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.

Human endometrial MAIT cells are transiently tissue resident and respond to Neisseria gonorrhoeae.

Mucosa-associated invariant T (MAIT) cells are non-classical T cells important in the mucosal defense against microbes. Despite an increasing interest in the immunobiology of the endometrial mucosa, little is known regarding human MAIT cells in this compartment. The potential role of MAIT cells as a tissue-resident local defense against microbes in the endometrium is largely unexplored. Here, we performed a high-dimensional flow cytometry characterization of MAIT cells in endometrium from pre- and postmenopausal women, and in decidua from first-trimester pregnancies. Furthermore, we assessed MAIT cell function by stimulation with Neisseria gonorrhoeae (N. gonorrhoeae). Endometrial MAIT (eMAIT) cells represented a stable endometrial immune cell population as limited dynamic changes were observed during the menstrual cycle, post menopause, or in response to pregnancy. Furthermore, eMAIT cells exhibited an activated tissue-resident phenotype. Despite expressing CD69 and CD103, eMAIT cells were replenished over time by circulating MAIT cells, as assessed using human uterus transplantation as a model. Finally, functional experiments revealed the capability of MAIT cells to respond to the sexually transmitted and tissue-relevant pathogen, N. gonorrhoeae. In conclusion, our study provides novel insight into human MAIT cell dynamics and anti-microbial properties in the human uterus.

The serogroup B meningococcal outer membrane vesicle-based vaccine 4CMenB induces cross-species protection against Neisseria gonorrhoeae.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.

Modulation of phagocytosis-induced cell death of human neutrophils by Neisseria gonorrhoeae.

Optimal innate immune response to infection includes eradication of potential pathogens, resolution of associated inflammation, and restitution of homeostasis. Phagocytosing human polymorphonuclear leukocytes (hPMN) undergo accelerated apoptosis, a process referred to as phagocytosis-induced cell death (PICD) and an early step in their clearance from inflammatory sites. Among human pathogens that modulate hPMN apoptosis, Neisseria gonorrhoeae delays PICD, which may contribute to the exuberant neutrophilic inflammation that characterizes gonorrhea. To elucidate the mechanisms underlying delayed PICD, we compared features of hPMN cell death that followed phagocytosis of N. gonorrhoeae FA1090 wild-type (GC) or serum-opsonized zymosan (OPZ), a prototypical stimulus of PICD. Phosphatidylserine externalization required NADPH oxidase activity after ingestion of GC or OPZ, and annexin V staining and DNA fragmentation were less after phagocytosis of GC compared to OPZ. Caspase 3/7 and caspase 9 activities after phagocytosis of GC were less than that seen after ingestion of OPZ, but caspase 8 activity was the same after ingestion of GC or OPZ. When hPMN sequentially ingested GC followed by OPZ, both caspase 3/7 and 9 activities were less than that seen after OPZ alone, and the inhibition was dose dependent for GC, suggesting that ingestion of GC actively inhibited PICD. Sequential phagocytosis did not block caspase 8 activity, mitochondrial depolarization, or annexin V/propidium iodide staining compared to responses of hPMN fed OPZ alone, despite inhibition of caspases 3/7 and 9. Taken together, these data suggest that active inhibition of the intrinsic pathway of apoptosis contributes to the delay in PICD after hPMN ingestion of N. gonorrhoeae.

Inhibition of TLR2/TLR4 alleviates the Neisseria gonorrhoeae infection damage in human endometrial epithelial cells via Nrf2 and NF-Kßsignaling.

