. gonorrhoeae isolates in this study, inactivating mutations render PorA a pseudogene in N. gonorrhoeae (127). Analysis of the additional recombinant antigens present in 4CMenB indicates that NadA is absent in N. gonorrhoeae (127) and although orthologs of NHBA, fHbp, GNA2091, and GNA1030 are present in N. gonorrhoeae strains, fHbp (239), GNA2091 (240), and GNA1030 (241) are not thought to be surface exposed. Importantly, the 4CMenB NZ98/254 N. meningitidis strain NHBA antigen shares 67% mean amino acid sequence similarity to N. gonorrhoeae (128), suggesting the presence of this antigen in the 4CMenB vaccine may provide an additive protective effect against N. gonorrhoeae infection.

In addition, analysis of the antibody response of rabbits, mice, and humans after immunization with 4CMenB, or the OMV present in 4CMenB has demonstrated the induction of cross-reacting gonorrhoea-specific antibodies (128, 242). For example, in rabbits immunized with the OMV present in 4CMenB, several cross-reactive proteins were detected by Western blot analysis of whole-cell lysates comprising three different N. gonorrhoeae strains, and an elevated enzyme-linked immunosorbent assay (ELISA) titer to N. gonorrhoeae strain 1291 OMVs was observed (128). Similar findings were observed in a serology study of humans who had received three doses of 4CMenB, with a significant rise in the ELISA titer against N. gonorrhoeae whole-cell lysates between pre- and post-vaccination. Western blot analysis of human post-vaccination sera also demonstrated reactivity to several gonococcal and meningococcal proteins (128). Further investigation in the estradiol-treated female mouse model demonstrated that subcutaneous and intraperitoneal immunization of mice with 4CMenB induced serum and vaginal antibodies to whole-cell lysates of six different N. gonorrhoeae strains, as well as serum and vaginal antibodies that cross-react with several OMV proteins, including promising N. gonorrhoeae vaccine targets such as MtrE and PilQ. Furthermore, vaccination with 4CMenB significantly reduced N. gonorrhoeae bacterial load and accelerated clearance of infection after N. gonorrhoeae vaginal inoculation in the estradiol-treated mouse model (242).

A number of investigators are currently undertaking studies to further characterize the immunological responses to a two-dose schedule of the 4CMenB vaccine. These include a study comprising up to 15 male and female participants conducted at the University of North Carolina, Chapel Hill in which the change in anti-gonococcal OMV- specific IgG, IgA, and immunoglobulin M (IgM) concentrations and the mean change in the proportion of CD4+ T lymphocytes expressing at least two different activation markers (interferon-gamma, tumor necrosis factor-alpha, and interleukin-2) will be measured after in vitro stimulation with N. gonorrhoeae strain FA1090 OMVs in participants after vaccination with two doses of 4CMenB (https://clinicaltrials.gov/study/NCT04094883). In another study, investigators at the University of Oxford and KEMRI-Wellcome Trust Collaborative Research Program aim to recruit approximately 50 male and female participants, including HIV-uninfected and HIV-infected individuals from existing follow-up cohorts in Mtwapa, Kenya. These investigators will also measure serum humoral and T-cell responses to N. gonorrhoeae before and after two doses of 4CMenB (https://clinicaltrials.gov/study/NCT04297436). Furthermore, in a study at the National Institute of Allergy and Infectious Diseases, 50 male and female participants will be recruited and serum and mucosal antibody responses at oropharyngeal, rectal, and vaginal sites will be measured before and after vaccination with two doses of 4CMenB (https://clinicaltrials.gov/study/NCT04722003). In addition, a number of the randomized two-dose 4CMen B vaccine efficacy studies described above will investigate serum (https://clinicaltrials.gov/study/NCT04350138) or serum and mucosal immune responses (https://clinicaltrials.gov/study/NCT04415424, 234).

