Genotypic and Phenotypic Characterization of the O-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates.

Species within the genus Neisseria display significant glycan diversity associated with the O-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail the pgl genotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. The O-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All major pgl locus polymorphisms identified in Neisseria meningitidis to date were present in our isolate collection, with the variable presence of pglG and pglH, both in combination with either pglB or pglB2 We identified significant changes and diversity in the pgl genotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and the O-acetyltransferase. To our knowledge, this is the first characterization of the pgl genotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity in N. meningitidis and into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described in Neisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

Molecular characterization of clinical and environmental isolates of Legionella pneumophila in Norway, 2001-2008.

BACKGROUND: The aims of the study were to determine the molecular characteristics of a collection of Legionella pneumophila isolates from 45 cases with Legionnaires' disease and from 96 environmental samples, received by the national reference laboratory in Norway between 2001 and 2008, to use these characteristics to identify links between cases and suspected sources of infection, and to compare the isolate characteristics with those in other European countries. METHODS: The isolates were characterized by 7-gene locus sequence-based typing and dot-blotting with monoclonal antibodies to various serogroups and subgroups. RESULTS: The clinical isolates represented 12.6% of the 357 cases notified in Norway between 2001 and 2008, during which 3 outbreaks of L. pneumophila serogroup 1 occurred. Outbreak cases constituted 62.2% of the cases, followed by travel-associated (24.4%) and sporadic cases (11.1%). Forty-two (93.3%) of the clinical and 69 (71.9%) of the environmental isolates were serogroup 1, and 39 (86.7%) and 50 (52.1%) isolates, respectively, carried the monoclonal antibody (Mab) 3/1 virulence-associated epitope. The clinical isolates belonged to 17 sequence types and the environmental isolates to 19 sequence types. neuA was not detected in 23 environmental isolates. CONCLUSIONS: Matching characteristics of sequence types and monoclonal subgroups for case and environmental isolates were obtained for all 3 outbreaks and for 2 of 5 cases of sporadic disease. Sampling during the outbreaks accounted for the higher proportion of serogroup 1 and Mab 3/1-positive environmental isolates in comparison with other European strain collections.

Interlaboratory standardization of the sandwich enzyme-linked immunosorbent assay designed for MATS, a rapid, reproducible method for estimating the strain coverage of investigational vaccines.

The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or "relative potency" (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

Specificity of subcapsular antibody responses in Ethiopian patients following disease caused by serogroup A meningococci.

Dissecting the specificities of human antibody responses following disease caused by serogroup A meningococci may be important for the development of improved vaccines. We performed a study of Ethiopian patients during outbreaks in 2002 and 2003. Sera were obtained from 71 patients with meningitis caused by bacteria of sequence type 7, as confirmed by PCR or culture, and from 113 Ethiopian controls. Antibody specificities were analyzed by immunoblotting (IB) against outer membrane antigen extracts of a reference strain and of the patients' own isolates and by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) levels against lipooligosaccharide (LOS) L11 and the proteins NadA and NspA. IB revealed that the main antigens targeted were the proteins PorA, PorB, RmpM, and Opa/OpcA, as well as LOS. MenA disease induced significant increases in IgG against LOS L11 and NadA. The IgG levels against LOS remained elevated following disease, whereas the IgG anti-NadA levels returned to acute-phase levels in the late convalescent phase. Among adults, the anti-LOS IgG levels were similar in acute-phase patient sera as in control sera, whereas anti-NadA IgG levels were significantly higher in acute-phase sera than in controls. The IgG antibody levels against LOS and NadA correlated moderately but significantly with serum bactericidal activity against MenA strains. Future studies on immune response during MenA disease should take into account the high levels of anti-MenA polysaccharide IgG commonly found in the population and seek to clarify the role of antibodies against subcapsular antigens in protection against MenA disease.

Serum antibody responses in Ethiopian meningitis patients infected with Neisseria meningitidis serogroup A sequence type 7.