BACKGROUND: Neisseria gonorrhoeae (N.g) is Gram-negative bacteria and can lead to endometritis in female. Toll-like receptors regulate immune response in various diseases. However, the roles of TLR2 and TLR4 in. Neisseria gonorrhoeae-induced infection damage in human endometrial epithelia were investigated. METHODS: hEECs were infected with N.g (MOI 10 and 100) and cell viability and apoptosis were measured by CCK8 and flow cytometry assays in both infected groups with the uninfected normal hEECs as negative control. TLR2/TLR4 proteins were measured by ELISA method. Pro-inflammatory markers NLRP3, PGES (PGE2) and TNF-α were assessed by RT-qPCR (mRNA expression) and Elisa (protein concentrations). Transfection assays were performed to up- or down- regulate expression of TLR2 and TLR4 so as to study the functions of TLR2/TLR4 in. N.g-infected hEECs, followed by apoptosis and inflammation assessment. Similarly, we explored the interactions between TLR2/TLR4 and Nrf2/NF-κB/p65 by knocking down TLR2/TLR4 to detect the signaling and further regulating the signaling to evaluate TLR2/ TLR4, apoptosis and inflammation in cells. RESULTS: N.g suppressed cell viabilities and induced cell apoptosis and inflammation. TLR2/TLR4 downregulation inhibited the infection damage. Nrf2 was activated while NF-κB/p65 was depleted as TLR2/ TLR4 was knocked down. Activation of Nrf2 and inhibition of NF-κB resulted in decrease of TLR2/TLR4, which could retard apoptosis and inflammation induced by N.g infection. CONCLUSION: TLR2/TLR4 depletion could alleviate the N.g-infected hEECs via Nrf2/NF-kB signaling, suggesting that TLR2/TLR4 inhibitors might serve as a treatment to reduce N.g infection in human endometrial epithelia.

Complement interactions with the pathogenic Neisseriae: clinical features, deficiency states, and evasion mechanisms.

Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, while Neisseria meningitidis is an important cause of bacterial meningitis and sepsis. Complement is a central arm of innate immune defenses and plays an important role in combating Neisserial infections. Persons with congenital and acquired defects in complement are at a significantly higher risk for invasive Neisserial infections such as invasive meningococcal disease and disseminated gonococcal infection compared to the general population. Of note, Neisseria gonorrhoeae and Neisseria meningitidis can only infect humans, which in part may be related to their ability to evade only human complement. This review summarizes the epidemiologic and clinical aspects of Neisserial infections in persons with defects in the complement system. Mechanisms used by these pathogens to subvert killing by complement and preclinical studies showing how these complement evasion strategies may be used to counteract the global threat of meningococcal and gonococcal infections are discussed.

Progress Toward a Gonococcal Vaccine: The Way Forward.

The concept of immunizing against gonorrhea has received renewed interest because of the recent emergence of strains of Neisseria gonorrhoeae that are resistant to most currently available antibiotics, an occurrence that threatens to render gonorrhea untreatable. However, despite efforts over many decades, no vaccine has yet been successfully developed for human use, leading to pessimism over whether this goal was actually attainable. Several factors have contributed to this situation, including extensive variation of the expression and specificity of many of the gonococcal surface antigens, and the ability of N. gonorrhoeae to resist destruction by complement and other innate immune defense mechanisms. The natural host restriction of N. gonorrhoeae for humans, coupled with the absence of any definable state of immunity arising from an episode of gonorrhea, have also complicated efforts to study gonococcal pathogenesis and the host's immune responses. However, recent findings have elucidated how the gonococcus exploits and manipulates the host's immune system for its own benefit, utilizing human-specific receptors for attachment to and invasion of tissues, and subverting adaptive immune responses that might otherwise be capable of eliminating it. While no single experimental model is capable of providing all the answers, experiments utilizing human cells and tissues in vitro, various in vivo animal models, including genetically modified strains of mice, and both experimental and observational human clinical studies, have combined to yield important new insight into the immuno-pathogenesis of gonococcal infection. In turn, these have now led to novel approaches for the development of a gonococcal vaccine. Ongoing investigations utilizing all available tools are now poised to make the development of an effective human vaccine against gonorrhea an achievable goal within a foreseeable time-frame.

Preclinical Efficacy of a Lipooligosaccharide Peptide Mimic Candidate Gonococcal Vaccine.

