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Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.

The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.

The human nasopharynx is colonized by commensal bacteria and pathobionts, which comprise a complex microbial ecosystem capable of generating primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica results in safe, sustained colonization. Herein is described a novel technology to chromosomally transform N. lactamica with heterologous antigen, for the purpose of safe delivery to the mucosal surface and the generation of an antigen-specific immune response. N. lactamica was transformed to express the meningococcal vaccine antigen Neisseria Adhesin A (NadA) and was inoculated intranasally into humans at a dose of 105 colony-forming units. NadA-expressing N. lactamica colonized these individuals and was carried asymptomatically for 3 months. Colonization with NadA-expressing N. lactamica generated NadA-specific IgG-secreting plasma cells within 14 days of colonization and both NadA-specific IgG and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells circulate in the bloodstream of colonized participants for at least 90 days. Genetically transformed N. lactamica has the potential to be a safe bacterial vehicle to generate beneficial immune responses to a wide range of heterologous antigens during sustained pharyngeal carriage.

Purpose of review Nonpathogenic commensal Neisseria are rarely considered in the clinical setting despite evidence that they can cause invasive opportunistic infections. In contrast, they may offer protection against pathogenic Neisseria, and such relationships are being actively explored in experimental studies. Recent findings Recent case reports are presented of invasive infection caused by nonpathogenic Neisseria in patients on novel biologic therapies. On the other hand, Neisseria lactamica, a nonpathogenic commensal, has been shown in human challenge studies to inhibit colonization by Neisseria meningitidis. Experimental mouse models have also explored the inhibitory effects of nonpathogenic Neisseria on Neisseria gonnhoreae infection. Cutting-edge advances in metagenomics and microbiomics are being used to understand the mechanisms underpinning these effects. Summary Clinicians should have increased awareness of nonpathogenic Neisseria. First, as new immunomodulating therapies become licenced, the interactions that maintain balance between commensals and their human hosts may be altered. Second, these bacteria are showing promise in their capacity to exclude pathogenic Neisseria species from their anatomical niches.

Although acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is a manageable disease for many, it is still a source of significant morbidity and economic hardship for many others. The predominant mode of transmission of HIV/AIDS is sexual intercourse, and measures to reduce transmission are needed. Previously, we showed that caerin 1 antimicrobial peptides (AMPs) originally derived from Australian amphibians inhibited in vitro transmission of HIV at relatively low concentrations and had low toxicity for T cells and an endocervical cell line. The use of AMPs as part of microbicidal formulations would expose the vaginal microbiome to these agents and cause potential harm to protective lactobacilli. Here, we tested the effects of caerin 1 peptides and their analogs on the viability of two species of common vaginal lactobacilli (Lactobacillus rhamnosus and Lactobacillus crispatus). Several candidate peptides had limited toxicity for the lactobacilli at a range of concentrations that would inhibit HIV. Three AMPs were also tested for their ability to inhibit growth of Neisseria lactamica, a close relative of the sexually transmissible Neisseria gonorrhoeae. Neisseria lactamica was significantly more sensitive to the AMPs than the lactobacilli. Thus, several candidate AMPs have the capacity to inhibit HIV and possible N. gonorrhoeae transmission at concentrations that are significantly less harmful to the resident lactobacilli.