To elucidate critical components of protective immune responses induced during the natural course of serogroup A meningococcal disease, we studied acute-, early-convalescent-, and late-convalescent-phase sera from Ethiopian patients during outbreaks in 2002 to 2003. Sera were obtained from laboratory-confirmed patients positive for serogroup A sequence type 7 (ST-7) meningococci (A:4/21:P1.20,9) (n = 71) and from Ethiopian controls (n = 113). The sera were analyzed using an enzyme-linked immunosorbent assay to measure levels of immunoglobulin G (IgG) against serogroup A polysaccharide (APS) and outer membrane vesicles (OMVs) and for serum bactericidal activity (SBA) using both rabbit and human complement sources. Despite relatively high SBA titers and high levels of IgG against APS and OMVs in acute-phase patient sera, significant increases were seen in the early convalescent phase. Antibody concentrations returned to acute-phase levels in the late convalescent phase. Considering all patients' sera, a significant but low correlation (r = 0.46) was observed between SBA with rabbit complement (rSBA) using an ST-5 reference strain and SBA with human complement (hSBA) using an ST-7 strain from Ethiopia. While rSBA demonstrated a significant linear relation with IgG against APS, hSBA demonstrated significant linear relationships with IgG against both APS and OMV. This study indicates that antibodies against both outer membrane proteins and APS may be important in providing the protection induced during disease, as measured by hSBA. Therefore, outer membrane proteins could also have a role as components of future meningococcal vaccines for the African meningitis belt.

Development and characterisation of outer membrane vesicle vaccines against serogroup A Neisseria meningitidis.

Neisseria meningitidis bacteria of serogroup A are causing recurring meningitis epidemics on the African continent. An outer membrane vesicle (OMV) vaccine against serogroup A meningococci made from a subgroup III serogroup A meningococcal strain was previously shown to induce antibodies with serum bactericidal activity (SBA) in mice. We have here further investigated the properties of OMV vaccines made from five different subgroup III serogroup A meningococcal strains grown in a synthetic medium with low iron content. In addition to the major outer membrane proteins (PorA, PorB, RmpM, Opa and OpcA), small amounts of the NadA, TdfH, Omp85, FetA, FbpA and NspA outer membrane proteins, as well as lipooligosaccharides, were detected in the vaccines. The OMV vaccines were used to immunise mice. Anti-meningococcal IgG antibodies in the mouse sera were analysed by immunoblotting and by enzyme-linked immunosorbent assay against OMVs, and against live meningococcal cells in SBA and a flow-cytometric assay. The vaccines induced antibodies with high SBA and opsonophagocytic activity. The strongest IgG responses were directed against PorA. Significant SBA responses were also observed against a subgroup III strain, which did not express PorA, whereas no SBA was observed against a clone IV-1 serogroup A strain. An OMV vaccine from serogroup A meningococci may be an alternative to polysaccharide and conjugate polysaccharide vaccines for Africa.

Immunogenicity and bactericidal activity in mice of an outer membrane protein vesicle vaccine against Neisseria meningitidis serogroup A disease.

Serogroup A Neisseria meningitidis organisms of the subgroup III have caused epidemics of meningitis in sub-Saharan Africa since their introduction into the continent in 1987. The population structure of these bacteria is basically clonal, and these meningococci are strikingly similar in their major outer membrane antigens PorA and PorB. Protein-based vaccines might be an alternative to prevent epidemics caused by these meningococci; thus, we developed an outer membrane vesicle (OMV) vaccine from a serogroup A meningococcal strain of subgroup III. The serogroup A OMV vaccine was highly immunogenic in mice and elicited significant bactericidal activity towards several other serogroup A meningococci of subgroup III. The IgG antibodies generated were in immunoblot shown to be mainly directed towards the PorA outer membrane protein. The results presented demonstrate the potential of an OMV vaccine as an optional strategy to protect against meningococcal disease caused by serogroup A in Africa.

Complement activation induced by purified Neisseria meningitidis lipopolysaccharide (LPS), outer membrane vesicles, whole bacteria, and an LPS-free mutant.

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.