Invasive meningococcal capsular group Y disease, England and Wales, 2007-2009.

Enhanced national surveillance for invasive meningococcal disease in England and Wales identified an increase in laboratory-confirmed capsular group Y (MenY) disease from 34 cases in 2007 to 44 in 2008 and 65 in 2009. For cases diagnosed in 2009, patient median age at disease onset was 60 years; 39% of patients had underlying medical conditions, and 19% died. MenY isolates causing invasive disease during 2007-2009 belonged mainly to 1 of 4 clonal complexes (cc), cc23 (56% of isolates), cc174 (21%), cc167 (11%), and cc22 (8%). The 2009 increase resulted primarily from sequence type 1655 (cc23) (22 cases in 2009, compared with 4 cases each in 2007 and 2008). cc23 was associated with lpxL1 mutations and meningitis in younger age groups (<25 years); cc174 was associated with nonmeningitis, particularly pneumonia, in older age groups (>65 years). The increase in MenY disease requires careful epidemiologic and molecular monitoring.

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.

Bacterial genomic detection within cerebrospinal fluid of patients with meningococcal disease is influenced by microbial and host characteristics.

Among 384 patients with confirmed meningococcal disease, the likelihood of detecting Neisseria meningitidis DNA in cerebrospinal fluid (CSF) increased with age, serogroup B infection, and prehospitalization antibiotic treatment. Plasma and CSF genomic bacterial loads of non-B N. meningitidis serogroups correlated significantly. Serogroup B-infected patients with genotype TNF2 (-308A) had significantly higher CSF bacterial loads.

Severity of meningococcal disease associated with genomic bacterial load.

BACKGROUND: Diagnostic polymerase chain reaction (PCR) detection of Neisseria meningitidis has enabled accurate quantification of the bacterial load in patients with meningococcal disease. METHODS: Quantification of the N. meningitidis DNA level by real time-PCR was conducted on whole-blood samples obtained from patients presenting with meningococcal disease to hospitals throughout England and Wales over a 3-year period. Levels were correlated with clinical outcome, infecting serogroup, and host factors including, interleukin-1 genotype (IL-1). RESULTS: Bacterial loads were available for 1045 patients and were not associated with the age of the patient, delay in sample submission, or administration of antibiotics prior to admission. The median log bacterial load was higher in 95 patients who died (5.29 log(10)copies/mL; interquartile range, 4.41-6.30 log(10)copies/mL) than in 950 patients who survived (3.79 log(10)copies/mL; interquartile range, 2.87-4.71 log(10)copies/mL). Logistic regression revealed that age (odds ratio, 1.04 per 1-year increase in age) and bacterial load (odds ratio, 2.04 per log(10)-copies/mL increase) had a statistically significant effect on the risk of death. Infection with N. meningitidis serogroup C was associated with increased risk of death and an increased bacterial load. Also associated with a higher bacterial load were prolonged hospitalization (duration, >10 days); digit, limb, or soft-tissue loss; and requirement of hemodialysis. Carriage of IL-1RN(+2018) was associated with increased mortality (odds ratio, 2.14; P=.07) but not with a higher bacterial load. CONCLUSIONS: In meningococcal disease, bacterial load is associated with likelihood of death, development of permanent disease sequelae, and prolonged hospitalization. The bacterial load was relatively higher in patients infected with N. meningitidis serogroup C than in those infected with other serogroups. The effects of age and IL-1 genotype on mortality are independent of a high genomic bacterial load.

Characterization of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales.

Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster.

The tumor necrosis factor polymorphism TNF (-308) is associated with susceptibility to meningococcal sepsis, but not with lethality.

OBJECTIVE: To determine whether the promoter polymorphism tumor necrosis factor (TNF) (-308) is associated with susceptibility to or death from meningococcal sepsis. DESIGN, SETTING, PATIENTS, AND PARTICIPANTS: Association study involving 1321 patients with microbiologically proven invasive meningococcal disease presenting to hospitals throughout United Kingdom during 1998-2001, among whom 134 died. Controls were derived from 1280 northern English blood donors. MEASUREMENTS: DNA from patients and controls was genotyped at TNF (-308). After analysis, DNA was subsequently genotyped at eight other markers in strong linkage disequilibrium with TNF (-308); these markers were IkappaBL (-62), BAT3, LST1, NOTCH4 (+1297), NOTCH4 (+3061), CCHCR1 (+436), CCHCR1 (+2271), and LTalpha. To confirm functional relevance of TNF (-308) in the context of meningococcal disease, TNF secretion by, and TNF messenger RNA expression of macrophages derived from volunteers with known TNF (-308) genotype after exposure to Neisseria meningitidis were measured. MAIN RESULTS: Among cases of meningococcal disease, likelihood of death was shown to be influenced by the age of the affected individual and also with the infecting serogroup, but was not influenced by genotype at TNF (-308) or the other linked markers. However, patients with meningococcal disease, irrespective of whether they died, were more likely to be homozygous for the rare allele at TNF (-308) (odds ratio = 1.93, 95% confidence interval 1.08-3.46), and less likely to be heterozygous for this marker (odds ratio = 0.79, 95% confidence interval 0.64-0.97), compared with the control cohort. There was no association of susceptibility to disease with the other markers studied. Macrophages derived from volunteers homozygous for the rare allele at TNF (-308) expressed higher levels of TNF messenger RNA and secreted higher concentrations of TNF compared with common homozygotes after exposure to N. meningitidis. CONCLUSIONS: Genotype at TNF (-308) modifies cellular TNF secretion in response to N. meningitidis and may influence susceptibility to meningococcal disease, but does not influence the likelihood of death after infection.