The genus Neisseria includes three major species of importance to human health and disease ( Neisseria gonorrhoeae , Neisseria meningitidis , and Neisseria lactamica ) that express broad-spectrum O -linked protein glycosylation (Pgl) systems. The potential for related Pgl systems in other species in the genus, however, remains to be determined. Using a strain of Neisseria elongata subsp. glycolytica , a unique tetrasaccharide glycoform consisting of di- N -acetylbacillosamine and glucose as the first two sugars followed by a rare sugar whose mass spectrometric fragmentation profile was most consistent with di- N -acetyl hexuronic acid and a N -acetylhexosamine at the nonreducing end has been identified. Based on established mechanisms for UDP-di- N -acetyl hexuronic acid biosynthesis found in other microbes, we searched for genes encoding related pathway components in the N. elongata subsp. glycolytica genome. Here, we detail the identification of such genes and the ensuing glycosylation phenotypes engendered by their inactivation. While the findings extend the conservative nature of microbial UDP-di- N -acetyl hexuronic acid biosynthesis, mutant glycosylation phenotypes reveal unique, relaxed specificities of the glycosyltransferases and oligosaccharyltransferases to incorporate pathway intermediate UDP-sugars into mature glycoforms. IMPORTANCE Broad-spectrum protein glycosylation (Pgl) systems are well recognized in bacteria and archaea. Knowledge of how these systems relate structurally, biochemically, and evolutionarily to one another and to others associated with microbial surface glycoconjugate expression is still incomplete. Here, we detail reverse genetic efforts toward characterization of protein glycosylation mutants of N. elongata subsp. glycolytica that define the biosynthesis of a conserved but relatively rare UDP-sugar precursor. The results show both a significant degree of intra- and transkingdom conservation in the utilization of UDP-di- N -acetyl-glucuronic acid and singular properties related to the relaxed specificities of the N. elongata subsp. glycolytica system

Background Neisseria meningitidis are common colonizers of the human nasopharynx. In some circumstances, N. meningitidis becomes an opportunistic pathogen that invades tissues and causes meningitis. While a vaccine against a number of serogroups has been in effective use for many years, a vaccine against N. meningitidis group B has not yet been universally adopted. Bacterial heat shock protein complex (HSPC) vaccines comprise bacterial HSPs, purified with their chaperoned protein cargo. HSPC vaccines use the intrinsic adjuvant activity of their HSP, thought to act via Toll-like receptors (TLR), to induce an immune response against their cargo antigens. This study evaluated HSPC vaccines from N. meningitidis and the closely related commensal N. lactamica . Results The protein composition of N. lactamica and N. meningitidis HSPCs were similar. Using human HEK293 cells we found that both HSPCs can induce an innate immune response via activation of TLR2. However, stimulation of TLR2 or TLR4 deficient murine splenocytes revealed that HSPCs can activate an innate immune response via multiple receptors. Vaccination of wildtype mice with the Neisseria HSPC induced a strong antibody response and a Th1-restricted T helper response. However, vaccination of mice deficient in the major TLR adaptor protein, MyD88, revealed that while the Th1 response to Neisseria HSPC requires MyD88, these vaccines unexpectedly induced an antigen-specific antibody response via a MyD88-independent mechanism. Conclusions N. lactamica and N. meningitidis HSPC vaccines both have potential utility for immunising against neisserial meningitis without the requirement for an exogenous adjuvant. The mode of action of these vaccines is highly complex, with HSPCs inducing immune responses via both MyD88-dependent and -independent mechanisms. In particular, these HSPC vaccines induced an antibody response without detectable T cell help.

The three species Neisseria meningitidis , Neisseria gonorrheae , and Neisseria lactamica are often regarded as highly recombining bacteria. N . meningitidis has been considered a paradigmatic case of the “semiclonal model” or of “epidemic clonality,” demonstrating occasional bouts of clonal propagation in an otherwise recombining species. In this model, occasional clonality generates linkage disequilibrium in the short term. In the long run, however, the effects of clonality are countered by recombination. We show that many data are at odds with this proposal and that N . meningitidis fits the criteria that we have proposed for predominant clonal evolution (PCE). We point out that ( i ) the proposed way to distinguish epidemic clonality from PCE may be faulty and ( ii ) the evidence of deep phylogenies by microarrays and whole-genome sequencing is at odds with the predictions of the semiclonal model. Last, we revisit the species status of N . meningitidis , N . gonorrheae , and N . lactamica in the light of the PCE model.

We present the high quality, complete genome assembly of Neisseria lactamica Y92–1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

We present the high quality, complete genome assembly of Neisseria lactamica Y92–1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary 'omic' platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide 'universal' Neisseria vaccines.

Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to Nl, Nm occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive Nm strains and one Nm carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that Nm strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited - © 2006 Snyder and Saunders; licensee BioMed Central Ltd. Background: Neisseria meningitidis causes the life-threatening diseases meningococcal meningitis and meningococcal septicemia. Neisseria gonorrhoeae is closely related to the meningococcus, but is the cause of the very different infection, gonorrhea. A number of genes have been implicated in the virulence of these related yet distinct pathogens, but the genes that define and differentiate the species and their behaviours have not been established. Further, a related species, Neisseria lactamica is not associated with either type of infection in normally healthy people, and lives as a harmless commensal. We have determined which of the genes so far identified in the genome sequences of the pathogens are also present in this non-pathogenic related species. Results: Thirteen unrelated strains of N. lactamica were investigated using comparative genome hybridization to the pan-Neisseria microarray-v2, which contains 2845 unique gene probes. The presence of 127 'virulence genes' was specifically addressed; of these 85 are present in N. lactamica. Of the remaining 42 'virulence genes' only 11 are present in all four of the sequenced pathogenic Neisseria. Conclusion: Assessment of the complete dataset revealed that the vast majority of genes present in the pathogens are also present in N. lactamica. Of the 1,473 probes to genes shared by all four pathogenic genome sequences, 1,373 hybridize to N. lactamica. These shared genes cannot include genes that are necessary and sufficient for the virulence of the pathogens, since N. lactamica does not share this behaviour. This provides an essential context for the interpretation of gene complement studies of the pathogens. This study is supported by the Wellcome Trust.

Measuring cell proliferation and cell death during bacterial infection involves performing end-point assays that represent the response at a single time point. A new technology from Roche Applied Science and ACEA Biosciences allows continuous monitoring of cells in real-time using specialized cell culture microplates containing micro-electrodes. The xCELLigence system enables continuous measurement and quantification of cell adhesion, proliferation, spreading, cell death and detachment, thus creating a picture of cell function during bacterial infection. Furthermore, lag and log phases can be determined to estimate optimal times to infect cells. In this study we used this system to provide valuable insights into cell function in response to several virulence factors of the meningitis causing pathogen Neisseria meningitidis, including the lipopolysaccharide (LPS), the polysaccharide capsule and the outer membrane protein Opc. We observed that prolonged time of infection with pathogenic Neisseria strains led to morphological changes including cell rounding and loss of cell-cell contact, thus resulting in changed electrical impedance as monitored in real-time. Furthermore, cell function in response to 14 strains of apathogenic Neisseria spp. (N. lactamica and N. mucosa) was analyzed. In contrast, infection with apathogenic N. lactamica isolates did not change electrical impedance monitored for 48 h. Together our data show that this system can be used as a rapid monitoring tool for cellular function in response to bacterial infection and combines high data acquisition rates with ease of handling.

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation ( pgl ) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro . These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.

SinceNeisseria gonorrhoeaeandNeisseria meningitidisare obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensalNeisseriagenome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. OnlyNeisseria mucosacontained a nitrate reductase (Nar), and onlyNeisseria lactamica,Neisseria cinerea,Neisseria subflava,Neisseria flavescensandNeisseria siccacontained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested exceptN. mucosahad a two- to fourfold lower nitrite reductase activity thanN. gonorrhoeae, whileN. meningitidisand most of the commensalNeisseriaspecies had a two- to fourfold higher nitric oxide (NO) reductase activity. ForN. meningitidisand most of the commensalNeisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared withN. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that ofN. gonorrhoeae. The greatest difference between theNeisseriaspecies was the lack of a functional Nos system in the pathogenic speciesN. gonorrhoeaeandN. meningitidis.

