Profiling IgG and IgA antibody responses during vaccination and infection in a high-risk gonorrhoea population.

Development of a vaccine against gonorrhoea is a global priority, driven by the rise in antibiotic resistance. Although Neisseria gonorrhoeae (Ng) infection does not induce substantial protective immunity, highly exposed individuals may develop immunity against re-infection with the same strain. Retrospective epidemiological studies have shown that vaccines containing Neisseria meningitidis (Nm) outer membrane vesicles (OMVs) provide a degree of cross-protection against Ng infection. We conducted a clinical trial (NCT04297436) of 4CMenB (Bexsero, GSK), a licensed Nm vaccine containing OMVs and recombinant antigens, comprising a single arm, open label study of two doses with 50 adults in coastal Kenya who have high exposure to Ng. Data from a Ng antigen microarray established that serum IgG and IgA reactivities against the gonococcal homologs of the recombinant antigens in the vaccine peaked at 10 but had declined by 24 weeks. For most reactive OMV-derived antigens, the reverse was the case. A cohort of similar individuals with laboratory-confirmed gonococcal infection were compared before, during, and after infection: their reactivities were weaker and differed from the vaccinated cohort. We conclude that the cross-protection of the 4CMenB vaccine against gonorrhoea could be explained by cross-reaction against a diverse selection of antigens derived from the OMV component.

Vaccine effectiveness and impact of meningococcal vaccines against gonococcal infections: A systematic review and meta-analysis.

OBJECTIVES: To systematically review and synthesis the evidence of vaccine effectiveness (VE) and impact (VI) of meningococcal vaccines in preventing gonorrhoea. METHODS: We systematically evaluated studies. Literature searches were conducted in PubMed, Embase, Cochrane Library, CINAHL, Google Scholar, clinical trial registries, and major health and immunisation conferences. Meta-analysis was performed with the DerSimonian-Laird random-effects model to estimate the pooled VE. RESULTS: Twelve studies met the criteria for inclusion. VE of meningococcal B (MenB) outer membrane vesicle (OMV) vaccines was evaluated in nine studies, with one study evaluating a non-OMV vaccine, MenB-FHbp. The majority of studies targeted individuals aged 15-30 years. Adjusted VE for OMV vaccines against gonorrhoea ranged from 22% to 46%. MenB-FHbp did not show protection against gonorrhoea. The pooled VE estimates of OMV vaccines against any gonorrhoea infection following the full vaccine series were 33-34%. VI was assessed for 4CMenB in Canada and Australia, for VA-MENGOC-BC in Cuba; and for MenBvac in Norway. VI ranged from a 30% to 59% reduction in gonorrhoea incidence. CONCLUSIONS: 4CMenB and other MenB-OMV vaccines show moderate effectiveness against gonorrhoea. Further research is required to explore the factors associated with vaccine protection, informing more effective vaccination strategies for the management of gonococcal infections.

Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria.

The genus Neisseria, which colonizes mucosal surfaces, includes both commensal and pathogenic species that are exclusive to humans. The two pathogenic Neisseria species are closely related but cause quite different diseases, meningococcal sepsis and meningitis (Neisseria meningitidis) and sexually transmitted gonorrhea (Neisseria gonorrhoeae). Although obvious differences in bacterial niches and mechanisms for transmission exists, pathogenic Neisseria have high levels of conservation at the levels of nucleotide sequences, gene content and synteny. Species of Neisseria express broad-spectrum O-linked protein glycosylation where the glycoproteins are largely transmembrane proteins or lipoproteins localized on the cell surface or in the periplasm. There are diverse functions among the identified glycoproteins, for example type IV biogenesis proteins, proteins involved in antimicrobial resistance, as well as surface proteins that have been suggested as vaccine candidates. The most abundant glycoprotein, PilE, is the major subunit of pili which are an important colonization factor. The glycans attached can vary extensively due to phase variation of protein glycosylation (pgl) genes and polymorphic pgl gene content. The exact roles of glycosylation in Neisseria remains to be determined, but increasing evidence suggests that glycan variability can be a strategy to evade the human immune system. In addition, pathogenic and commensal Neisseria appear to have significant glycosylation differences. Here, the current knowledge and implications of protein glycosylation genes, glycan diversity, glycoproteins and immunogenicity in pathogenic Neisseria are summarized and discussed.

Characterization of Neisseria gonorrhoeae colonization of macrophages under distinct polarization states and nutrients environment.

