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The sexually transmitted infection gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, can cause urethritis, cervicitis, and systemic disease, among other manifestations. N. gonorrhoeae has rapidly rising incidence along with increasing levels of antibiotic resistance to a broad range of drugs including first-line treatments. The rise in resistance has led to fears of untreatable gonorrhea causing substantial disease globally. In this review, we will describe multiple approaches being undertaken to slow and control this spread of resistance. First, a number of old drugs have been repurposed and new drugs are being developed with activity against Neisseria gonorrhoeae. Second, vaccine development, long an important goal, is advancing. Third, new diagnostics promise rapid detection of antibiotic resistance and a shift from empiric to tailored treatment. The deployment of these new tools for addressing the challenge of antibiotic resistance will require careful consideration to provide optimal care for all patients while extending the lifespan of treatment regimens., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The 10 human-restricted Neisseria species all colonize mucosal surfaces, but show a spectrum of pathogenicity. The commensal Neisseria do not normally cause pathology, while the two pathogenic species, Neisseria meningitidis and Neisseria gonorrhoeae, straddle the border between commensalism and pathogenicity. Why the pathogenic Neisseria continue to mediate host damage after thousands of years of co-evolution with their human host, and why the commensal species have not acquired the ability to damage the host, if this capability provides a selective advantage, is not understood. One way the pathogenic species are different from the commensal species is by their ability to induce PMN inflammation, which is dependent on the site of colonization. I discuss how the site of colonization dictates whether copious inflammation occurs with both pathogenic species. I put forth a model that posits that an ancestor of both pathogenic species changed colonization site from the oral cavity to the genital tract of a human or humanoid and had to evolve multiple, new traits - to induce PMN inflammation and avoid adaptive immunity - to allow efficient sexual transmission. This model predicts that PMN inflammation produces the serious sequelae of gonorrhea and increases the probability that N. meningitidis might exit the oral cavity to produce systemic disease. In both cases, the pathology produced by these host-adapted species is an unintended by product of the inflammation but host damage does not provide any selective advantage for these organisms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4 + T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Objectives: Resistance to the currently recommended extended-spectrum cephalosporins, which is used to treat Gonorrhea, is increasing continuously and leading to a threat of untreatable infection. It is, therefore, becoming extremely essential to search for new therapeutic strategies to control Gonorrhea. Vaccination may be considered as an effective control measure to control this disease, which is caused by Neisseria gonorrhoeae., Methods: In-silico hierarchical approach was used to help identify candidate proteins of N. gonorrhoeae that might contribute significantly in vaccine research. In contrast to the conventional vaccine research which requires at least 10-12 years, the present approach would reduce the time period drastically and help to identify Potential Universal Vaccine Candidates (PUVCs). These proteins were further analyzed for the presence of T-cell and linear B-cell epitopes, by using HLAPred and ABCpred servers respectively, in order to facilitate the identification of Multi Epitope Peptide Vaccine Constructs., Results: We have identified 23 non-host candidate proteins, using the proteomic information of four sequenced strains of N. gonorrhoeae namely FA 1090, TCDC_NG08107, NCCP11945 and MS11 and labeled them as PUVCs. Since all these identified 23 PUVCs contained both T cell and B cell epitopes, these have been further reiterated as PUVCs which could be used as promising leads for vaccine development., Conclusions: This hierarchical approach is the first comprehensive study to identify potential vaccine candidates which once utilized for vaccine development would surely serve as promising tools for effective control of Gonorrhea., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Women with asymptomatic Neisseria gonorrhoeae infection are at risk of developing pelvic inflammatory disease (PID) if the bacteria ascend from the endocervix into the uterus and oviducts. Factors that affect disease severity, ranging from mild discomfort to severe inflammation, pain, and infertility, remain elusive. Herein we perform direct transcervical inoculation of N. gonorrhoeae into the uterus of mice to establish an infection that leads to PID. Profoundly different disease outcomes were apparent at different stages of the reproductive cycle. Mice that were infected during the diestrus stage of the reproductive cycle displayed extensive gonococcal penetration into the submucosa, severe inflammation, and clinical signs reflecting discomfort. Meanwhile, infection during the intervening estrus stage showed only modest effects. Furthermore, a gonococcal-specific humoral response was only elicited following the penetrative upper genital tract (UGT) infection during diestrus but not estrus. Strikingly, the potential for antibodies to contribute to protection during re-infection also depends upon the reproductive stage, as antigonococcal antibodies within the genital tract were markedly higher when mice were in diestrus. Combined, this work establishes a robust new model reflecting gonococcal PID in humans and reveals how the reproductive cycle determines the pathogenic outcome of gonococcal infections of the UGT.