The global spread of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a public health emergency. With limited antibiotic treatment options, there is an urgent need for development of a safe and effective vaccine against gonorrhea. Previously, we constructed a prototype vaccine candidate comprising a peptide mimic (mimitope) of a glycan epitope on gonococcal lipooligosaccharide (LOS), recognized by monoclonal antibody 2C7. The 2C7 epitope is (i) broadly expressed as a gonococcal antigenic target in human infection, (ii) a critical requirement for gonococcal colonization in the experimental setting, and (iii) a virulence determinant that is maintained and expressed by gonococci. Here, we have synthesized to >95% purity through a relatively facile and economical process a tetrapeptide derivative of the mimitope that was cyclized through a nonreducible thioether bond, thereby rendering the compound homogeneous and stable. This vaccine candidate, called TMCP2, when administered at 0, 3, and 6 weeks to BALB/c mice at either 50, 100 or 200 µg/dose in combination with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE; a Toll-like receptor 4 and TH1-promoting adjuvant), elicited bactericidal IgG and reduced colonization levels of gonococci in experimentally infected mice while accelerating clearance by each of two different gonococcal strains. Similarly, a 3-dose biweekly schedule (50 µg TMCP2/dose) was also effective in mice. We have developed a gonococcal vaccine candidate that can be scaled up and produced economically to a high degree of purity. The candidate elicits bactericidal antibodies and is efficacious in a preclinical experimental infection model.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics. The incidence of gonorrhea is also sharply increasing. A safe and effective antigonococcal vaccine is urgently needed. Lipooligosaccharide (LOS), the most abundant outer membrane molecule, is indispensable for gonococcal pathogenesis. A glycan epitope on LOS that is recognized by monoclonal antibody (MAb) 2C7 (called the 2C7 epitope) is expressed almost universally by gonococci in vivo Previously, we identified a peptide mimic (mimitope) of the 2C7 epitope, which when configured as an octamer and used as an immunogen, attenuated colonization of mice by gonococci. Here, a homogenous, stable tetrameric derivative of the mimitope, when combined with a TH1-promoting adjuvant and used as an immunogen, also effectively attenuates gonococcal colonization of mice. This candidate peptide vaccine can be produced economically, an important consideration for gonorrhea, which affects socioeconomically underprivileged populations disproportionately, and represents an important advance in the development of a gonorrhea vaccine.

C4BP-IgM protein as a therapeutic approach to treat Neisseria gonorrhoeae infections.

Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 porin B1a (PorB1a) and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding substantially correlated with the ability to evade complement-dependent killing (r = 0.78). We designed 2 chimeric proteins that fused C4BP domains to the backbone of IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (KD C4BP-IgM = 2.4 nM; KD C4BP-IgG 980.7 nM), but only hexameric C4BP-IgM efficiently outcompeted heptameric C4BP from the bacterial surface, resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM substantially attenuated the duration and burden of colonization of 2 C4BP-binding gonococcal isolates but not a non-C4BP-binding strain in a mouse vaginal colonization model using human factor H/C4BP-transgenic mice. Our preclinical data present C4BP-IgM as an adjunct to conventional antimicrobials for the treatment of gonorrhea.

Advancing vaccine development for gonorrhoea and the Global STI Vaccine Roadmap.

Efforts to develop vaccines against Neisseria gonorrhoeae have become increasingly important, given the rising threat of gonococcal antimicrobial resistance (AMR). Recent data suggest vaccines for gonorrhoea are biologically feasible; in particular, epidemiological evidence shows that vaccines against a closely related pathogen, serogroup B Neisseria meningitidis outer membrane vesicle (OMV) vaccines, may reduce gonorrhoea incidence. Vaccine candidates using several approaches are currently in preclinical development, including meningococcal and gonococcal OMV vaccines, a lipooligosaccharide epitope and purified protein subunit vaccines. The Global STI Vaccine Roadmap provides action steps to build on this technical momentum and advance gonococcal vaccine development. Better quantifying the magnitude of gonorrhoea-associated disease burden, for outcomes like infertility, and modelling the predicted role of gonococcal vaccines in addressing AMR will be essential for building a full public health value proposition, which can justify investment and help with decision making about future vaccine policy and programs. Efforts are underway to gain consensus on gonorrhoea vaccine target populations, implementation strategies and other preferred product characteristics that would make these vaccines suitable for use in low- and middle-income, as well as high-income, contexts. Addressing these epidemiological, programmatic and policy considerations in parallel to advancing research and development, including direct assessment of the ability of meningococcal B OMV vaccines to prevent gonorrhoea, can help bring about the development of viable gonococcal vaccines.