Neisseria gonorrhoeae (Ng) is a uniquely adapted human pathogen and the etiological agent of gonorrhea, a sexually transmitted disease. Ng has developed numerous mechanisms to avoid and actively suppress innate and adaptive immune responses. Ng successfully colonizes and establishes topologically distinct colonies in human macrophages and avoids phagocytic killing. During colonization, Ng manipulates the actin cytoskeleton to invade and create an intracellular niche supportive of bacterial replication. The cellular reservoir(s) supporting bacterial replication and persistence in gonorrhea infections are poorly defined. The manner in which gonococci colonize macrophages points to this innate immune phagocyte as a strong candidate for a cellular niche during natural infection. Here we investigate whether nutrients availability and immunological polarization alter macrophage colonization by Ng. Differentiation of macrophages in pro-inflammatory (M1-like) and tolerogenic (M2-like) phenotypes prior to infection reveals that Ng can invade macrophages in all activation states, albeit with lower efficiency in M1-like macrophages. These results suggest that during natural infection, bacteria could invade and grow within macrophages regardless of the nutrients availability and the macrophage immune activation status.

IL-17C is a driver of damaging inflammation during Neisseria gonorrhoeae infection of human Fallopian tube.

The human pathogen Neisseria gonorrhoeae ascends into the upper female reproductive tract to cause damaging inflammation within the Fallopian tubes and pelvic inflammatory disease (PID), increasing the risk of infertility and ectopic pregnancy. The loss of ciliated cells from the epithelium is thought to be both a consequence of inflammation and a cause of adverse sequelae. However, the links between infection, inflammation, and ciliated cell extrusion remain unresolved. With the use of ex vivo cultures of human Fallopian tube paired with RNA sequencing we defined the tissue response to gonococcal challenge, identifying cytokine, chemokine, cell adhesion, and apoptosis related transcripts not previously recognized as potentiators of gonococcal PID. Unexpectedly, IL-17C was one of the most highly induced genes. Yet, this cytokine has no previous association with gonococcal infection nor pelvic inflammatory disease and thus it was selected for further characterization. We show that human Fallopian tubes express the IL-17C receptor on the epithelial surface and that treatment with purified IL-17C induces pro-inflammatory cytokine secretion in addition to sloughing of the epithelium and generalized tissue damage. These results demonstrate a previously unrecognized but critical role of IL-17C in the damaging inflammation induced by gonococci in a human explant model of PID.

Comprehensive observational study evaluating the enduring effectiveness of 4CMenB, the meningococcal B vaccine against gonococcal infections in the Northern Territory and South Australia, Australia: study protocol.

INTRODUCTION: The effectiveness of antibiotics for treating gonococcal infections is compromised due to escalating antibiotic resistance; and the development of an effective gonococcal vaccine has been challenging. Emerging evidence suggests that the licensed meningococcal B (MenB) vaccine, 4CMenB is effective against gonococcal infections due to cross-reacting antibodies and 95% genetic homology between the two bacteria, Neisseria meningitidis and Neisseria gonorrhoeae, that cause the diseases. This project aims to undertake epidemiological and genomic surveillance to evaluate the long-term protection of the 4CMenB vaccine against gonococcal infections in the Northern Territory (NT) and South Australia (SA), and to determine the potential benefit of a booster vaccine doses to provide longer-term protection against gonococcal infections. METHODS AND ANALYSES: This observational study will provide long-term evaluation results of the effectiveness of the 4CMenB vaccine against gonococcal infections at 4-7 years post 4CMenB programme implementation. Routine notifiable disease notifications will be the basis for assessing the impact of the vaccine on gonococcal infections. Pathology laboratories will provide data on the number and percentage of N. gonorrhoeae positive tests relative to all tests administered and will coordinate molecular sequencing for isolates. Genome sequencing results will be provided by SA Pathology and Territory Pathology/New South Wales Health Pathology, and linked with notification data by SA Health and NT Health. There are limitations in observational studies including the potential for confounding. Confounders will be analysed separately for each outcome/comparison. ETHICS AND DISSEMINATION: The protocol and all study documents have been reviewed and approved by the SA Department for Health and Well-being Human Research Ethics Committee (HREC/2022/HRE00308), and the evaluation will commence in the NT on receipt of approval from the NT Health and Menzies School of Health Research Human Research Ethics Committee. Results will be published in peer-reviewed journals and presented at scientific meetings and public forums.

