Search

The recent entry of multiple vaccines targeting Neisseria gonorrhoeae (Ng) into the clinical development pathway has highlighted the need to establish methods of adequately assessing anti-gonococcal immune responses. Serum bactericidal activity (SBA) is utilized as a measurement of efficacy in licensure of meningococcal vaccines, but the importance of functional antibodies in preventing and/or clearing gonococcal infections remains largely unknown. In an effort to elucidate the utility of SBA as an immune correlate of protection, we sought to develop a standardized high-throughput human complement SBA (hSBA) assay for which any strain of interest could be tested under uniform conditions, with minimal screening of complement required. Utilization of IgG/IgM-depleted pooled human complement serum permitted testing of diverse serum resistant and sensitive gonococcal and unencapsulated meningococcal strains when bacteria were cultured to induce lipooligosaccharide sialylation; only one of nine randomly selected lots demonstrated intrinsic toxicity. The hSBA assay was well suited for use with multiple test serum sources, including rabbit, mouse, and human serum samples, suggesting widespread applicability., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Brian Bonk, Chris Lyle and Bob Natuk report a relationship with Pel-Freez Biologicals that includes employment. Brian Bonk reports a relationship with Pel-Freez Biologicals that includes board membership, equity and stock. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Background: Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea., Methods: We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets., Results: Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB., Conclusions: Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine., (Published by Oxford University Press for the Infectious Diseases Society of America 2021.)

Detergent-extracted detoxified outer membrane vesicle (dOMV) vaccines are effective at preventing invasive serogroup B meningococcal (MenB) disease caused by the homologous Neisseria meningitidis strain from which they are produced, but offer limited protection from heterologous strains. Differences in vaccine efficacy are partially due to strain-specific variations in the antigenic sequence types and expression levels of outer membrane proteins (OMPs), including the immunodominant OMP PorA. In this study, dOMV vaccines deficient in major OMPs, including PorA, PorB, and RmpM were isolated and used to immunize rabbits and mice. Serum samples were obtained from each animal and tested for antibody responses against five MenB strains. Immunization with wild type dOMVs elicited antibodies to major antigens including PorA, PorB, RmpM, and lipooligosaccharide (LOS), and demonstrated limited bactericidal activity against heterologous strains. In contrast, OMP-deficient dOMV vaccines elicited broadly cross-reactive bactericidal antibodies, with PorA/PorB-dual deficient dOMVs inducing antibodies exhibiting the greatest cross-reactivity. Enhanced killing of heterologous strains correlated with binding to unique protein bands in immunoblots, suggestive of improved immunogenicity of antigens under-represented in the wild type vaccine., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [K. A. M. and M. C. B. are named as inventors on patent applications in the area of meningococcal and gonococcal vaccines. Rights to these inventions have been assigned to the U.S. Food and Drug Administration. All other authors report no potential conflicts of interest. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the U.S. Food and Drug Administration, the Uniformed Services University of the Health Sciences, or the Department of Defense]., (Published by Elsevier Ltd.)

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp-CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to G M1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease., (Published by Elsevier Ltd.)

PorB is a well-characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface-exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non-epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non-adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane-associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB-containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion., (© 2017 John Wiley & Sons Ltd.)

Background: PsA-TT (MenAfriVac) is a conjugated polysaccharide vaccine developed to eliminate group A meningococcal disease in Africa. Vaccination of African study participants with 1 dose of PsA-TT led to the production of anti-A polysaccharide antibodies and increased serum bactericidal activity measured using rabbit complement (rSBA). Bactericidal responses measured with human complement (hSBA) are presented here., Methods: Sera collected before and at 28 days and 1 year after vaccination with either PsA-TT or quadrivalent polysaccharide vaccine (PsACWY) from a random, age-distributed 360-subject subset of the Meningitis Vaccine Project study of PsA-TT in Africans aged 2-29 years were tested for hSBA. Geometric mean titer, fold-rise, and threshold analyses were compared between vaccine groups and age groups. hSBA, rSBA, and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) results were compared and assay correlation and agreement determined., Results: hSBA responses to PsA-TT were substantially higher than those to PsACWY at 28 days and 1 year following immunization, similar to previously reported rSBA and IgG results. The hSBA and IgG ELISA results identified differences between age groups that were not evident by rSBA. The rSBA data indicated sustained high titers 1 year after immunization, whereas hSBA GMTs at 1 year approached 4 in young children., Conclusions: The high level of protection following PsA-TT immunization campaigns is consistent with the strong hSBA immune responses observed here. Future implementation decisions will likely depend on immunologic data and their long-term correlation with disease and carriage prevention. Expanded immunologic and epidemiologic surveillance may improve the interpretation of differences between these immunoassays., (Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC') was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC' and then used the SBA assay with hC' (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC' lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC'. hSBA titers determined using three independent lots of pooled hC' were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC' is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