Infection with Neisseria gonorrhoeae triggers an intense inflammatory response characterized by an influx of neutrophils in the genital tract, yet natural gonococcal infection does not induce a state of protective immunity. Our previous studies in a mouse model of N. gonorrhoeae infection demonstrated that transforming growth factor-β (TGF-β) is involved in the suppression of adaptive immunity by this organism, but complete inhibition of TGF-β activity only partially reverses N. gonorrhoeae-mediated suppression of T helper type 1 (Th1) and Th2 responses. In this study, we show that N. gonorrhoeae strongly induced the production of interleukin (IL)-10 and type 1 regulatory T (Tr1) cells. Blockade of IL-10 and Tr1 cell activity enhanced both Th1/Th2-dependent adaptive immune responses and Th17-governed innate responses to N. gonorrhoeae. Treatment of mice with anti-IL-10 antibody during gonococcal challenge led to faster clearance of infection and induced protection against secondary infection, with the generation of circulating and vaginal anti-gonococcal antibodies. Our results suggest that inhibition of IL-10 and Tr1 cells affords a new approach to the treatment of gonorrhea and facilitates the development of specific protective immunity.

In most female patients, the symptoms of genital infection due to Neisseria gonorrhoeae tend to be slight or even absent. Our previous studies suggested that progesterone might play a role in female asymptomatic gonococcal infection. In this study, we demonstrated that progesterone induced the expression of thymic stromal lymphopoietin (TSLP) and regulatory T cells (Treg)-related transcription factor Foxp3, and inhibited the expression of Th17 related transcription factor RORγt, and reduced the influx of neutrophils in murine vaginal gonococcal infection. Blockade of TSLP with antibody partially reversed the effects of progesterone on the murine model of gonococcal vaginal infection. In in vitro experiments, progesterone induced a rapid up-regulation of TSLP in vaginal epithelial cells stimulated with N. gonorrhoeae. Blocking thymic stromal lymphopoietin receptor (TSLPR) with a TSLPR monoclonal antibody partially prevented progesterone suppression of IL-17-producing T cells differentiation, and progesterone promotion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells differentiation. Altogether, our results indicate that the progesterone suppresses Th17 cell responses, and enhances the development of Treg cells, through TSLP-dependent mechanisms, and play a role in female asymptomatic gonococcal infections., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in Neisseria gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Infection with Neisseria gonorrhoeae does not induce specific immunity or immune memory. Our previous studies in a murine model of vaginal gonococcal infection showed that innate immunity governed by Th17 cells was a critical aspect of the immune response elicited by this pathogen. Herein we show that N. gonorrhoeae selectively inhibited Th1 and Th2 cells and enhanced Th17 cell development through the induction of TGF-β. Whereas Th17 responses depended on gonococcal lipooligosaccharide acting through TLR4, the inhibitory effect of N. gonorrhoeae on Th1/Th2 responses involved gonococcal Opa proteins. In vitro Th17 responses to N. gonorrhoeae could be diverted to Th1/Th2 by blockade of TGF-β, but not by blockade of IL-17. The results reveal that N. gonorrhoeae suppresses Th1/Th2-mediated adaptive immune response through mechanisms dependent on TGF-β, and that this effect can be manipulated to promote the development of adaptive immunity.

Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens that threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical, and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), human immunodeficiency virus 1 (HIV-1), and Lactobacillus crispatus before being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. An analysis of cytokines and chemokines in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans.

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and non-protective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses, which trigger innate defense mechanisms, including neutrophils and antimicrobial proteins, that it can resist. We found that N. gonorrhoeae induced the production of interleukin-17 (IL-17) in mouse T-cells and Th17-inducing cytokines in mouse and human APCs in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor (IL-17R) in IL-17RA(KO) mice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RA(KO) mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.