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5'-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea. IMPORTANCE: Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Use of a meningococcal group B vaccine (4CMenB) in populations at high risk of gonorrhoea in the UK.

The meningococcal group B vaccine, 4CMenB, is a broad-spectrum, recombinant protein vaccine that is licensed for protection against meningococcal group B disease in children and adults. Over the past decade, several observational studies supported by laboratory studies have reported protection by 4CMenB against gonorrhoea, a sexually transmitted infection caused by Neisseria gonorrhoeae. Gonorrhoea is a major global public health problem, with rising numbers of diagnoses and increasing resistance to multiple antibiotics. In England, more than 82 000 cases of gonorrhoea were diagnosed in 2022, with nearly half of the cases diagnosed among gay, bisexual, and other men who have sex with men. There are currently no licensed vaccines against gonorrhoea but 4CMenB is estimated to provide 33-47% protection against gonorrhoea. On Nov 10, 2023, the UK Joint Scientific Committee on Vaccination and Immunisation agreed that a targeted programme should be initiated using 4CMenB to prevent gonorrhoea among individuals at higher risk of infection attending sexual health services in the UK. This decision was made after reviewing evidence from retrospective and prospective observational studies, laboratory and clinical data, national surveillance reports, and health economic analyses. In this Review, we summarise the epidemiology of invasive meningococcal disease and gonorrhoea in England, the evidence supporting the use of 4CMenB for protection against gonorrhoea, and the data needed to inform long-term programme planning and extension to the wider population.

Review of mathematical models of Neisseria gonorrhoeae vaccine impact: Implications for vaccine development.

An effective prophylactic vaccine for prevention of Neisseria gonorrhoeae infection would have a major impact on sexual and reproductive health worldwide. Interest in developing gonorrhoea vaccines is growing due to the reported high rates of N. gonorrhoeae infections globally, and the threat of antimicrobial resistance. Several gonorrhoea vaccine candidates are currently under evaluation and various mathematical models have been used to assess the potential population-level impact a gonorrhoea vaccine may have once available. Here we review key aspects of gonorrhoea vaccine mathematical modelling studies, including model structures, populations considered, and assumptions used as well as vaccine characteristics and implementation scenarios investigated. The predicted vaccine impact varied between studies, ranging from as little as ∼17 % reduction in N. gonorrhoeae prevalence after 30 years up to 100 % reduction after 5 years. However, all studies predicted that even a partially effective gonorrhoea vaccine could have a substantial impact in reducing N. gonorrhoeae prevalence or incidence, particularly when high coverage is achieved within either important risk groups or the overall sexually active population. As expected, higher vaccine efficacy against acquisition of N. gonorrhoeae and longer duration of protection were linked to greater reductions in infections. A vaccine that alters onward transmission could also substantially reduce infections. Several gaps and research needs have been identified by researchers in the field and via this narrative literature review. For example, future modelling to inform gonorrhoea vaccine development and implementation should consider additional populations that are at high risk of N. gonorrhoeae infection, especially in low- and middle-income settings, as well as the impact of vaccination on the potential adverse sexual and reproductive health outcomes of infection. In addition, more detailed and robust epidemiological, biological, and behavioural data is needed to enable more accurate and robust modelling of gonorrhoea vaccine impact to inform future scientific and public health decision-making.

Vaccination May Be Economically and Epidemiologically Advantageous Over Frequent Screening for Gonorrhea Prevention.

BACKGROUND: Gonorrhea's rapid development of antimicrobial resistance underscores the importance of new prevention modalities. Recent evidence suggests that a serogroup B meningococcal vaccine may be partially effective against gonococcal infection. However, the viability of vaccination and the role it should play in gonorrhea prevention are an open question. METHODS: We modeled the transmission of gonorrhea over a 10-year period in a heterosexual population to find optimal patterns of year-over-year investment of a fixed budget in vaccination and screening programs. Each year, resources could be allocated to vaccinating people or enrolling them in a quarterly screening program. Stratifying by mode (vaccination vs. screening), sex (male vs. female), and enrollment venue (background screening vs. symptomatic visit), we consider 8 different ways of controlling gonorrhea. We then found the year-over-year pattern of investment among those 8 controls that most reduced the incidence of gonorrhea under different assumptions. A compartmental transmission model was parameterized from existing literature in the US context. RESULTS: Vaccinating men with recent symptomatic infection, which selected for higher sexual activity, was optimal for population-level gonorrhea control. Given a prevention budget of $3 per capita, 9.5% of infections could be averted ($299 per infection averted), decreasing gonorrhea sequelae and associated antimicrobial use by similar percentages. These results were consistent across sensitivity analyses that increased the budget, prioritized incidence or prevalence reductions in women, or lowered screening costs. Under a scenario where only screening was implemented, just 5.5% of infections were averted. CONCLUSIONS: A currently available vaccine, although only modestly effective, may be superior to frequent testing for population-level gonorrhea control.