Sexually Transmitted Infection Positivity Among Adolescents With or at High-Risk for Human Immunodeficiency Virus Infection in Los Angeles and New Orleans.

BACKGROUND: Gay, bisexual, and transgender youth and homeless youth are at high risk for sexually transmitted infections (STIs). However, little recent data exist describing STI positivity by anatomical site among those groups. We determined the positivity of Chlamydia trachomatis (CT) infection, Neisseria gonorrhoeae (NG) infection, and syphilis antibody reactivity among lesbian, gay, bisexual, transgender, and homeless youth. METHODS: We recruited 1,264 adolescents with high risk behavior aged 12 to 24 years from homeless shelters, lesbian, gay, bisexual, and transgender organizations, community health centers, and using social media and online dating apps in Los Angeles, California and New Orleans, Louisiana from May 2017 to February 2019. Participants received point-of-care pharyngeal, rectal, and urethral/vaginal CT and NG testing and syphilis antibody testing. We calculated STI positivity by anatomical site and compared positivity by participant subgroups based on human immunodeficiency virus (HIV) status, sex assigned at birth, and gender identity. RESULTS: CT and NG positivity and syphilis antibody reactivity was higher among HIV-infected adolescent men who have sex with men (MSM) than HIV-uninfected adolescent MSM (40.2% vs. 19%, P < 0.05), particularly CT or NG rectal infection (28% vs. 12.3%, P < 0.05). Of participants with positive CT or NG infections, 65% had extragenital-only infections, 20% had both extragenital and urogenital infections, and 15% had urogenital-only infections. CONCLUSIONS: Sexually transmitted infection positivity was high, particularly among transgender women and MSM. The high proportion of rectal and pharyngeal infections highlights the importance of both urogenital and extragenital STI screening. More accessible STI testing is necessary for high-risk adolescent populations.

Mechanistic Insight Into the Activation of the NLRP3 Inflammasome by Neisseria gonorrhoeae in Macrophages.

Gonorrhea is a type III legal communicable disease caused by Neisseria gonorrhoeae (NG), one of the most common sexually transmitted bacteria worldwide. NG infection can cause urethritis or systemic inflammation and may lead to infertility or other complications. The NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome is a protein complex composed of NLRP3, apoptosis-associated speck-like protein and caspase-1 and is an important part of the cellular machinery controlling the release of interleukin (IL)-1ß and IL-18 and the pathogenesis of numerous infectious diseases. It has been reported that NG infection activates the NLRP3 inflammasome; however, the underlying mechanism remain unclear. In this report, the signaling pathways involved in the regulation of NG-mediated NLRP3 inflammasome activation in macrophages were studied. The results indicated that viable NG, but not heat-killed or freeze/thaw-killed NG, activated the NLRP3 inflammasome in macrophages through toll-like receptor 2, but not toll-like receptor 4. NG infection provided the priming signal to the NLRP3 inflammasome that induced the expression of NLRP3 and IL-1ß precursor through the nuclear factor kappa B and mitogen-activated protein kinase pathways. In addition, NG infection provided the activation signal to the NLRP3 inflammasome that activated caspase-1 through P2X7 receptor-dependent potassium efflux, lysosomal acidification, mitochondrial dysfunction, and reactive oxygen species production pathways. Furthermore, we demonstrated that NLRP3 knockout increased phagocytosis of bacteria by macrophages and increases the bactericidal activity of macrophages against NG. These findings provide potential molecular targets for the development of anti-inflammatory drugs that could ameliorate NG-mediated inflammation.

Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody.