O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.

Triggering receptor expressed on myeloid cells-2 (TREM-2) is an innate immune receptor that initiates cellular activation upon ligation. In this study, we examined the interaction of TREM-2 with Neisseria gonorrhoeae using murine TREM-2A, as it has been reported to recognize bacterial ligands. Using a whole-bacteria enzyme-linked immunosorbent assay (ELISA), TREM-2A bound to all six strains in variable degrees. Far-western blots of gonococcal outer membranes revealed TREM-2A binding to lipooligosaccharide (LOS) and opacity (Opa) protein, with predominant binding to LOS. Binding of TREM-2A to LOS was confirmed by ELISA and surface plasmon resonance. O-deacylation of the lipid A significantly reduced binding. Flow cytometry and reporter cell assays showed that gonococci bound to TREM-2A-transfected cells and induced transmembrane signaling. In humans, TREM-2 was constitutively expressed by genitourinary and fallopian tube epithelial cells, both of which are primary targets of gonococcal invasion. Ligation of TREM-2 by LOS induced interleukin-6 production in HeLa cervical carcinoma cells. To our knowledge, this is the first report of the expression of human TREM-2 by cells deriving from a non-myeloid lineage. We conclude that gonococci can interact with TREM-2 receptors through binding to LOS and Opa protein and initiate cell signaling and cytokine production.

Reliable data concerning the incidence and phenotypic and genotypic characteristics of the Neisseria gonorrhoeae population in many eastern European countries are lacking. Clinically significant N. gonorrhoeae isolates (n = 76) from 76 consecutive patients in Arkhangelsk, Russia were characterised by antimicrobial susceptibility testing, serovar determination, porB gene sequencing and N. gonorrhoeae multi-antigen sequence typing (NG-MAST). The isolates were assigned to 12 different serovars, displayed 35 divergent porB sequences, and belonged to 40 different sequence types (STs). All the serovars, but only seven of the STs, had been identified previously in other countries. Twelve ST clusters of between two and 14 isolates were identified, which indicated that many multiple transmission networks exist in Arkhangelsk. The high number of unique STs (n = 28) may be a consequence of sub-optimal diagnostic procedures, ineffective partner tracing, local emergence of new STs, import of strains via sexual tourists, or foreign travel. The N. gonorrhoeae population circulating in Arkhangelsk was highly diverse and differed from the N. gonorrhoeae populations disseminated in some western European countries. Thorough knowledge concerning the incidence of gonorrhoea, antibiotic susceptibility and other phenotypic and genotypic characteristics of the N. gonorrhoeae strains circulating in eastern Europe is crucial.

Purpose: To identify attitudes and behavioral factors associated with parental intent to vaccinate their adolescent children against sexually transmitted infections (STI) and adolescent intent to accept vaccination for the prevention of STI., Methods: In this cross-sectional study, 320 parents and their adolescent children (aged 12-17 years) were recruited from urban adolescent health clinics and private practice pediatric offices to complete audio, computer-assisted self-interviews (A-CASI). Parents and their adolescents were asked about acceptability of gonorrhea, genital herpes, and human immumodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) vaccines. These three items were summed to create an STI vaccine acceptability scale, the primary outcome variable. Potential predictors measured included health beliefs, sociodemographic factors, and health and sexual behaviors., Results: A substantial majority of parents and their adolescent children rated the three STI vaccines as very acceptable. Parental health beliefs and parental history of STI diagnosis were significant independent predictors of intent to vaccinate adolescent children against STI. Parental intent to vaccinate and having a friend who had engaged in sexual intercourse were significant independent predictors of adolescents' intent to accept STI vaccination., Conclusions: The majority of these parents and their adolescent children found STI vaccination very acceptable, suggesting that there will be great interest in these vaccines once they become available. Interventions designed to address parental health beliefs may prove successful at maintaining or increasing interest in STI vaccines. Adolescents are likely to look to their parents for guidance around acceptance of these vaccines, but personal experiences also may play a role.