In summary, there is substantial evidence of an association between meningococcal serogroup B OMV vaccines and reduced N. gonorrhoeae infection. This includes human ecological and observational trial data, evidence of overlap in important vaccine targets in meningococcal serogroup B OMV vaccines and N. gonorrhoeae and induction of cross-reactive antibody responses. Lacking are data defining the impact of meningococcal serogroup B OMV vaccines on N. gonorrhoeae infection at various anatomical sites and in different population groups. With a number of randomized-controlled studies assessing the vaccine efficacy of 4CMenB currently underway, further information will become available. Given the promising findings of meningococcal serogroup B OMV vaccines against N. gonorrhoeae infection to date, as well as the widespread availability and demonstrated safety data of vaccines such as 4CMenB (243), implementation of this vaccine in settings with particularly high N. gonorrhoeae prevalence should be considered.

IN THE PIPELINE: NEISSERIA GONORRHOEAE OUTER MEMBRANE VESICLE VACCINES

Importantly, several N. gonorrhoeae-specific OMV vaccines are in preclinical or clinical development (227, 244, https://clinicaltrials.gov/study/NCT05630859). These include the NGoXIM (227) and dmGC_0817560 (226, 244) native OMV vaccine candidates which are in the late stages of preclinical development (245), and generalized modules for membrane antigens (GMMA) vaccine, which is currently recruiting participants into a phase 1/2 study (https://clinicaltrials.gov/study/NCT05630859). The NGoXIM vaccine is being developed by Intravacc and TherapyX in the Netherlands and the United States, with funding from the US National Institute of Allergy and Infectious Diseases (246). This vaccine is a N. gonorrhoeae native OMV vaccine formulated for intranasal mucosal delivery combined with a sustained-release microsphere encapsulated IL-12 adjuvant (245). Studies have demonstrated that intravaginal and intranasal administration of this vaccine induced Th1-driven responses that accelerated the clearance of N. gonorrhoeae genital tract infection in mice (209, 227). Intranasal administration of this experimental vaccine generated antigonococcal serum IgG, salivary IgA and vaginal IgG, and IgA antibodies in female mice and antigonococcal serum IgG and salivary IgA antibodies in male mice. In addition, female mice that received intranasal immunization with this experimental vaccine demonstrated accelerated clearance of homologous and heterologous strains of N. gonorrhoeae infection. Further, various adaptations to this vaccine have been made to optimise its performance, including detergent-extraction of OMVs to reduce LOS content, and production of OMVs from N. gonorrhoeae strains with deleted rmp and lpxl1 genes to eliminate anti-Rmp blocking antibodies and reduce LOS endotoxicity. Studies of these modified vaccines have demonstrated accelerated clearance of vaginal gonococcal infection in the female mouse model (227).

The dmGC_0817560 vaccine is being developed by the Jenner Institute and Oxford Vaccine Group in the United Kingdom, with funding from CARB-X (245). This vaccine is also a native OMV vaccine formulated from a Chilean N. gonorrhoeae strain in which genes for Rmp and LpxL1 have been deleted, combined with an aluminum hydroxide adjuvant. Preclinical studies demonstrate that parenteral delivery of this experimental vaccine induced anti-gonococcal serum and vaginal mucosal IgG and IgA antibodies and gonococcal-specific Th1/Th17 CD4+ T-cell responses in the female mouse model. In addition, female mice immunized with the candidate vaccine demonstrated accelerated clearance of genital N. gonorrhoeae infection with a heterologous strain and cleared infection significantly faster than mice immunized with 4CMenB (226).

The intramuscular NgG generalized modules for membrane antigens (GMMA) vaccine is developed by GlaxoSmithKline in the United States (https://clinicaltrials.gov/study/NCT05630859). GMMA vaccines are OMV vaccines that have been produced from bacterial strains that have been genetically modified to increase the production of OMVs and reduce endotoxin levels (247). To our knowledge, preclinical studies of this experimental vaccine have not been published; however, a phase 1/2 study of this experimental vaccine has commenced recruitment, aiming to evaluate the safety, reactogenicity, immunogenicity, and efficacy of this experimental vaccine in a randomized, observer-blind, placebo-controlled multi-center study in an estimated 774 participants aged 18–50 years of age (https://clinicaltrials.gov/study/NCT05630859). The phase 1 dose-escalation safety study for this vaccine is now complete and the study has entered phase 2; furthermore, the US Food and Drug Administration (FDA) has granted a Fast Track designation to accelerate its path to US FDA submission (248).