The availability of complete genome sequence of Neisseria meningitidis serogroup B strain MC58 and reverse vaccinology has allowed the discovery of several novel antigens. Here, we have explored the potential of N. meningitidis lipoprotein NMB0938 as a vaccine candidate, based on investigation of gene sequence conservation and the antibody response elicited after immunization in mice. This antigen was previously identified by a genome-based approach as an outer membrane lipoprotein unique to the Neisseria genus. The nmb0938 gene was present in all 37 Neisseria isolates analyzed in this study. Based on amino acid sequence identity, 16 unique sequences were identified which clustered into three variants with identities ranging from 92 to 99%, with one cluster represented by the Neisseria lactamica strains. Recombinant protein NMB0938 (rNMB0938) was expressed in Escherichia coli and purified after solubilization of the insoluble fraction. Antisera produced in mice against purified rNMB0938 reacted with a range of meningococcal strains in whole-cell ELISA and western blotting. Using flow cytometry, it was also shown that anti-rNMB0938 antibodies bound to the surface of the homologous meningococcal strain and activated complement deposition. Moreover, antibodies against rNMB0938 elicited complement-mediated killing of meningococcal strains from both sequence variants and conferred passive protection against meningococcal bacteremia in infant rats. According to our results, NMB0938 represents a promising candidate to be included in a vaccine to prevent meningococcal disease.

The report describes developments in meningococcal disease vaccines presented at the 16th International Pathogenic Neisseria Conference, Rotterdam, 7-12 September 2008. Great progress has been made by the Meningitis Vaccine Project to provide an affordable and effective serogroup A conjugate vaccine for use in the meningitis belt of Sub-Saharan Africa. The vaccine has been shown to be safe and to produce excellent immune response in phase 2 clinical trials in India and Africa in the target populations and will be rolled out to the worst affected countries from 2009. This vaccine has the potential to make a huge impact on public health in this region. This conference heard that the use of an epidemic strain-specific outer membrane vesicle (OMV) vaccine in New Zealand has been discontinued. Views for and against this decision were presented. Several MenB vaccines have progressed to clinical evaluation. The most advanced are the Novartis five recombinant protein variants and the Wyeth vaccine based on two factor H binding protein variants. Promising results from both vaccines with genetically-detoxified lipooligosaccharide and overexpressed heterologous antigens, OMV's from Neisseria lactamica and recombinant Opa proteins.

Serogroup B strains are now responsible for over 80% of meningococcal disease in the UK and no suitable vaccine is available that confers universal protection against all serogroup B strains. Neisseria lactamica shares many antigens with the meningococcus, except capsule and the surface protein PorA. Many of these antigens are thought to be responsible for providing cross-protective immunity to meningococcal disease. We have developed an N. lactamica vaccine using methods developed for meningococcal outer membrane vesicle (OMV) vaccines. The major antigenic components were identified by excision of 11 major protein bands from an SDS-PAGE gel, followed by mass spectrometric identification. These bands contained at least 22 proteins identified from an unassembled N. lactamica genome, 15 of which having orthologues in published pathogenic Neisseria genomes. Western blotting revealed that most of these bands were immunogenic, and antibodies to these proteins generally cross-reacted with N. meningitidis proteins. Sera from mice and rabbits immunized with either N. lactamica or N. meningitidis OMVs produced comparable cross-reactive ELISA titres against OMVs prepared from a panel of diverse meningococcal strains. Mice immunized with either N. meningitidis or N. lactamica OMVs showed no detectable serum bactericidal activity against the panel of target strains except N. meningitidis OMV sera against the homologous strain. Similarly, rabbit antisera to N. lactamica OMVs elicited little or no bactericidal antibodies against the panel of serogroup B meningococcal strains. However, such antisera did mediate opsonophagocytosis, suggestingthat this may did mediate opsonophagocytosis, suggesting that this may be a mechanism by which this vaccine protects in a mouse model of meningococcal bacteraemia.