Vaccine value profile for Neisseria gonorrhoeae.

Neisseria gonorrhoeae infection (gonorrhoea) is a global public health challenge, causing substantial sexual and reproductive health consequences, such as infertility, pregnancy complications and increased acquisition or transmission of HIV. There is an urgency to controlling gonorrhoea because of increasing antimicrobial resistance to ceftriaxone, the last remaining treatment option, and the potential for gonorrhoea to become untreatable. No licensed gonococcal vaccine is available. Mounting observational evidence suggests that N. meningitidis serogroup B outer membrane vesicle-based vaccines may induce cross-protection against N. gonorrhoeae (estimated 30%-40% effectiveness using the 4CMenB vaccine). Clinical trials to determine the efficacy of the 4CMenB vaccine against N. gonorrhoeae are underway, as are Phase 1/2 studies of a new gonococcal-specific vaccine candidate. Ultimately, a gonococcal vaccine must be accessible, affordable and equitably dispensed, given that those most affected by gonorrhoea are also those who may be most disadvantaged in our societies, and most cases are in less-resourced settings. This vaccine value profile (VVP) provides a high level, holistic assessment of the current data to inform the potential public health, economic and societal value of pipeline vaccines. This was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships and multi-lateral organizations. All contributors have extensive expertise on various elements of the N. gonorrhoeae VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using published data obtained from peer-reviewed journals or reports.

Development of a Tag/Catcher-mediated capsid virus-like particle vaccine presenting the conserved Neisseria gonorrhoeae SliC antigen that blocks human lysozyme.

Virus-like particles (VLPs) are promising nanotools for the development of subunit vaccines due to high immunogenicity and safety. Herein, we explored the versatile and effective Tag/Catcher-AP205 capsid VLP (cVLP) vaccine platform to address the urgent need for the development of an effective and safe vaccine against gonorrhea. The benefits of this clinically validated cVLP platform include its ability to facilitate unidirectional, high-density display of complex/full-length antigens through an effective split-protein Tag/Catcher conjugation system. To assess this modular approach for making cVLP vaccines, we used a conserved surface lipoprotein, SliC, that contributes to the Neisseria gonorrhoeae defense against human lysozyme, as a model antigen. This protein was genetically fused at the N- or C-terminus to the small peptide Tag enabling their conjugation to AP205 cVLP, displaying the complementary Catcher. We determined that SliC with the N-terminal SpyTag, N-SliC, retained lysozyme-blocking activity and could be displayed at high density on cVLPs without causing aggregation. In mice, the N-SliC-VLP vaccines, adjuvanted with AddaVax or CpG, induced significantly higher antibody titers compared to controls. In contrast, similar vaccine formulations containing monomeric SliC were non-immunogenic. Accordingly, sera from N-SliC-VLP-immunized mice also had significantly higher human complement-dependent serum bactericidal activity. Furthermore, the N-SliC-VLP vaccines administered subcutaneously with an intranasal boost elicited systemic and vaginal IgG and IgA, whereas subcutaneous delivery alone failed to induce vaginal IgA. The N-SliC-VLP with CpG (10 µg/dose) induced the most significant increase in total serum IgG and IgG3 titers, vaginal IgG and IgA, and bactericidal antibodies.

Assessment of Serum Bactericidal and Opsonophagocytic Activity of Antibodies to Gonococcal Vaccine Targets.

There is no vaccine available to prevent Neisseria gonorrhoeae infection, however there is currently a high level of interest in developing gonococcal vaccines due to the increasing number of cases and continuing emergence of antimicrobial resistance worldwide. A key aspect of vaccine development is the investigation of the functional immune response raised to the vaccine targets under investigation. Here, we describe two assays used to assess the functional immune response raised against gonococcal vaccine targets: the serum bactericidal assay (SBA) and the opsonophagocytic assay (OPA).

Preclinical Testing of Vaccines and Therapeutics for Gonorrhea in Female Mouse Models of Lower and Upper Reproductive Tract Infection.