These vaccines represent the next generation of anti-gonococcal OMV vaccines that have been specifically engineered to build on the scientific advances in understanding N. gonorrhoeae pathogenesis and host immune response, as well as the significant progress made in the past decade to explore the association between serogroup B meningococcal OMV vaccines and reduced N. gonorrhoeae infection. These include (i) the use of a N. gonorrhoeae strain to produce OMVs for use in next-generation vaccines, potentially increasing the specificity of the immune responses induced by this multi-antigen vaccine technology; (ii) the inclusion of adjuvants that stimulate a Th1 response; (iii) genetically modifying selected gonococcal strains to reduce the endotoxicity associated with LOS and blocking antibodies induced by Rmp; and (iv) evaluation of mucosal administration to increase the immune response at the mucosal sites of gonorrhoea infection.

POTENTIAL PUBLIC HEALTH IMPACT OF A NEISSERIA GONORRHOEAE VACCINE

Determining the potential public health impact of a N. gonorrhoeae vaccine requires consideration of the health, economic, and societal value of future N. gonorrhoeae vaccines. The WHO convened an international panel of experts in 2019 to define the public health value and preferred product characteristics of N. gonorrhoeae vaccines (37, 38). At this meeting, prevention of poor sexual and reproductive health outcomes and addressing the threat of AMR were identified as the key goals of future N. gonorrhoeae vaccines. Important considerations to define the target product profile of a N. gonorrhoeae vaccine include (i) defining the target endpoint for assessment of vaccine efficacy (e.g., prevention of infection, vs prevention of symptomatic disease, vs prevention of AMR); (ii) the target population for the vaccine (e.g., all individuals prior to sexual activity or high-risk populations; whether to include both females and males); and (iii) the target programmatic delivery program (e.g., schools or sexual health clinics). Notably, the preferred product characteristics of a potential vaccine may also vary according to the epidemiology of N. gonorrhoeae infection and AMR in the target population. The promotion of a vaccine against a sexually transmitted infection may also require adaptation to the specific socio-cultural context to maximize acceptability.

Modeling studies are important to understanding the potential impact of N. gonorrhoeae vaccines on gonococcal infection and AMR and to aid policy development and program delivery. The public health impact and cost-effectiveness of potential N. gonorrhoeae vaccines have been modeled in several studies, including various target population groups, vaccine program strategies, and levels of vaccine coverage. In addition, the effects of various levels of vaccine efficacy and duration of protection have been investigated.

Modeling the impact of Neisseria gonorrhoeae vaccines in heterosexual populations

The impact of gonococcal vaccines delivered prior to the commencement of sexual activity has been estimated in a number of heterosexual population model studies. Craig et al. used an individual-based, epidemiological simulation model of a N. gonorrhoeae vaccine delivered prior to the commencement of sexual activity in a heterosexual population of 100,000 individuals using theoretical vaccines of 10–100% efficacy and 2.5- to 20-year duration of protection (249). The model output predicted that N. gonorrhoeae prevalence could be reduced by at least 90% after 20 years by a non-waning vaccine with 50% efficacy and universal vaccination coverage. The duration of protection of a theoretical vaccine had a significant effect on the prevalence of N. gonorrhoeae in the model; a vaccine with 100% efficacy that waned after 7.5 years was predicted to reduce N. gonorrhoeae prevalence by at least 90% after 20 years, one whose protection waned after 5 years by 50% and one with 2.5 years protection having minimal impact on prevalence. Similarly, vaccine coverage played a key role in predicted vaccine impact, with 50% vaccine coverage of a N. gonorrhoeae vaccine with 50% efficacy predicted to reduce N. gonorrhoeae prevalence by 50% after 20 years, compared to at least 90% reduction if the same vaccine had universal vaccine coverage (249).