Background Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensalNeisseria lactamicaand meningococcal disease, we investigated whether controlled infection of human volunteers withN. lactamicaprevents colonization byNeisseria meningitidis. Methods In a block-randomized human challenge study, 310 university students were inoculated with 10^4 colony-forming units ofN. lactamicaor were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated withN. lactamicaand resampled 2 weeks later. Results At baseline, naturalN. meningitidiscarriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation ofN. lactamica, 33.6% (48/143) of the challenge group became colonized withN. lactamica. In this group, meningococcal carriage reduced from 24.2% (36/149) at inoculation to 14.7% (21/143) 2 weeks after inoculation (−9.5%;P= .006). The inhibition of meningococcal carriage was only observed in carriers ofN. lactamica, was due both to displacement of existing meningococci and to inhibition of new acquisition, and persisted over at least 16 weeks. Crossover inoculation of controls withN. lactamicareplicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types. Results The inhibition of meningococcal carriage byN. lactamicais even more potent than after glycoconjugate meningococcal vaccination.Neisseria lactamicaor its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage ofN. lactamica.

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.

The muscosal delivery of vaccines has many advantages including ease of administration and the induction of a mucosal immune response at the natural site of infection for many pathogens. Mice were immunised with outer membrane vesicles (OMV) prepared from Neisseria lactamica or Neisseria meningitidis by subcutaneous (SC) or intranasal (IN) routes, or live cells of N. lactamica given IN or by SC injection. A systemic IgG and mucosal IgA response was demonstrated and N. lactamica OMV induced antibodies cross-reactive with N. meningitidis; however, a cross-reactive response following IN administration was only evident after three doses of vaccine. OMV from both organisms were also an effective intranasal adjuvant for a co-administered model antigen, hepatitis B surface antigen (HBsAg), inducing systemic IgG against HBsAg and IgA in lung and vaginal washes. IN administration of N. meningitidis OMV elicited serum antibodies that were bactericidal for meningococci and provided passive protection in an infant rat model of meningococcal bacteraemia. The antibody response to N. lactamica OMV given IN was only weakly bactericidal but still afforded passive protection. Thus, OMV from N. lactamica given IN elicit immune responses cross-reactive with N. meningitidis and act as an effective mucosal adjuvant.

Neisseria gonorrhoeae is a human pathogen causing the sexually transmitted disease gonorrhoeae. The bacteria preferentially attach to and invade epithelial cells of the genital tract. As these cells previously have been shown to express the human cathelicidin LL-37, we wanted to investigate the role of LL-37 during N. gonorrhoeae infection. The cervical epithelial cell line ME180 was utilized and the expression of LL-37 was confirmed on both peptide and transcriptional levels. Moreover, LL-37 exhibited potent in vitro activity against N. gonorrhoeae. Interestingly, the transcript and peptide levels of LL-37 were downregulated during infection, according to quantitative real-time polymerase chain reaction (PCR) and immunocyto-chemistry. The downregulation was most prominent with pathogenic strains of Neisseria, while non-pathogenic strains such as Neisseria lactamica and Escherichia coli only exhibited moderate effects. Heat-killed N. gonorrhoeae had no impact on the downregulation, emphasizing the importance of live bacteria. The results in this study suggest that pathogenic Neisseria may gain a survival advantage in the female genital tract by downregulating LL-37 expression.

Serum samples from 31 childrenor = 4 years old who were convalescing after meningococcal disease were used in a quantitative hybridization assay to establish antibody reactivity to 94 candidate meningococcal vaccine antigens. Genes encoding 22 of 23 strongly recognized proteins were found inor = 94% of the patients' meningococcal strains, and most were also widely prevalent in Neisseria lactamica and other commensal Neisseria species. Similar antibody reactivity was found in serum samples from healthy control children, suggesting that these antibodies arose from asymptomatic colonization. The 23rd protein, NadA, elicited strong reactivity solely in convalescent patients previously infected with a nadA+ strain. nadA was not present in any of 29 diverse N. lactamica strains, suggesting that reactivity in these children arose from meningococcal infection. In contrast, serum samples from healthy adults contained anti-NadA immunoglobulin G at high levels. The correlation of NadA antibody level with natural acquisition of protective immunity suggests that NadA may be a valuable component of a childhood antimeningococcal vaccine.