Gonorrhea occurs at high incidence throughout the world and significantly impacts reproductive health and the spread of human immunodeficiency virus. Current control measures are inadequate and seriously threatened by the rapid emergence of antibiotic resistance. Progress on gonorrhea vaccines has been slow; however, recent advances justify significant effort in this area. Conserved vaccine antigens have been identified that elicit bactericidal antibodies and, or play key roles in pathogenesis that could be targeted by a vaccine-induced response. A murine genital tract infection model is available for systematic testing of antigens, immunization routes and adjuvants, and transgenic mice exist to relieve some host restrictions. Furthermore, mechanisms by which Neisseria gonorrhoeae avoids inducing a protective adaptive response are being elucidated using human cells and the mouse model. Induction of a Th1 response in mice clears infection and induces a memory response, which suggests Th1-inducing adjuvants may be key in vaccine-induced protection. Continued research in this area should include human testing and clinical studies to confirm or negate findings from experimental systems and to define protective host factors., (Copyright © 2013. Published by Elsevier Ltd.)

Background: Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA(91/95) and parC86 mutations on gonococcal fitness., Methods: Mutant gyrA(91/95) and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR(-79) mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection., Results: In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA(91/95) mutation conferred an in vivo fitness benefit to wild-type and mtrR(-79) mutant gonococci. The gyrA(91/95), parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA(91/95), parC86, mtrR(-79) mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (Cip(R)) mutant was selected during infection with the gyrA(91/95), parC86, mtrR(-79) mutant in which the mtrR(-79) mutation was repaired and the gyrA(91) mutation was altered. This in vivo-selected mutant grew as well as the wild-type strain in vitro., Conclusions: gyrA(91/95) mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain Cip(R).

Background: Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988-2006) for study (n = 372)., Methods: We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA., Results: In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1., Conclusions: A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.

Capsular polysaccharide (CP) plays an important role in the pathogenicity and immunogenicity of Staphylococcus aureus, yet the common serotypes of S. aureus isolated from US pediatric patients have not been reported. We investigated capsular serotype as well as methicillin susceptibility, presence of Panton-Valentine leukocidin (PVL), and clonal relatedness of pediatric S. aureus isolates. Clinical isolates were tested for methicillin susceptibility, presence of mecA, lukS-PV and lukF-PV, cap5 and cap8 genes by PCR, and for capsular or surface polysaccharide expression (CP5, CP8, or 336 polysaccharide) by agglutination. Genetic relatedness was determined by pulsed-field gel electrophoresis. All S. aureus isolates encoded cap5 or cap8. Sixty-nine percent of 2004-2005 isolates were methicillin-susceptible (MSSA) and most expressed a detectable capsule. The majority of MRSA isolates (82%) were unencapsulated, exposing an expressed cell wall techoic acid antigen 336. Pulsed-field type USA300 were MRSA, PVL-positive, unencapsulated strains that were associated with deep skin infections and recurrent disease. Over half (58%) of all isolates from invasive pediatric dermatologic infections were USA300. All pediatric isolates contained either capsule type 5 or capsule type 8 genes, and roughly half of the S. aureus clinical disease isolates from our population were diverse MSSA-encapsulated strains. The majority of the remaining pediatric clinical disease isolates were unencapsulated serotype 336 strains of the PVL(+) USA300 community-associated-MRSA clone., (FEMS Immunology & Medical Microbiology © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.)

A double-blind, randomized, controlled phase I study to assess the safety, immunogenicity, and antibody persistence of a new group A conjugate vaccine (PsA-TT) in volunteers aged 18 to 35 years was previously performed. Subjects received one dose of either the PsA-TT conjugate vaccine, meningococcal A/C polysaccharide vaccine (PsA/C), or tetanus toxoid vaccine. The conjugate vaccine was shown to be safe and immunogenic as demonstrated by a standardized group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and by a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA). This report details further analysis of the sera using four additional immunologic assays to investigate the relationship between the different immunoassays. The immunoassays used were an SBA assay that used human complement (hSBA), a group A-specific IgG multiplexed bead assay, and two opsonophagocytic antibody (OPA) assays which used two different methodologies. For each vaccine group, geometric mean concentrations or geometric mean titers were determined for all assays before and 4, 24, and 48 weeks after vaccination. Pearson's correlation coefficients were used to assess the relationship between the six assays using data from all available visits. An excellent correlation was observed between the group A-specific IgG concentrations obtained by ELISA and those obtained by the multiplexed bead assay. hSBA and rSBA titers correlated moderately, although proportions of subjects with putatively protective titers and those demonstrating a > or = 4-fold rise were similar. The two OPA methods correlated weakly and achieved only a low correlation with the other immunoassays. The correlation between hSBA and group A-specific IgG was higher for the PsA-TT group than for the PsA/C group.

Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, beta-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered.

Background: Distinguishing the recent introduction of quinolone resistant gonococci into a population from diversification of resistant strains already in the population is important for planning effective infection control strategies. We applied molecular evolutionary analyses to DNA sequences from 9 housekeeping genes and gyrA, parC and porB of 24 quinolone resistant N. gonorrhoeae (QRNG) and 24 quinolone sensitive isolates collected in Israel during 2000-2001., Results: Phylogenetic and eBURST analyses and estimates of divergence time indicated QRNG were introduced on 3 separate occasions and underwent limited diversification by mutation, deletion and horizontal gene transfer. Reconstruction of N. gonorrhoeae demography showed a slowly declining effective strain population size from 1976 to 1993, rapid decline between 1994 and 1999, and an increase from 1999 to 2001. This is partially attributable to declining gonorrhea case rates from 1973 to 1994. Additional contributing factors are selective sweeps of antibiotic resistant gonococci and increased transmission from sex workers. The abrupt decline in the mid-1990s heralded an increased incidence of gonorrhea from 1997 to the present. The subsequent increase in effective strain population size since 1999 reflects the increased gonococcal census population and introduction of quinolone resistance strains., Conclusion: Our study demonstrates the effective use of population genetic approaches to assess recent and historical population dynamics of N. gonorrhoeae.

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests., (Copyright (c) 2007 Wiley-Liss, Inc.)

Neisseria gonorrhoeae is a common cause of genitourinary sexually transmitted infections. N. gonorrhoeae is an obligate human pathogen that has evidence of tissue-specific host interactions and diverse extragenital manifestations of infection both in adult and pediatric populations. The clinical presentation of extragenital gonorrhea, diagnostic methods, treatment and preventive measures are reviewed.

The porin gene (porB) of Neisseria gonorrhoeae encodes the major outer membrane protein identified as PI or Por. To examine the utility of por variable-region (VR) typing, porB from 206 isolates was characterized by using oligonucleotide probes in a checkerboard hybridization assay that identifies the sequence types of five VRs of both PIA and PIB porB alleles. The strains represented temporally and geographically distinct isolates, isolates from a large cluster, epidemiologically linked partner isolates, and a collection of strains from disseminated gonococcal infections. By using rigorous epidemiologic criteria for transmission of infection between sex partners, por VR typing was more discriminatory than serovar typing in classifying isolates from both members of 43 epidemiologically linked pairs: 39 of 43 pairs were classified as coinciding by por VR typing compared to 43 of 43 by serovar determination (P = 0.058). porB sequence data confirmed the accuracy of the por VR method. Relationships between VR type and serovar typing monoclonal antibodies were observed for all six PIB and three of six PIA antibodies. por VR typing is a molecular tool that appears to have broad applicability. This method can be adapted to a wide range of technologies from simple hybridization to microarray and may allow for typing from noncultured clinical specimens.

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Quinolone resistance is increasing rapidly in Neisseria gonorrhoeae and is a significant public health problem that requires ongoing surveillance. To examine the feasibility of molecular surveillance of quinolone resistance, and to further characterize an outbreak of resistant N. gonorrhoeae in Israel, the quinolone resistance-determining region (QRDR) sequences and the por types of 80 N. gonorrhoeae isolates were determined using molecular techniques. QRDRs of gyrA and parC were amplified by polymerase chain reaction and were sequenced directly. The por type was determined by checkerboard hybridizations performed using oligonucleotide probes to regions encoding 5 variable loops of the porin protein. All 42 ciprofloxacin-resistant (CipR) isolates had mutations in QRDRs of both gyrA and parC, and identical mutations were found in 93% of these isolates. One intermediately resistant isolate had 1 mutation in gyrA, and susceptible isolates showed no mutations. Forty isolates had 1 of 2 por types that differed only by an in-frame deletion in variable region 5; all but 1 of these isolates were CipR. QRDR sequencing and por type determination showed that the outbreak of CipR N. gonorrhoeae in Israel was clonal. QRDR mutations were consistent with those previously characterized; this indicates that DNA probes can be developed for rapid detection and surveillance of quinolone-resistant N. gonorrhoeae in settings in which nonculture diagnostic methods are used.

This article provides a brief summary of recent US epide-miology, antimicrobial resistance, and treatment of Neisseria gonorrhoeae infections. Selected research regarding infections caused by N. gonorrhoeae is described, with particular emphasis on the advances made by new molecular methods.