The impact of a 4CMenB adolescent vaccine on N. gonorrhoeae prevalence has been estimated in a number of studies using transmission models of N. gonorrhoeae infection among heterosexual populations. Carey et al. developed a heterosexual transmission model of 15- to 24-year-olds in the United States using Approximate Bayesian Computation analysis to account for uncertainty in key transmission factors (rates of natural clearance, rates of screening, proportion of symptomatic infections, and annual number of sexual contacts). The results of this analysis estimated that a vaccine with 30% efficacy and 2-year duration of protection would result in a 12.2–39.4% reduction in N. gonorrhoeae prevalence if 50% vaccine coverage was achieved in this population, and 4.8–14.3% reduction in prevalence if 20% vaccine coverage was achieved (250). Looker et al. developed a deterministic transmission-dynamic model of heterosexual 13- to 64-year-olds in England and estimated the impact a vaccinating 14-year-olds with a vaccine with 31% efficacy, 6-year duration of protection and 85% of vaccine uptake (251). The results of this analysis indicated that 10% (95% CrI 8–13%), 18% (95%CrI 13–23%), and 25% (95%CrI 17–33%) of cases of N. gonorrhoeae infections would be prevented in this population over a 10-, 20-, and 70-year period, respectively (251). Regnier et al. modeled the potential health and economic impact of a 4CMenB adolescent vaccination on N. gonorrhoeae infection with an estimated 20% vaccine efficacy, 10-year duration of protection, and 70.5% vaccination rate using a decision-analysis model developed using published US healthcare utilization and cost data (252). This model predicted that vaccination could prevent 83,167 lifetime N. gonorrhoeae infections and 55 lifetime HIV infections per vaccinated birth cohort in the United States. This was predicted to reduce the direct medical costs of N. gonorrhoeae infection by US$28.7 million and reduce income and productivity losses by US$40.0 million (252).

Modeling the impact of Neisseria gonorrhoeae vaccines in men who have sex with men populations

The impact of gonococcal vaccines within a male population of MSM has been modeled in four studies. Using a stochastic transmission-dynamic model that incorporated heterogeneous sexual behavior and symptomatic and asymptomatic infection in an MSM population based on surveillance data from England, Whittles et al. assessed the potential N. gonorrhoeae vaccination impact and the feasibility of achieving the WHO target of reducing N. gonorrhoeae incidence by 90% by 2030 (253). This study estimated that the WHO target is achievable even if the worst-case scenario where untreatable AMR infection emerges if all MSM attending sexual health clinics receive a vaccine with ≥52% efficacy and ≥6 years of protection; or ≥70% efficacy and ≥3 years protection (253). Heinje et al. developed a compartmental model of N. gonorrhoeae transmission among a population of MSM with heterogeneous sexual behavior and symptomatic and asymptomatic infection. This model also incorporated AMR as a stepwise increase in minimum inhibitory concentration (MIC) and eventual resistance to ceftriaxone. The impact of a partially protective vaccine with 30% efficacy that provided 2 years of protection delivered to high-risk MSM (with baseline gonorrhoea prevalence of 12.5%) on N. gonorrhoeae prevalence and AMR was assessed. The modeling output indicated that a vaccine with 30% vaccine effectiveness could not prevent AMR despite high uptake or long-term protection but would increase the time to development of AMR by several years (254).

More recent modeling studies of N. gonorrhoeae vaccines within male populations of MSM have added increasing layers of complexity to their models. Hui et al. simulated anatomical site-specific data into their individual-based mathematical model of N. gonorrhoeae transmission in an urban population of 10,000 MSM with heterogenous sexual behavior and symptomatic and asymptomatic infection (238). Three types of vaccine efficacy were investigated, including (i) “protective efficacy,” the protection of a vaccinated individual against acquiring N. gonorrhoeae infection; (ii) “transmission suppression efficacy,” the reduction of N. gonorrhoeae transmission from a vaccinated individual; and (iii) “symptom suppression efficacy,” the reduction of symptoms of N. gonorrhoeae infection in the setting of infection in a vaccinated individual. It was estimated that N. gonorrhoeae elimination may be possible within the population in this model in 8 years with vaccines with ≥50% efficacy and 2 years of protection if 30% of MSM presenting for sexually transmitted infection testing were vaccinated and underwent a booster vaccination every 3 years. Importantly, it was estimated that vaccine impact may be substantially reduced if a N. gonorrhoeae vaccine is not effective at the oropharynx and that prevalence may actually increase if a vaccine prevents symptoms but does not prevent infection or transmission. In addition, this study estimated that N. gonorrhoeae vaccines that reduced transmission without conferring protection from N. gonorrhoeae infection would have a similar impact on N. gonorrhoeae prevalence as vaccines with protective efficacy and that the impact of vaccines with both transmission suppression and protective efficacy would be additive (238).