We evaluated a rapid antigen detection method with an immunochromatographic assay for Neisseria gonorrhoeae (NOW Gonorrhea Test) by using urine samples from patients with urethritis to diagnose gonococcal infection in males. Among 58 male patients who underwent urethral swab culture, 34 cases (58.6%) were found to have N. gonorrhoeae infection. The sensitivity and specificity of the NOW Test compared with the results of standard culture were 94.1% (32/34) and 95.8% (23/24), respectively. The predictive values of positive NOW and negative NOW were 96.9% (32/33) and 92.0% (23/25), respectively. The detection limit of this assay was determined as 5 x 10(4) cfu/ml using N. gonorrhoeae suspension as an antigen. In contrast to standard cultures, gonococcal antigens in specimens were still detectable by this method up to 45 h of storage at either room temperature or 4 degrees C. Considering the rapidity and ease of this method, our results suggest that the NOW method might be a useful and reliable diagnostic screening tool for gonococcal urethritis.

Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria. These glycolipid antigens are in the M(r) range of 3-7 kDa, and SDS/PAGE has been used as an analytical tool. Although we are able to separate relatively higher M(r) LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations. In the present study, we selected PID2 LOS consisting of six LOS components of 3.0-5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller M(r) LOS components but also to retain resolving capability of higher LOS components. We found that mini-PAGE with stepwise and linear gradient gels (glycine-SDS) resolved smaller M(r) LOS components. Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0-5.1 kDa were separated as tight and even bands. Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels. Our study also showed that preformed tricine-SDS (TSDS) minigels such as 16.5 and 10-20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels. By using glycine-SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller M(r) LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past., (Copyright 2000 Academic Press.)

The first high-level tetracycline resistance (MIC > or = 16 mg/l) isolates of Neisseria gonorrhoeae (TRNG) were reported in 1990 from patients attending a Sexual Transmitted Disease (STD) Center in Lisbon. The TRNG prevalence was 4% in 1991, 5.3% in 1992 and 10,8% in 1994, exploding to 52.2% in 1995. The tet M determinant was evaluated by PCR. The digests of PCRP using HpaII produced the restriction pattern 2 for all the strains, except one (pattern 3). 78.3% of the TRNG strains were beta-lactamase producers and the 4.5 MDa penicillinase plasmid was the dominant (83%), 90% and 93.3% of the TRNG strains belonged to the auxotype NR and to the serogroup IA, respectively. The IA-8/NR class represented 58.3% of the TRNG isolates, suggesting a clonal spreading.

We investigated the fate of the major outer membrane protein of Neisseria gonorrhoeae, P.IA, during gonococcal infection of Chang conjunctiva epithelial cells by using immunoelectron microscopy. Probing of P.IA epitopes with mono- and polyclonal antibodies revealed variable, fixation-dependent P.IA epitope exposure in the gonococci used as an inoculum in the infection experiments. Detection of invariable exposed P.IA epitopes in cryosections of infected epithelial cells with a polyclonal antiserum revealed unaltered P.IA exposure on the bacterial membranes during early attachment of the bacteria to the eukaryotic cells. Upon entry of the bacteria into the host cells, however, labelling was extended to membraneous structures that intercalated between the bacteria and the host cell surface, and, occasionally, to the host cell plasma membrane. The latter observation is consistent with the suggested active role of P.I. in the uptake process (as shown in 1985 by E.C. Gotschlich). Once inside the epithelial cells, both morphologically intact and disintegrating bacteria could be distinguished. The disintegration of the bacteria was accompanied by a loss of P.IA immunoreactivity.

The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide.

The prevalence of sexually transmitted disease (STD), pelvic inflammatory disease (PID), and cervical cancer, and the relation between these conditions were studied in 2111 Ethiopian women. Early sexual activity was associated with an increase in prevalence rates of STD and PID; possible aetiological factors include physical and immunological immaturity of the female genital tract and the number of sexual partners.

On the basis of bacterial typing, pyocin typing, and antibiotic sensitivity tests, two consorts appeared to have been reinfected 34 and 41 days later, respectively, with the same gonococcal strain, suggesting a failure in these patients to develop effective immunity to reinfection. However, these tests do not measure antigens which mediate attachment, a function which may correlate with virulence. When the above infecting strains were retested in an inhibition-of-attachment assay using rabbit gonococcal antisera, the antigens mediating attachment were found to be different. Homologous antisera inhibited attachment of the homologous strain at a high titer. Absorbing the antisera with the initial infecting strain did not remove any of the blocking activity of the antisera raised to the reinfecting strai, but the latter strain did share some attachment antigens in common with the initial infecting strain. Homologous antisera also bound preferentially to pili purified from the homologous strains.

