Margaret C. Bash

Vaccines against gonorrhea: Current status and future challenges

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.

Impact of fluoroquinolone resistance mutations on gonococcal fitness and in vivo selection for compensatory mutations.

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).

Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species.

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.

Quinolone Resistance–Determining Region Mutations and por Type of Neisseria gonorrhoeae Isolates: Resistance Surveillance and Typing by Molecular Methodologies

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.

Molecular Epidemiology of Neisseria meningitidis Serogroup B in Brazil

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.

A Typing System for Neisseria gonorrhoeae Based on Biotinylated Oligonucleotide Probes to PIB Gene Variable Regions

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.

Phenotypic and Genotypic Analyses of Neisseria gonorrhoeae Isolates That Express Frequently Recovered PorB PIA Variable Region Types Suggest that Certain P1a Porin Sequences Confer a Selective Advantage for Urogenital Tract Infection

ABSTRACT 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, β-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.

Distinguishing importation from diversification of quinolone-resistant Neisseria gonorrhoeae by molecular evolutionary analysis

BackgroundDistinguishing 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.ResultsPhylogenetic 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.ConclusionOur study demonstrates the effective use of population genetic approaches to assess recent and historical population dynamics of N. gonorrhoeae.

Heterogeneity in non-epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitidis : PorB interactions alter antibody binding

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.

Human Complement Bactericidal Responses to a Group A Meningococcal Conjugate Vaccine in Africans and Comparison to Responses Measured by 2 Other Group A Immunoassays

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.