Whittles et al.’s most recent study incorporated a cost-effectiveness analysis into their transmission-dynamic model that incorporated heterogeneous sexual behavior and symptomatic and asymptomatic infection in an MSM population based on surveillance data from England (255). The impact and cost-effectiveness of four different vaccination strategies were assessed in this study. It was estimated that vaccination of adolescents in schools would have little impact on N. gonorrhoeae prevalence, whereas vaccination of individuals on attendance for STI testing at sexual health clinics would have the largest impact. Vaccination on the diagnosis of N. gonorrhoeae infection at sexual health clinics would have a moderate impact but require fewer doses than a vaccination on attendance approach, while vaccination of sexual health clinic attendees according to risk (defined as individuals diagnosed with N. gonorrhoeae infection in the past 12 months or with >5 sexual partners per year) was estimated to have a similar impact as vaccination of all STI clinic attendees, however, required administration of fewer vaccine doses. The most cost-effective strategy for vaccines with moderate efficacy or duration of protection was vaccination according to risk, whereas vaccination on diagnosis of N. gonorrhoeae infection was most cost-effective for highly efficacious and long-lasting vaccines. The impact of 4CMenB vaccination against N. gonorrhoeae infection, assuming a vaccine efficacy of 31% and protection lasting 18 months after two-dose primary vaccination and 36 months after single-dose booster vaccination, was also evaluated. A strategy comprising 4CMenB vaccination administered according to risk was estimated to prevent 110,200 cases, gaining a mean of 100.3 QALYs and saving a mean of £7.9 million over 10 years (255).

Modeling the impact of Neisseria gonorrhoeae vaccines in low- and middle-income settings

The use of modeling to assess the impact of N. gonorrhoeae vaccines in a high-prevalence LMIC setting was reported in a recent study (256). Using a compartmental model of N. gonorrhoeae transmission among a 15- to 49-year-old heterosexual population in a high-prevalence LMIC setting similar to South Africa, Padeniya et al. modeled the impact of vaccines with varying levels of protective and transmission suppression efficacy on the prevalence N. gonorrhoeae infection. In addition, the impact of vaccination programs delivered to various age- and sexual-activity groups was assessed. Vaccination of 15- to 49-year-olds with a vaccine with protective efficacy of 25%, a 5-year duration of protection, and 10% annual vaccine uptake would have the greatest impact on N. gonorrhoeae prevalence, with the model predicting that a 50% reduction in prevalence would be achieved, compared to 25% reduction in prevalence if only 15- to 24-year-olds were vaccinated. Vaccination of only individuals with high sexual activity was predicted to achieve an almost equivalent reduction in N. gonorrhoeae prevalence to vaccinating the entire 15- to 49-year-old population using theoretical vaccines with the same efficacy, duration of protection, and uptake characteristics over the same time period but was able to achieve this more efficiently, requiring approximately three times fewer vaccinations. Similar to the findings of the modeling study by Hui et al.’s of an urban MSM population, this study estimated that a vaccine with both protective and transmission suppression efficacy would have an additive impact on reducing N. gonorrhoeae prevalence (256).