Extracts of gonococcal surface antigens, examined by crossed affinity electrophoresis (CAE) with wheat germ agglutinin (WGA), yielded a single antigen-lectin precipitate in agarose gels. This precipitate induced in rabbits a potent antiserum specific for the gonococcal antigen which reacted with WGA. Immune electron microscopy on whole gonococci showed that this antiserum reacted with outer membrane vesicles. Lipopolysaccharide (LPS) purified by phenol-water extraction of whole gonococci reacted with WGA and also with the antiserum to the WGA-antigen complex. This indicated that the antiserum was specific for antigen(s) of the outer membrane complex.

The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay.

The authors evaluated the Gonosticon Dri-Dot system as a tool for the determination of the prevalence of gonococcal serum antibody in two female populations. This paper discusses their findings.

The chemical and immunochemical properties of lipopolysaccharides (LPS) isolated from pyocin 103-sensitive and -resistant Neisseria gonorrheae were investigated. Marked differences were found in immunochemical behavior of LPS from pyocin-sensitive gonococcal strain JW31 and its isogenic pyocin-resistant variant JW31R. JW31 LPS readily precipitated wheat-germ agglutinin, soybean lectin, and rabbit anti-Streptococcus faecalis or horse anti-type 14 pneumococcal antibody. In contrast, JW31R LPS precipitated only soybean lectin. The combining-site specificity of anti-S. faecalis cross-precipitated by JW31 LPS, or type 14 pneumococcal capsular polysaccharide, was examined by hapten inhibition, and lactose found to be the most potent inhibitor. Horse anti-pneumococcal type 14 antibodies, cross-precipitated by JW31 LPS and streptococcal lactose polymer, exhibited heterogeneity with respect to combining site specificity. Gel filtration of LPS-derived core oligosaccharide showed both strain JW31 and JW31 R to possess R-type lipopolysaccharide with cores having a Mr approximately 1800. JW31R LPS contains more galactose but less hexosamine than JW31 LPS. Both JW31 and JW31R core oligosaccharides possess D-glucosamine and D-galactosamine, probably N-acetylated, as the only nonreducing end-groups, and (1 leads to 4)-linked D-glucose residues. Chemical data support immunochemical findings which indicate that lactose units occur as a structural feature of JW31 gonococcal LPS.

Oligosaccharide was isolated from the lipopolysaccharide of Neisseria gonorrhoeae P9 following partial acid hydrolysis. The exposed terminal keto-deoxyoctonic acid group of the oligosaccharide was convert into the reactive phenylisothiocyanato derivative by reaction with 2-(4-aminophenyl)-ethylamine followed by reduction with sodium cyanoborohydride and treatment with thiophosgene. This intermediate was used to prepare an oligosaccharide-protein conjugate with either bovine serum albumin or purified gonococcal pili. Immunisation of rabbits with protein conjugates produced antibodies reactive with both pure protein and intact lipopolysaccharide.

If protective immunity were to develop following an episode of gonococcal pelvic inflammatory disease (salpingitis), PID should recur with organisms bearing antigens that do not react with these immune mechanisms. To test this hypothesis, gonococci from 15 women with gonococcal PID, who experienced 19 subsequent episodes of gonococcal infection, were serotyped for their principal outer membrane protein (Protein I) antigens. Of nine cases in which the initial and subsequent infections involved the same Protein I serotype, none was associated with recurrence of PID. Of 10 cases in which the initial and subsequent infections involved different Protein I types, five were associated with recurrent PID (p = 0.02). These data suggest that an episode of gonococcal PID produces some immunity to repeated episodes of salpingitis with the same Protein I serotype, while not preventing reinfection with the same Protein I serotype. The immune response to Protein I antigen may thus provide serotype-specific protection against gonococcal salpingitis.

