Dlawer A. A. Ala'Aldeen

Impact of meningococcal serogroup C conjugate vaccines on carriage and herd immunity

BACKGROUND In 1999, meningococcal serogroup C conjugate (MCC) vaccines were introduced in the United Kingdom for those under 19 years of age. The impact of this intervention on asymptomatic carriage of meningococci was investigated to establish whether serogroup replacement or protection by herd immunity occurred. METHODS Multicenter surveys of carriage were conducted during vaccine introduction and on 2 successive years, resulting in a total of 48,309 samples, from which 8599 meningococci were isolated and characterized by genotyping and phenotyping. RESULTS A reduction in serogroup C carriage (rate ratio, 0.19) was observed that lasted at least 2 years with no evidence of serogroup replacement. Vaccine efficacy against carriage was 75%, and vaccination had a disproportionate impact on the carriage of sequence type (ST)-11 complex serogroup C meningococci that (rate ratio, 0.06); these meningococci also exhibited high rates of capsule expression. CONCLUSIONS The impact of vaccination with MCC vaccine on the prevalence of carriage of group C meningococci was consistent with herd immunity. The high impact on the carriage of ST-11 complex serogroup C could be attributed to high levels of capsule expression. High vaccine efficacy against disease in young children, who were not protected long-term by the schedule initially used, is attributed to the high vaccine efficacy against carriage in older age groups.

CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.

Changing carriage rate of Neisseria meningitidis among university students during the first week of term: cross sectional study.

Abstract Objective: To determine the rates of, and risk factors for, meningococcal carriage and acquisition among university students. Design: Repeated cross sectional study. Participants: 2507 students in their first year at university. Main outcome measures: Prevalence of carriage of meningococci and risk factors for carriage and acquisition of meningococci. Results: Carriage rates for meningoccoci increased rapidly in the first week of term from 6.9% on day 1, to 11.2% on day 2, to 19.0% on day 3, and to 23.1% on day 4. The average carriage rate during the first week of term in October among students living in catered halls was 13.9%. By November this had risen to 31.0% and in December it had reached 34.2%. Independent associations for acquisition of meningococci in the autumn term were frequency of visits to a hall bar (5-7 visits: odds ratio 2.7, 95% confidence interval 1.5 to 4.8), active smoking (1.6, 1.0 to 2.6), being male (1.6, 1.2 to 2.2), visits to night clubs (1.3, 1.0 to 1.6), and intimate kissing (1.4, 1.0 to 1.8). Lower rates of acquisition were found in female only halls (0.5, 0.3 to 0.9). The most commonly acquired meningococcal strain was C2a P1.5 (P1.2), which has been implicated in clusters of invasive meningococcal disease at other UK universities. Conclusions: Carriage rates of meningococci among university students increase rapidly in the first week of term, with further increases during the term. The rapid rate of acquisition may explain the increased risk of invasive meningococcal disease and the timing of cases and outbreaks in university students.

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram‐negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti‐App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.

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

ABSTRACT 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.

The moonlighting protein fructose-1, 6-bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion

Fructose‐1, 6‐bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non‐glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life‐threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane‐localized FBA was surface‐accessible to FBA‐specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA‐deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp‐2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells

BackgroundGlyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.ResultsIn this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant.ConclusionsOur data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.

Characterization of Humoral and Cellular Immune Responses Elicited by Meningococcal Carriage

ABSTRACT In order to study the immune response elicited by asymptomatic carriage of Neisseria meningitidis, samples of serum, peripheral blood mononuclear cells (PBMCs), and saliva were collected from a cohort of more than 200 undergraduate students in Nottingham, United Kingdom, who were subject to high rates of acquisition and carriage of meningococci. Serum immunoglobulin G levels were elevated following increases in the rate of carriage, and these responses were specific for the colonizing strains. In order to investigate T-cell responses, PBMCs from 15 individuals were stimulated with a whole-cell lysate of the H44/76 meningococcal strain (B:15:P1.7,16), stained to detect cell surface markers and intracellular cytokines, and examined by flow cytometry. The cells were analyzed for expression of CD69 (to indicate activation), gamma interferon (IFN-γ) (a representative T-helper 1 subset [Th1]-associated cytokine), and interleukin-5 (IL-5) (a Th2-associated cytokine). Following a brief meningococcal stimulation, the numbers of CD69+ IFN-γ+ CD56/16+ NK cells were much higher than cytokine-positive CD4+ events. Both IFN-γ+ and IL-5+ events were detected among the CD69+ CD4+ population, leading to the conclusion that an unbiased T-helper subset response was elicited by meningococcal carriage.

Persistence, Replacement, and Rapid Clonal Expansion of Meningococcal Carriage Isolates in a 2008 University Student Cohort

ABSTRACT A study of meningococcal carriage dynamics was performed with a cohort of 190 first-year students recruited from six residential halls at Nottingham University, United Kingdom. Pharyngeal swabs were obtained on four occasions between November 2008 and May 2009. Direct plating and culture on selective media were succeeded by identification and characterization of meningococci using PCR-based methodologies. Three serogroup Y clones and one serogroup 29E clone were highly prevalent in particular residential halls in November 2008, which is indicative of rapid clonal expansion since the start of the academic year. Persistent carriage of the same meningococcal strain for at least 5 to 6 months was observed in 45% of carriers, with infrequent evidence of antigenic variation in PorA. Sequential carriage of heterologous meningococcal strains occurred in 36% of carriers and involved strains with different capsules and antigenic variants of PorA and FetA in 83% of the cases. These clonal replacement strains also exhibited frequent differences in the presence and antigenic structures of two other surface proteins, NadA and HmbR. This study highlights the low level of antigenic variation associated with persistent carriage but, conversely, the importance of alterations in the repertoire of antigenic variants for sequential carriage of meningococcal strains. Rapid clonal expansion of potentially pathogenic strains in residential halls has implications for the implementation of public health interventions in university populations.

Neisserial TonB-dependent outer-membrane proteins: Detection, regulation and distribution of three putative candidates identified from the genome sequences

Computer searches were carried out of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative non-contiguous genes were found and three of these (identified in gonococcal strain FA1090) were chosen for further study. Consensus motif analysis of the peptide sequences was consistent with the three genes encoding TonB-dependent receptors. In view of the five previously characterized TonB-dependent proteins of pathogenic neisseriae, the putative genes were labelled tdfF, tdfG and tdfH. TdfF had homology with the siderophore receptors FpvA of Pseudomonas aeruginosa and FhuE of Escherichia coli, whereas TdfG and TdfH had homology with the haemophore receptor HasR of Serratia marcescens. The aim of this project was to characterize these proteins and determine their expression, regulation, distribution and surface exposure. Strain surveys of iron-stressed commensal and pathogenic neisseriae revealed that TdfF is unlikely to be expressed, TdfG is expressed by gonococci only and that TdfH is expressed by both meningococci and gonococci. Expression of TdfH was unaffected by iron availability. Susceptibility of TdfH to cleavage by proteases in live gonococci was consistent with surface exposure of this protein. TdfH may function as a TonB-dependent receptor for a non-iron nutrient source. Furthermore, TdfH is worthy of future investigation as a potential meningococcal vaccine candidate as it is a highly conserved, widely distributed and surface-exposed outer-membrane protein.