Genomic analysis of serogroup Y Neisseria meningitidis isolates reveals extensive similarities between carriage and disease-associated organisms

Background. Neisseria meningitidis is a frequent colonizer of the human nasopharynx with asymptomatic carriage providing the reservoir for invasive, disease-causing strains. Serogroup Y (MenY) strains are a major cause of meningococcal disease. High resolution genetic analyses of carriage and disease isolates can establish epidemiological relationships and identify potential virulence factors. Methods. Whole genome sequence data were obtained from UK MenY carriage isolates from 1997-2010 (n=99). Sequences were compared to those from MenY invasive isolates from 2010 and 2011 (n=73) using a gene-by-gene approach. Results. Comparisons across 1,605 core genes resolved 91% of isolates into one of eight clusters containing closely related disease and carriage isolates. Six clusters contained carried meningococci isolated in 1997-2001 suggesting temporal stability. One cluster of isolates, predominately sharing the designation Y: P1.5-1,10-1: F4-1: ST-1655 (cc23), was resolved into a sub-cluster with 86% carriage isolates and a second with 90% invasive isolates. These subclusters were defined by specific allelic differences in five core genes encoding glycerate kinase (glxK), valine-pyruvate transaminase (avtA), superoxide dismutase (sodB) and two hypothetical proteins. Conclusions. High resolution genetic analyses detected long-term temporal stability and temporally-overlapping carriage and disease populations for MenY clones but also evidence of a disease-associated clone

Changes in serogroup and genotype prevalence among carried meningococci in the United Kingdom during vaccine implementation.

BACKGROUND: Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population. METHODS: Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed. RESULTS: A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear. CONCLUSIONS: Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, “phasotypes,” was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence

Pathogen Proteins Eliciting Antibodies Do Not Share Epitopes with Host Proteins: A Bioinformatics Approach

The best way to prevent diseases caused by pathogens is by the use of vaccines. The advent of genomics enables genome-wide searches of new vaccine candidates, called reverse vaccinology. The most common strategy to apply reverse vaccinology is by designing subunit recombinant vaccines, which usually generate an humoral immune response due to B-cell epitopes in proteins. A major problem for this strategy is the identification of protective immunogenic proteins from the surfome of the pathogen. Epitope mimicry may lead to auto-immune phenomena related to several human diseases. A sequence-based computational analysis has been carried out applying the BLASTP algorithm. Therefore, two huge databases have been created, one with the most complete and current linear B-cell epitopes, and the other one with the surface-protein sequences of the main human respiratory bacterial pathogens. We found that none of the 7353 linear B-cell epitopes analysed shares any sequence identity region with human proteins capable of generating antibodies, and that only 1% of the 2175 exposed proteins analysed contain a stretch of shared sequence with the human proteome. These findings suggest the existence of a mechanism to avoid autoimmunity. We also propose a strategy for corroborating or warning about the viability of a protein linear B-cell epitope as a putative vaccine candidate in a reverse vaccinology study; so, epitopes without any sequence identity with human proteins should be very good vaccine candidates, and the other way around

Epidemiology, Molecular Characterization and Antibiotic Resistance of Neisseria meningitidis from Patients ≤15 Years in Manhiça, Rural Mozambique

BACKGROUND: The epidemiology of meningococcal disease in Mozambique and other African countries located outside the "meningitis belt" remains widely unknown. With the event of upcoming vaccines microbiological and epidemiological information is urgently needed. METHODS: Prospective surveillance for invasive bacterial infections was conducted at the Manhiça District hospital (rural Mozambique) among hospitalized children below 15 years of age. Available Neisseria meningitidis isolates were serogrouped and characterized by Multilocus Sequence Typing (MLST). Antibiotic resistance was also determined. RESULTS: Between 1998 and 2008, sixty-three cases of confirmed meningococcal disease (36 meningitis, 26 sepsis and 1 conjunctivitis) were identified among hospitalized children. The average incidence rate of meningococcal disease was 11.6/100,000 (8/100,000 for meningitis and 3.7/100,000 for meningococcemia, respectively). There was a significant rise on the number of meningococcal disease cases in 2005-2006 that was sustained till the end of the surveillance period. Serogroup was determined for 43 of the 63 meningococcal disease cases: 38 serogroup W-135, 3 serogroup A and 2 serogroup Y. ST-11 was the most predominant sequence type and strongly associated with serogroup W-135. Two of the three serogroup A isolates were ST-1, and both serogroup Y isolates were ST-175. N. meningitidis remained highly susceptible to all antibiotics used for treatment in the country, although the presence of isolates presenting intermediate resistance to penicillin advocates for continued surveillance. CONCLUSIONS: Our data show a high rate of meningococcal disease in Manhiça, Mozambique, mainly caused by serogroup W-135 ST-11 strains, and advocates for the implementation of a vaccination strategy covering serogroup W-135 meningococci in the country

Characterization of MspA, an Immunogenic Autotransporter Protein That Mediates Adhesion to Epithelial and Endothelial Cells in Neisseria meningitidis

A novel putative autotransporter protein (NMB1998) was identified in the available genomic sequence of meningococcal strain MC58 (ET-5; ST-32). The mspA gene is absent from the genomic sequences of meningococcal strain Z2491 (ET-IV; ST-4) and the gonococcal strain FA1090. An orthologue is present in the meningococcal strain FAM18 (ET-37; ST-11), but the sequence contains a premature stop codon, suggesting that the protein may not be expressed in this strain. MspA is predicted to be a 157-kDa protein with low cysteine content, and it exhibits 36 and 33% identity to the meningococcal autotransporter proteins immunoglobulin A1 (IgA1) protease and App, respectively. Search of the Pfam database predicts the presence of IgA1 protease and autotransporter β-barrel domains. MspA was cloned, and a recombinant protein of the expected size was expressed and after being affinity purified was used to raise rabbit polyclonal monospecific antiserum. Immunoblot studies showed that ca. 125- and 95-kDa fragments of MspA are secreted in meningococcal strain MC58, which are absent from the isogenic mutant. Secretion of MspA was shown to be modified in an AspA isogenic mutant. A strain survey showed that MspA is expressed by all ST-32 and ST-41/44 (lineage 3) strains, but none of the ST-8 (A4) strains examined. Sera from patients convalescing from meningococcal disease were shown to contain MspA-specific antibodies. In bactericidal assays, anti-MspA serum was shown to kill the homologous strain (MC58) and another ST-32 strain. Escherichia coli-expressing recombinant MspA was shown to adhere to both human bronchial epithelial cells and brain microvascular endothelial cells