Recent studies have shown that human seminal plasma contains chemically and biologically distinct factors which inhibit lymphocyte functions and the serum bactericidal and opsonic activities associated with the killing of gram-negative organisms. Because of the direct association between complement action and serum bactericidal and opsonic activities, inhibition of complement may be one of the possible mechanisms of action of seminal plasma immunoinhibitory factors. Complement hemolytic activity was measured for C3 and C4 in serum Neisseria gonorrhoeae and Escherichia coli bactericidal reaction mixtures with and without addition of seminal plasma. In the presence of seminal plasma, where there was no bactericidal action, C3 and titers were reduced to approximately 50% of the titers in the reactions with complement donor serum. The C3 titers were lower than in the reaction mixtures with immune serum and complement donor serum, where N. gonorrhoeae bactericidal activity occurred. Individual human seminal plasma specimens depressed CH50 activity of pooled normal human sera up to 50% of normal levels. There were no differences in inhibition by seminal plasma specimens from normal or vasectomized men. Treatment with seminal plasma depressed the functional activity of complement components C1 and C3 by more than 50%. Seminal plasma also inhibited alternate pathway activity. Cleavage of factor B was demonstrated. The seminal plasma factor which inhibited complement was of low molecular weight. DPF blocked the seminal plasma complement-inhibitory factor. However, amidolytic activity for serine protease substrates could not be demonstrated. It is likely that the seminal plasma complement inhibitor is a protease inhibitor acting singly or in combination.

A solid phase radioimmunoassay (SPRIA) has been developed for detection of IgG antibodies to gonococcal outer membrane components. Gonococcal antigens were immobilized on a solid support by covalent coupling to CNBr-activated Sepharose in the presence of the detergent Triton X-100. Binding of specific antibody to the Sepharose-antigen complex was detected using radiolabelled Protein A as the antiglobulin. Protein A was labelled by radioacetylation with tritiated acetic anhydride, yielding a product of high specific activity and high stability. No detectable loss of activity was observed over a ten month period. The entire assay was performed on Mitex teflon hydrophobic membrane filters which held the Sepharose beads and aqueous supernatant as a discrete drop of liquid. The supernatants and incubation media were easily and rapidly removed from the beads by suction on a specially designed manifold system. This procedure removed the need for repeated and time-consuming centrifugations. Titres were obtained graphically from double log plots of cpm bound versus antiserum dilution by extrapolation of the straight line to a point corresponding to twice the control level of radioactivity binding. The assay proved to be a very reliable and simple procedure for the detection of IgG antibodies to gonococcal surface antigens.

A method for production of purified and concentrated mouse monoclonal antibodies was developed. The rationale for the procedure is, firstly, to expand hybridoma cell number in culture medium-containing serum; secondly, to transfer the cells to serum-free medium for production of antibodies; and finally, to harvest antibodies from the conditioned medium by means of cation exchange chromatography on SP-Sephadex C-50, followed by gel filtration on Superose 6B. When compared with chromatography of monoclonal antibodies on protein A-Sepharose our results suggest that the method is particularly useful for purification of antibodies of the IgG1 subclass. Experience from production and purification of 15 various monoclonal antibodies is reported.

We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins.

Direct recording of the kinetics of early phagocytic events, recognition or adhesion and ingestion, may better characterize some forms of polymorphonuclear leukocyte (PMN) dysfunction. This report describes a method and criteria to discriminate concurrently between adhesion and ingestion of Staphylococcus aureus by human PMNs at the level of the light microscope. The criteria were confirmed by several lines of direct evidence: low temperature and cytochalasin b treatment of PMNs; lysostaphin digestion of target Staphylococcus aureus after PMN incubation; and differential ingestibility of colony types I and III N. gonorrheae. Concurrent determinations of the initial kinetics of adhesion and ingestion of bacteria by PMNs demonstrated generalized first order kinetics, sensitive to bacterial challenge ratio and total particle density. Most importantly, the method may be applied to determine if phagocytically defective PMNs actually fail to recognize opsonized bacteria.

A reproducible gonococcal serum bactericidal microassay is described which has easily determined endpoints and lends itself to large scale tests. The microassay may be used to assess sensitivity to normal human serum among strains of gonococci, and to define the antigens conferring this sensitivity. It may be readily adapted to test for bactericidal activity in hyperimmune antisera and to evaluate antigens which inhibit serum bactericidal activity.