A novel antigen that induces cross-reactive bactericidal antibodies against a number ofNeisseria meningitidisstrains is described. This antigen, a ∼28-kDa lipoprotein called LP2086, was first observed within a complex mixture of soluble outer membrane proteins (sOMPs) following a series of fractionation, protein purification, and proteomics steps. Approximately 95 different neisserial isolates tested positive by Western blotting and PCR screening methods for the presence of the protein and the gene encoding LP2086. The strains tested included isolates ofN. meningitidisserogroups A, B, C, W135, and Y,Neisseria gonorrhoeae, andNeisseria lactamica. To better understand the microheterogeneity of this protein, the 2086 genes from 63 neisserial isolates were sequenced. Two different subfamilies of LP2086 were identified based on deduced amino acid sequence homology. A high degree of amino acid sequence similarity exists within each 2086 subfamily. The highest degree of genetic diversity was seen between the two subfamilies which share approximately 60 to 75% homology at the nucleic acid level. Flow cytometry (fluorescence-activated cell sorting) analyses and electron microscopy indicated that the LP2086 is localized on the outer surface ofN. meningitidis. Antiserum produced against a single protein variant was capable of eliciting bactericidal activity against strains expressing different serosubtype antigens. Combining one recombinant lipidated 2086 (rLP2086) variant from each subfamily with two rPorA variants elicited bactericidal activity against all strains tested. The rLP2086 family of antigens are candidates worthy of further vaccine development.

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P0.005) and military recruits (37.4%) (chi2=21.11, P0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.

Three of the 18 open reading frames in the division and cell wall synthesis cluster of the pathogenic Neisseria spp. are not present in the clusters of other bacterial species. The region containing two of these, dcaB and dcaC, displays interstrain and interspecies variability uncharacteristic of such clusters. 3' of dcaB is a Correia repeat enclosed element (CREE), which is only present in some strains. It has been suggested that this CREE is a transcriptional terminator, although we demonstrate otherwise. A gearbox-like promoter within this CREE is active in Escherichia coli but not in Neisseria meningitidis. There is an active promoter 5' of dcaC, although its sequence is not conserved. The presence of similarly located promoters has not been demonstrated in other species. In Neisseria lactamica, this promoter involves another dcw-associated CREE, the first demonstration of active promoter generation at the 5' end of this common intergenic, apparently mobile, element. Upstream of this promoter is an inverted pair of neisserial uptake signal sequences, which are commonly considered to be transcriptional terminators. It has been proposed to terminate transcription in this location, although we have demonstrated transcript extending through this uptake signal sequence. dcaC contains a 108 bp tandem repeat, which is present in different copy numbers in the neisserial strains examined. This investigation reveals extensive sequence variation, disputes the presence of transcriptional terminators and identifies active internal promoters in this normally highly conserved cluster of essential genes, and addresses the transcriptional activity of two common neisserial intergenic components.

Background Neisseria lactamica as one of the main commensal in oropharynx during the childhood is related to the induction of a natural immunity against meningococcal meningitis. Also Moraxella catarrhalis in oropharynx of children is a predisposing factor for otitis media infection. Objectives The current study aimed to investigate the frequency of the N. lactamica, other nonpathogenic Neisseria spp. and M. catarrhalis in the oropharynx of young healthy children in Ahvaz, Iran by the two phenotypic tests and Polymerase Chain Reaction (PCR). Materials and methods A total of 192 oropharyngeal swab samples of the young healthy children were studied during four months. Swabs were plated onto enriched selective media and non-selective media. Gram-negative and oxidase-positive diplococci were identified by several conventional biochemical tests. The PCR and sequencing were used to confirm the accuracy of laboratory diagnosis to identify N. lactamica and M. catarrhalis. Results Among 192 young healthy children with the mean age of 5.93 ± 2.5903 years, authors identified: N. lactamica (21.9%) in the age group of one to nine years; N. mucosa (6.3%); N. sicca (7.8%); N. cinerea (1.6%); N. subflava (biovar subflava) (4.2%); N. subflava (biovar perflava) (28.1%); N. subflava (biovar flava) (7.3%) and M. catarrhalis (42.7%). Conclusions The young healthy children screening by colonization of N. lactamica and other nonpathogenic Neisseria spp. in oropharynx was the first report in Ahvaz, Iran. The study results demonstrated the high frequency of colonization of M. catarrhalis in the studied young healthy children other than Neisseria spp.

Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host-cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short-lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down-regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface-associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface-located, adhesion-activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.

iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria spp., particularly with N. lactamica . We report here that immunization with N. lactamica killed whole cells, outer membrane vesicles, or outer membrane protein (OMP) pools and protected mice against lethal challenge by a number of diverse serogroup B and C meningococcal isolates in a model of bacteremic infection. Sera raised to N. lactamica killed whole cells, OMPs, or protein pools were found to cross-react with meningococcal isolates of a diverse range of genotypes and phenotypes. The results confirm the potential of N. lactamica to form the basis of a vaccine against meningococcal disease.

Natural immunity against Neisseria meningitidis is acquired during childhood and youth through successive colonizations by commensal Neisseria, carrier N. meningitidis, and other bacterial genera sharing cross-reactive antigens with the meningococci. We have analyzed in mice the ability of Neisseria lactamica strains to induce immunological memory so that, upon a later contact with N. meningitidis, quickly raise protective responses against antigens that show cross-reactivity with meningococcal surface proteins. Sera obtained from mice immunized with N. lactamica and boosted with N. meningitidis were able to kill meningococci, with bactericidal activities variable depending on the immunizing strains used in the assays. Different mixtures of those sera resulted in higher killing activities, which agrees with the idea that successive colonizations by N. lactamica enhance the anti-meningococcal response. The existence of such outer membrane cross-reactive antigens has to be kept in mind when using outer membrane vesicle (OMV)-based anti-meningococcal vaccines because their use can affect colonization by N. lactamica and other species, hampering the natural mechanisms of acquisition of immunity to the meningococci, and leaving its ecological niche free for colonization by undesirable microorganisms.

Of 830 Neisseria meningitidis isolates obtained from healthy carriers in Bavaria, Germany, 136 (16.4%) lacked the operons necessary for the synthesis, lipid modification, and transport of capsular polysaccharide. These operons were replaced by a non-coding intergenic region either 113 or 114 bp in length, termed here the capsule null locus (cnl). Comparisons of the nucleotide sequence of this region in the meningococcus and its acapsulate relatives, Neisseria gonorrhoeae and Neisseria lactamica, revealed six distinct sequence variants (cnl-1 to cnl-6), with a total of 10 nucleotide substitutions and three indels. With the exception of one 4 bp insertion, which was unique to a gonococcal isolate, all of the individual sequence changes were present in the N. lactamica isolates examined. The meningococcal isolates with a cnl belonged to one of four otherwise genetically diverse genetic groupings: the ST-53 and ST-1117 complexes (75 isolates); the ST-845 complex (12 isolates); the ST-198 and 1136 complexes (46 isolates), and the ST-44 complex (one isolate). These data demonstrated that a substantial proportion of carried meningococci were incapable of capsule production, that the cnl circulated within Neisseria populations by horizontal genetic exchange, and that the expression of a polysaccharide capsule was not a requirement for person-to-person transmission of certain meningococcal lineages.

Colonization of the human nasopharynx is a feature of some species of Neisseria, and is a prerequisite of invasive meningococcal disease. The likelihood of colonization by Neisseria meningitidis varies widely between humans, and very few develop invasive disease. Explants of nasal mucosa derived from adult patients with non-allergic nasal obstruction were infected experimentally with Neisseria spp. At intervals over 18 h incubation, washed explants were homogenized, and viable bacteria were counted. To estimate bacterial invasion of mucosa, explants were exposed to 0·25% sodium taurocholate for 30 s prior to homogenization. N. meningitidis was recovered from the mucosa and the organism invaded and replicated within the tissue, in contrast to N. lactamica and N. animalis (n=9, P

We assessed the genetic diversity of 26 Neisseria lactamica strains from epidemiologically related sources, i.e., groups of kindergartens and primary schools in three Bavarian towns, by the partial sequencing of the argF, rho, recA , and 16S ribosomal genes. We found a total of 17 genotypes, of which 12 were found only in one strain. The genotypes comprised 5 alleles of the argF gene, 9 of rho , 8 of recA , and 10 of the 16S ribosomal DNA. Sequence analysis by determination of homoplasy ratios and split decomposition analysis revealed abundant recombination within N. lactamica .