The Neisseria gonorrhoeae porin protein (Por) is a potential vaccine target and is the antigenic determinant for serovar typing. Two classes of Por, PIA and PIB, and antigenically distinct variants within each class result from sequence variations in the por gene variable regions (VRs) encoding surface-exposed loops. Oligonucleotide probes to 5 VRs of each class were used in checkerboard hybridizations to type 282 clinical gonococcal isolates selected from strains collected over the course of 10 years. PIA strains (n=63) showed limited por diversity, with 90% having 1 of 4 por types. PIB strains (n=219) were more diverse, although several common por types were identified that persisted over time. Variation within individual VRs was found to be limited. The present study provides information about the diversity of Por in strains circulating in a single geographic region over time, illustrates the utility of a novel por typing method, and has implications for vaccine development.

The importance of O-acetyl groups to the immunogenicity of Neisseria meningitidis serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.

Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Nine lgt genes at three chromosomal loci (lgt-1, 2, 3) encoding the glycosyltransferases responsible for the biosynthesis of LOS oligosaccharide chains were examined in 26 Neisseria meningitidis, 51 Neisseria gonorrhoeae and 18 commensal Neisseria strains. DNA hybridization, PCR and nucleotide sequence data were compared to previously reported lgt genes. Analysis of the genetic organization of the lgt loci revealed that in N. meningitidis, the lgt-1 and lgt-3 loci were hypervariable genomic regions, whereas the lgt-2 locus was conserved. In N. gonorrhoeae, no variability in the composition or organization of the three lgt loci was observed. lgt genes were detected only in some commensal Neisseria species. The genetic organization of the lgt-1 locus was classified into eight types and the lgt-3 locus was classified into four types. Two types of arrangement at lgt-1 (II and IV) and one type of arrangement at lgt-3 (IV) were novel genetic organizations reported in this study. Based on the three lgt loci, 10 LOS genotypes of N. meningitidis were distinguished. Phylogenetic analysis revealed a gene cluster, lgtH, which separated from the homologous genes lgtB and lgtE. The lgtH and lgtE genes were mutually exclusive and were located at the same position in lgt-1. The data demonstrated that pathogenic and commensal Neisseria share a common lgt gene pool and horizontal gene transfer appears to contribute to the genetic diversity of the lgt loci in Neisseria.

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.

The porin proteins PIA and PIB of Neisseria gonorrhoeae are serotyping antigens for the serovar classification system and leading candidates for gonococcal vaccine development. Although serotyping has been a useful tool, this method can be insensitive to critical sequence changes in the por gene, including those in surface-exposed variable regions (VRs). A sensitive and specific typing system for N. gonorrhoeae has been developed that uses biotin-labeled oligonucleotide probes with chemiluminescence detection to type PIB gene VRs. The PIB VR types of geographically and temporally diverse gonococcal strains and sexual contact isolates were determined. por VR typing discriminated between most unrelated isolates and provided information about individual VR type that was not apparent from serovar designations. PIB VR typing avoids limited monoclonal antibody availability, interlaboratory variation, and the requirement for culture-based surveillance associated with gonococcal serotyping, and provides useful information about the molecular epidemiology of individual por gene VRs.

The class 3 porin proteins of Neisseria meningitidis stimulate bactericidal antibodies and express serotype-specific antigenic epitopes. Sequence analysis of porB genes for the class 3 proteins revealed regions of variability that map to surface-exposed loops. To evaluate the relationship between serotype and variable-region (VR) genotype, sequences from the 11 class 3-expressing serotype strains and 3 additional serotype 4 strains were analyzed by molecular techniques. Multiple-sequence alignment revealed a limited number of unique sequences at each of four VRs (VR1 to VR4), ranging from four unique sequences at VR1 to seven sequence patterns at VR2 and VR4. Serotype-specific VR sequences were found in each of the four VRs, suggesting that each VR has immunologic importance. Five serotypes had at least one VR sequence that was unique. Three serotypes which had sequences in common with other serotypes at each VR were distinguished by examining multiple VRs. Serotype 3 was identical to serotype 19 at each VR, and serotype 8 was identical to serotype 18 at each VR. Serotypes 4 and 21 were identical at VR1 and significantly different at VR3 and VR4. A subpopulation of serotype 4 strains with a unique VR2 sequence was identified. The serotypes which were grouped with closely related or identical sequences at one VR were grouped with different serotypes at other VRs consistent with the pattern of genetic mosaicism described for the porA (class 1 protein) gene. Hybridization assays demonstrated the ability to identify VR genotypes and distinguish serotypes using biotin-labelled oligonucleotide probes. This information may be useful in strain selection for vaccine development, in epidemiologic studies to determine the prevalence of the individual VR genotype (especially among nonserotypeable strains) and, combined with PCR, in the identification of culture-negative suspected meningococcal cases.

Ours is apparently the first reported pediatric case of Stomatococcus mucilaginosus bacteremia. It is the second reported case of S mucilaginosus catheter-associated infection, but the first case successfully treated without removing the central venous catheter. This unusual organism should be added to the list of opportunistic pathogens that can be isolated from immunocompromised pediatric patients.