The authors have titrated, by the Laurell technique, immunoglobulin A in vaginal washings ; the quantity of IgA found is higher for women having a pathogenic vaginal flora. Using immunofluorescence technique, and peroxydase-marked antibodies, the authors found specific IgA directed against Lactobacillus, N. gonorrhoeae and Candida albicans.

During the development of immunoassays to detect gram-negative bacteria, an effect of pH on the aggregation of some murine monoclonal antibodies directed to Neisseria gonorrhoeae was observed. By reacting positively charged primary amines on these antibodies with the neutral NHS-biotin (N-hydroxy-succinimidobiotin), the surface charge on the antibodies was altered and a concomitant change in the solubility of these antibodies noted. This derivatization produced not only a pH-dependent change in the solubility properties of the antibodies, but also affected the response of immunoassays in which these antibodies were used. Data presented suggests that the signal-to-noise (S/N) observed in these assays is maximized under conditions where the biotinylated antibody is introduced into the assay at a pH at least 2 U above its pI. Our hypothesis is that as the pH of the solution approaches the biotinylated antibodies' isoelectric point, they become 'stickier', perhaps by aggregation (which we have directly measured), leading to high non-specific binding and hence a lower S/N.

A previously described 70-Kd antigen present in all Neisseria gonorrhoeae strains tested and in most Neisseria species was characterized as a mercaptoethanol-and heat-stable protein. Using mouse polyclonal antisera specifically directed against this antigen, it was shown that it is a surface-exposed structure. The 70-Kd antigen was recovered from all gonococcal strains isolated from different anatomic sites in male and female partners, thus demonstrating its antigenic stability after in vivo transmission in humans.

Recent studies have demonstrated that virulent (Types 1 and 2) colonial variants of Neisseria gonorrhoeae are highly resistant to phagcytosis by normal human polymorphonuclear leukocytes. To pursue this observation, sera from prostitutes were compared with sera from Catholic nuns in terms of their ability to enhance phagocytosis of Type 1 gonococci. The phagocytic system consisted of a monolayer of normal human polymorphonuclear leukocytes and an inoculum of 107 to 108 colony-forming units of N. gonorrhoeae. In a representative test, the mean percentages of phagocytosis were: 13 nuns, 10 per cent; 32 prostitutes, 30 per cent (p less than 0.01). In a separate set of experiments, 5 (45 per cent) of 11 patients with acute gonococcal salpingitis had significant increases in serum opsonic activity between the acute and one month convalescent bleeding. A pool of prostitute sera retained detectable opsonic activity at dilutions as high as 1:320. The opsonic activity was heat-stable, demonstrable without complement, and resided in both the IgG and IgA fractions of serum. These studies demonstrate the presence of acquried opsonic antibody to N. gonorrhoeae in humans: the significance of such antibodies in proction against gonococcal disease requires further exploration.

In order to more fully understand the host defense mechanisms against gonococcal infections, we decided to define the role of the classic and alternate complement pathways in gonococcal BA. Sera and infecting isolates were collected from several patients with genital and disseminated gonococcal infections. Sera from two never-infected subjects and a hypogammaglobulinemic patient were also collected. Sera from patients with genital gonorrhea and never-infected controls demonstrated marked BA for gonococci after 30 min incubation. Chelation of these sera with MgEGTA delayed the expression of BA. Consumption of C3, but not C4, was observed in chelated samples. BA could not be demonstrated in any of the sera from DGI patients or the patient with hypogammaglobulinemia. Aliquots of fresh and chelated hypogammaglobulinemic serum to which IgM or IgG antigonococcal antibodies were added showed marked BA by 30 and 60 min, respectively. Absorption of chelated serum with a serum-sensitive isolate eliminated the previously observed delayed BA. The findings suggest that gonococcal serum BA is primarily associated with activation of the classic complement pathway. Activation of the alternate pathway also occurs; however, its expression is delayed and appears to be antibody-dependent.

In patients with acute gonorrhoea the antibody responses to envelope antigens of Neisseria gonorrhoea were investigated with a sensitive enzyme-linked immunosorbent assay. IgG antibodies predominated even early in the disease but IgM and IgA antibodies were present. Surprisingly there were circulating antibodies linked to secretory piece. These were largely but not entirely IgA and were presumed to have been absorbed from sites of local production.