Multidrug-resistant Neisseria gonorrhoeae is a global health problem. Monoclonal antibody (mAb) 2C7 recognizes a gonococcal lipooligosaccharide epitope that is expressed by >95% of clinical isolates and hastens gonococcal vaginal clearance in mice. Chimeric mAb 2C7 (human immunoglobulin G1 [IgG1]) with an E430G Fc modification that enhances Fc:Fc interactions and hexamerization following surface-target binding and increases complement activation (HexaBody technology) showed significantly greater C1q engagement and C4 and C3 deposition compared to mAb 2C7 with wild-type Fc. Greater complement activation by 2C7-E430G Fc translated to increased bactericidal activity in vitro and, consequently, enhanced efficacy in mice, compared with "Fc-unmodified" chimeric 2C7. Gonococci bind the complement inhibitors factor H (FH) and C4b-binding protein (C4BP) in a human-specific manner, which dampens antibody (Ab)-mediated complement-dependent killing. The variant 2C7-E430G Fc overcame the barrier posed by these inhibitors in human FH/C4BP transgenic mice, for which a single 1 µg intravenous dose cleared established infection. Chlamydia frequently coexists with and exacerbates gonorrhea; 2C7-E430G Fc also proved effective against gonorrhea in gonorrhea/chlamydia-coinfected mice. Complement activation alone was necessary and sufficient for 2C7 function, evidenced by the fact that (1) "complement-inactive" Fc modifications that engaged Fc gamma receptor (FcγR) rendered 2C7 ineffective, nonetheless; (2) 2C7 was nonfunctional in C1q-/- mice, when C5 function was blocked, or in C9-/- mice; and (3) 2C7 remained effective in neutrophil-depleted mice and in mice treated with PMX205, a C5a receptor (C5aR1) inhibitor. We highlight the importance of complement activation for antigonococcal Ab function in the genital tract. Elucidating the correlates of protection against gonorrhea will inform the development of Ab-based gonococcal vaccines and immunotherapeutics.

Biology of the Gonococcus: Disease and Pathogenesis.

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.

Isolation of Naturally Released Gonococcal Outer Membrane Vesicles as Vaccine Antigens.

The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.

Complement-Dependent Serum Bactericidal Assays for Neisseria gonorrhoeae.

The global spread of multidrug-resistant gonorrhea has spurred efforts to develop a safe and effective vaccine against gonorrhea. Complement plays an important role in host defenses against Neisseria infections. Complement-dependent bactericidal activity of antibodies (either natural antibodies or those elicited by immunization) is a well-established correlate of protection against meningococcal infections. Although correlates of protection against gonococcal infection have not been defined, there is evidence to suggest that complement-mediated killing may also predict vaccine efficacy against this disease. This chapter describes methods to prepare human complement sources and perform bactericidal assays against Neisseria gonorrhoeae.

Basic Methods for Examining Neisseria gonorrhoeae Interactions with Host Cells In Vitro.

The obligate human pathogen Neisseria gonorrhoeae colonizes primarily the mucosal columnar epithelium of the male urethra and the female endocervix. In addition, gonococci can infect the anorectal, pharyngeal, and gingival mucosae and epithelial cells of the conjunctiva. More rarely, the organism can disseminate through the bloodstream, which can involve interactions with other host cell types, including blood vessel endothelial cells and innate immune cells such as dendritic cells, macrophages, and neutrophils. "Disseminated gonococcal infection" is a serious condition with various manifestations resulting from the seeding of organs and tissues with the pathogen. The host response to gonococcal infection is inflammatory. Knowledge of the biology of gonococcal interactions has been served well through the use of a wide variety of ex vivo models using host tissues and eukaryotic cell monocultures. These models have helped identify bacterial surface adhesins and invasins and the corresponding cell surface receptors that play roles in gonococcal pathogenesis. Furthermore, they have been useful for understanding virulence mechanisms as well as innate and adaptive immune responses. In this chapter, readers are provided with protocols for examining the basic interactions between gonococci and a representative human cell line.