In summary, modeling studies undertaken in both heterosexual and MSM populations using data from various international settings have demonstrated that delivery of vaccines with efficacy and duration of protection derived from estimates of the currently available 4CMenB vaccine could have a significant impact on N. gonorrhoeae prevalence, and even be cost-saving when implemented in select high-risk populations (252, 255). In addition, such vaccines could delay the development of AMR, providing time for more efficacious vaccines and novel antimicrobials to be developed (254). Furthermore, even moderate improvements in N. gonorrhoeae vaccine efficacy and duration of protection may have a significant impact on N. gonorrhoeae infection prevalence, with some studies estimating that N. gonorrhoeae infection may be eliminated or prevalence reduced by 90% through the implementation of vaccines with approximately 50% efficacy and 2–6 years duration of protection (238, 253). Given the prediction that vaccine impact may be reduced if a vaccine is not effective at the oropharynx (238), further data regarding vaccine efficacy at different anatomical sites is pivotal in informing current and future vaccine implementation strategies. In addition, modeling studies simulating the epidemiological characteristics of N. gonorrhoeae infection in LMIC settings, where the burden of gonorrhoea infection is greatest, should be prioritized.

QUESTIONS REMAINING: RESEARCH PRIORITIES FOR GONOCOCCAL VACCINES

This is an exciting time for N. gonorrhoeae vaccine development, with evidence from observational studies suggesting that meningococcal B OMV vaccines may have efficacy against N. gonorrhoeae infection and multiple randomized trials underway. However, there are several key questions that remain unanswered about the currently available serogroup B meningococcal vaccines. These include (i) the major effector antigen/s responsible for the efficacy of OMV vaccines; (ii) the efficacy of vaccination on infection at various anatomical sites; (iii) the duration of protective immunity; and (iv) whether there is an immune correlate of protection that can be measured by laboratory tests. A number of these knowledge gaps, such as efficacy at various anatomical sites and further data on duration of protective immunity may be informed by currently recruiting clinical trials of the 4CMenB vaccine. In addition, a randomized trial of 4CMenB in a male urethritis gonorrhoea CHIM may provide more detailed data regarding immune responses to key serogroup B meningococcal OMV vaccine antigens.

The priority research areas outlined in the WHO Global STI Vaccine Roadmap and recently reviewed in the WHO stakeholder consultation regarding public health value and preferred product characteristics of gonococcal vaccines in 2019 remain pertinent today. These include (i) improving access to quality epidemiological data regarding infection including AMR; (ii) advancing the understanding of the natural history of gonorrhoea infection; (iii) modeling predicted gonorrhoea vaccine impact and cost-effectiveness; (iv) accelerating basic science, translational, immunobiologic, and clinical research; and (v), advocating for investment and planning for policy and implementation decisions (35, 37). Although there is much work to be done, there is significant momentum in N. gonorrhoeae vaccine development that is being fueled by the bench to bedside research described in this review.

CONCLUSION

In this review, we have described the unique challenges involved in the development of a N. gonorrhoeae vaccine. We have reviewed the breadth of data pertaining to N. gonorrhoeae vaccines, ranging from an overview of historical vaccines; to multi-omics vaccine antigen discovery and preclinical vaccine research; as well as contemporary clinical trials and modeling studies to inform potential vaccine implementation strategies. As we approach an important inflection point, with the imminent release of the results of six randomized trials of the efficacy of 4CMenB against N. gonorrhoeae infection, it is important to consider first how to best implement vaccination programs using currently available vaccines to protect against N. gonorrhoeae infection and second how to improve upon these technologies to develop the next generation of N. gonorrhoeae vaccines. The next generation of N. gonorrhoeae-specific OMV vaccines that include modifications of currently available vaccines may improve efficacy. However, alternative vaccines utilizing a range of gonococcal antigens that have shown promise in preclinical studies should also be pursued. Although these vaccine candidates are at a much earlier stage of development and their safety and efficacy in humans have not yet been demonstrated, there is good reason to hold optimism that they will confer improved protection over those currently available. As N. gonorrhoeae prevalence continues to increase and the threat of AMR to the treatment of gonorrhoea becomes increasingly urgent, expediting the development of highly efficacious N. gonorrhoeae vaccines and implementing high-coverage vaccine programs is a key priority for sexual and reproductive health.