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

Challenges and Controversies Concerning Neisseria gonorrhoeae-Neutrophil Interactions in Pathogenesis.

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

Neisseria gonorrhoeae Multivalent Maxibody with a Broad Spectrum of Strain Specificity and Sensitivity for Gonorrhea Diagnosis.

Gonorrhea is one of the most common, but still hidden and insidious, sexually transmitted diseases caused by Neisseria gonorrhoeae (gonococci). However, the diagnosis and treatment of gonorrhea are hampered by antigenic variability among gonococci, the lack of acquired immunity, and antimicrobial resistance. Further, strains resistant to cephalosporins, including ceftriaxone, the last line of defense, represent a growing threat, which prompted us to develop gonococci-specific diagnostic antibodies with broad-spectrum binding to gonococci strains to generate gonorrhea-detecting reagents. This study reports the identification of gonococci antibodies via bio-panning on gonococci cells using scFv-phage libraries. Reformatting the lead scFv-phage Clones 1 and 4 to a multivalent scFv1-Fc-scFv4 maxibody increased the sensitivity by up to 20-fold compared to the single scFv-Fc (maxibody) alone. Moreover, the multivalent maxibody showed broader cross-reactivity with clinical isolates and the ceftriaxone antibiotic-resistant World Health Organization (WHO) reference strain L. In contrast, the selected antibodies in the scFv-phage, maxibody, and multivalent maxibody did not bind to N. sicca, N. meningitides, and N. lactamica, suggesting the clinical and pharmaceutical diagnostic value of these selected antibodies for gonorrheal infections. The present study illustrates the advantages and potential application of multivalent maxibodies to develop rapid and sensitive diagnostic reagents for infectious diseases and cancer.

Synthetic DNA Delivery of an Optimized and Engineered Monoclonal Antibody Provides Rapid and Prolonged Protection against Experimental Gonococcal Infection.

Monoclonal antibody (MAb) 2C7 recognizes a lipooligosaccharide epitope expressed by most clinical Neisseria gonorrhoeae isolates and mediates complement-dependent bactericidal activity. We recently showed that a recombinant human IgG1 chimeric variant of MAb 2C7 containing an E430G Fc modification (2C7_E430G), which enhances complement activation, outperformed the parental MAb 2C7 (2C7_WT) in vivo Because natural infection with N. gonorrhoeae often does not elicit protective immunity and reinfections are common, approaches that prolong bacterial control in vivo are of great interest. Advances in DNA-based approaches have demonstrated the combined benefit of genetic engineering, formulation optimizations, and facilitated delivery via CELLECTRA-EP technology, which can induce robust in vivo expression of protective DNA-encoded monoclonal antibodies (DMAbs) with durable serum activity relative to traditional recombinant MAb therapies. Here, we created optimized 2C7-derived DMAbs encoding the parental Fc (2C7_WT) or complement-enhancing Fc variants (2C7_E430G and 2C7_E345K). 2C7 DMAbs were rapidly generated and detected throughout the 4-month study. While all complement-engaging 2C7 variants facilitated rapid clearance following primary N. gonorrhoeae challenge (day 8 after DMAb administration), the complement-enhancing 2C7_E430G variant demonstrated significantly higher potency against mice rechallenged 65 days after DMAb administration. Passive intravenous transfer of in vivo-produced, purified 2C7 DMAbs confirmed the increased potency of the complement-enhancing variants. This study highlights the ability of the DMAb platform to launch the in vivo production of antibodies engineered to promote and optimize downstream innate effector mechanisms such as complement-mediated killing, leading to hastened bacterial elimination.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics in clinical use. Currently, there is no safe and effective vaccine against gonorrhea. Measures to prevent the spread of gonorrhea are a global health priority. A monoclonal antibody (MAb) called 2C7, directed against a lipooligosaccharide glycan epitope expressed by most clinical isolates, displays complement-dependent bactericidal activity and hastens clearance of gonococcal vaginal colonization in mice. Fc mutations in a human IgG1 chimeric version of MAb 2C7 further enhance complement activation, and the resulting MAb displays greater activity than wild-type MAb 2C7 in vivo Here, we utilized a DNA-encoded MAb (DMAb) construct designed to launch production and assembly of "complement-enhanced" chimeric MAb 2C7 in vivo The ensuing rapid and sustained MAb 2C7 expression attenuated gonococcal colonization in mice at 8 days as well as 65 days postadministration. The DMAb system may provide an effective, economical platform to deliver MAbs for durable protection against gonorrhea.