Human immune serum recognition of outer membrane components from commensal and pathogenic Neisseria cultured under neutral and acidic conditions was investigated. Acid stress caused no detectable alterations in lipooligosaccharide migration and (or) staining, in outer membrane protein profiles, or in immune serum recognition of outer membrane components from Neisseria mucosa or Neisseria sicca. There was also no difference in the lipoologosaccharide electrophoretic pattern of acid- and neutral-grown Neisseria lactamica, but there were differences in outer membrane protein expression. The outer membrane protein alterations induced by acid stress in N. lactamica were not the same as those seen in isolates from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection. Many differences were detected in the immune serum recognition of outer membrane components from acid- and neutral-cultured N. lactamica and from the clinical isolates of Neisseria gonorrhoeae, and these should be considered in vaccine design.

A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli . Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXα and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica , Neisseria subflava , Neisseria flava , Neisseria polysacchareae , Neisseria kochii , and Neisseria cinerea . All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.

Summary Natural sequence variation was investigated among serogroup A subgroup IV-1 Neisseria meningitidis isolated from diseased patients and healthy carriers in The Gambia, West Africa. The frequencies of DNA import were analysed by sequencing fragments of four linked genes encoding the immunogenic outer membrane proteins TbpB (transferrin binding protein B) and OpaA (an adhesin) plus two housekeeping enzymes. Seventeen foreign tbpB alleles were independently imported into the 98 strains tested, apparently due to immune selection. The median size of the imported DNA fragments was 5 kb, resulting in the occasional concurrent import of linked housekeeping genes by hitchhiking. Sequences of tbpB from other strains of N. meningitidis as well as commensal Neisseria lactamica and Neisseria spp. isolated from the same geographical area revealed that these species share a common tbpB gene pool and identified several examples of interspecific genetic exchange. These observations indicate that recombination can be more frequent between related species than within a species and indicate that effective vaccination against serogroup B meningococcal disease may be difficult to achieve.

Carriage of non-serogroupable Neisseria meningitidis or Neisseria lactamica induces antibodies protective against meningococcal disease. Antibodies directed against outer membrane proteins are bactericidal and the serotype and subtype outer membrane protein antigens are being examined for their value as vaccine candidates for serogroup B disease. The aim of this study was to examine the effect of carriage of these two Neisseria species among children and young adults on induction of antibodies to outer membrane components from strains causing disease in Greece. Among 53 patients with meningococcal disease, IgG or IgM antibodies were detected by ELISA in 9 of 13 (69%) from whom the bacteria were isolated and 27 of 40 (67%) who were culture-negative. For military recruits (n = 604), the proportion of carriers of meningococci with IgM or IgG to outer membrane proteins was higher than non-carriers, P0.05 and P = 0.000000, respectively. Among school children (n = 319), the proportion with IgM or IgG to outer membrane proteins for carriers of meningococci was higher compared with non-carriers, P = 0.000000 and P = 0000043, respectively. Carriage of N. lactamica was not associated with the presence of either IgM or IgG to the outer membrane proteins in the children. The higher proportion of children (50%) with IgM to outer membrane proteins compared with recruits (10%) might reflect more recent exposure and primary immune responses to the bacteria. The lack of association between antibodies to outer membrane proteins and carriage of N. lactamica could reflect observations that the majority of N. lactamica isolates from Greece and other countries do not react with monoclonal typing reagents. Bactericidal antibodies to meningococci associated with high levels of IgG to N. lactamica were found in a previous study; these are thought to be directed to antigens other than outer membrane proteins or capsules and imply antigens such as lipo-oligosaccharide are involved in induction of antibodies cross-reactive with meningococci.