Biographies

Eloise Williams is an Infectious Diseases Physician and a Clinical Microbiologist at the Victorian Infectious Diseases Reference Laboratory, Melbourne, Australia, and is currently undertaking a Ph.D. through the Department of Infectious Diseases at the Peter Doherty Institute of Infection and Immunity at the University of Melbourne. She completed her medical studies (MBBS) at the University of Melbourne and a Master of Public Health and Tropical Medicine at James Cook University. Her research interests include public health, sexually transmitted infections, and blood-borne viruses. A primary aim of her Ph.D. is to develop a N. gonorrhoeae oropharyngeal controlled-human infection model to further characterize the pathogenesis of oropharyngeal N. gonorrhoeae infection and accelerate the development of novel vaccines and therapeutics.

Kate L. Seib is a Professor of Microbiology at Griffith University, where she is a Group Leader and an Associate Director of Research at the Institute for Glycomics. She completed a Ph.D. in microbiology in 2004 at the University of Queensland and was a Postdoctoral Fellow and Project Leader at Novartis Vaccines, where she was part of the team working on the meningococcal B vaccine, 4CMenB. Her research focuses on N. gonorrhoeae pathogenesis and host immune response, to identify therapeutic and preventative targets against N. gonorrhoeae infection. She has also led a number of studies modeling the impact of N. gonorrhoeae vaccines and is leading a multicenter randomized clinical trial evaluating the efficacy of the 4CMenB vaccine against N. gonorrhoeae infection.

Christopher K. Fairley is the Director of the Melbourne Sexual Health Centre and a Professor of Public Health at Monash University. His principle research interests are the public health control of sexually transmitted infections and the effectiveness of clinical services. He has been described by the Lancet as a "pioneer of sexually transmitted infection research." His work has substantially contributed to the understanding of the epidemiology, microbiology, and novel treatment and prevention approaches for N. gonorrhoeae infection. In particular, his work has been pivotal in identifying the significant role of the oropharynx in gonorrhoea transmission.

Georgina L. Pollock is a Post-Doctoral Researcher in the Department of Infectious Diseases at the Peter Doherty Institute for Infection and Immunity at the University of Melbourne. She completed a Ph.D. investigating the molecular mechanisms used by pathogenic Escherichia coli to evade the immune responses in the gut at the University of Melbourne in 2019 and is now part of a team that uses genomic approaches to investigate the dynamics of sexually transmitted infections. Her current research is focused on developing a N. gonorrhoeae oropharyngeal controlled human infection model.

Jane S. Hocking is a Professor of Epidemiology and implementation researcher at the Melbourne School of Population and Global Health at the University of Melbourne. She completed a Master of Public Health and Ph.D. at the University of Melbourne. Her research interests include the epidemiology and control of sexually transmitted infections, sexual health, and the implementation and evaluation of primary care interventions. Her work has substantially contributed to the understanding of the significant role of the oropharynx in gonorrhea transmission and novel approaches to gonorrhea prevention and prevention.

James S. McCarthy is a Director of the Victorian Infectious Diseases Service at the Royal Melbourne Hospital and a Professor of Medicine at the Doherty Institute. He received his medical degree from the University of Melbourne before undertaking clinical and research training in Australia, the UK, and the US at the University of Maryland and the Laboratory for Parasitic Diseases, National Institutes of Health, Bethesda, MD, before returning to Australia in 1997. His research has focused on the diagnosis and treatment of parasitic diseases, with a major recent focus on the development and application of controlled human infection models of malaria and other pathogenic organisms, including Neisseria gonorrhoeae. This has enabled the study of the host-pathogen interaction, the development of diagnostic biomarkers, and the evaluation of investigational drugs and vaccines.

Deborah A. Williamson is a member of the Royal College of Physicians and a Fellow of the Royal College of Pathologists. She has had a number of roles in clinical and public health microbiology in Australia, including as Deputy Director of the Microbiological Diagnostics Unit Public Health Laboratory, Director of Microbiology at Royal Melbourne Hospital, and Director of the Victorian Infectious Diseases Reference Laboratory. Her research has focused on public health microbiology, particularly sexually transmitted infections, microbial genomics, and antimicrobial resistance. Over the past decade, Deborah has undertaken numerous studies of the epidemiology, microbiology, and novel treatment and prevention strategies for N. gonorrhoeae infection.