Immune response to pneumococcal conjugate vaccination in asplenic individuals.

Asplenic individuals are at increased risk of infection with Streptococcus pneumoniae. The immune response to pneumococcal conjugate vaccine has not been investigated in this clinical risk group. We investigated immune responses to pneumococcal vaccination in asplenic individuals. Eligible subjects aged > or =4 years received one dose 7-valent pneumococcal conjugate vaccine (PCV7) and, if no prior 23-valent polysaccharide vaccine (PPV23) had been received within previous 5 years, one dose was given 6 months following PCV7. Pre- and post-vaccination blood samples were taken. Pneumococcal serotype-specific IgG levels were determined for 9 serotypes; the 7 in PCV7 plus serotypes1 and by standardized ELISA. One hundred and eleven asplenic individuals were recruited [median age 54.8 years, (18.1-81.8)]. Median age at splenectomy was 29.6 years (3.6-78.3); 108 (97.3%) individuals had previously received PPV23. Compliance with UK recommendations on immunization and prophylaxis in this group was poor, 91 (82%) subjects had received Haemophilus influenzae type b conjugate vaccine and only 68 (62%) had received meningococcal serogroup C conjugate vaccine. In total 61 (55%) subjects were taking antibiotic prophylaxis and 12 subjects had reported previous invasive pneumococcal disease, five episodes of which occurred post-splenectomy. High serotype-specific IgG concentrations were observed pre-PCV7, with significant increases (p < 0.01) in geometric mean concentrations pre- to post-PCV7 for the PCV7 serotypes. Post-PCV7, between 27% (serotype 14) and 69% (serotype 23F) of subjects had a > or =2-fold rise in IgG. Pre-PCV7, the percentage of individuals with levels > or =0.35 microg/mL ranged between 77% (serotype 4) and 97% (serotypes 14, 19F), whilst post-PCV7 this was 90% (serotype 6B) and 99% (serotype 14). No significant increases were observed post-PPV23. Asplenic individuals responded well to PCV7, though protective levels were demonstrated pre-PCV7 in majority of participants due to prior PPV23. Although immunogenic, there is insufficient evidence here to recommend routine PCV7 immunization over PPV23 immunization in adult asplenic individuals.

Opa protein repertoires of disease-causing and carried meningococci.

The meningococcal Opa proteins play an important role in pathogenesis by mediating invasion of human cells. The aim of this investigation was to determine whether carried and disease-associated meningococci possess different Opa repertoires and whether the diversity of these proteins is associated with clinical severity of disease. Opa repertoires in 227 disease-associated meningococci, isolated in the United Kingdom over a period of 6 years, were compared to the repertoires in 190 asymptomatically carried meningococci isolated in the United Kingdom from a contemporary, nonepidemic period. Multidimensional scaling (MDS) was employed to investigate the association between Opa repertoires and multilocus sequence typing (MLST) genotypes. Associations with clinical severity were also analyzed statistically. High levels of diversity were observed in opa alleles, variable regions, and repertoires, and MDS revealed that MLST genotypes were strongly associated with particular Opa repertoires. Individual Opa proteins or repertoires were not associated with clinical severity, though there was a trend toward an association with the opaD locus. Meningococcal Opa repertoire is strongly linked to MLST genotype irrespective of epidemiological sampling and therefore correlates with invasiveness. It is not, however, strongly associated with severity of meningococcal disease.

Molecular characterization of Neisseria meningitidis isolates using a resequencing DNA microarray.

Neisseria meningitidis is a major cause of both meningitis and septicemia. Typically, isolates are characterized by using a combination of immunological phenotyping, using monoclonal and polyclonal antisera, and Sanger nucleotide sequencing of epitope-encoding variable regions, although these methods can be both time-consuming and limited by reagent availability. Herein, we describe and evaluate a novel microarray to define the porB and porA serotypes of N. meningitidis by the resequencing of variable regions in a single hybridization reaction. PCR products for each gene were amplified, pooled in equimolar concentrations, hybridized to the microarray, and analyzed using Affymetrix GeneChip DNA Analysis Software. Resequencing of the microarray data was then validated by comparison with sequencing data. Molecular profiles were generated for 50 isolates that were combinations of phenotypically typeable (ie, PorA and PorB) and non-typeable (PorB only) isolates. Microarray-generated profiles from isolates with a PorB phenotype were concordant with predicted profiles compared with a previously described typing scheme. In addition, 42% (8 of 19) of previously non-typeable samples were assigned a PorB type when tested using the microarray. The remaining isolates were novel types for which no typing antisera are currently available. The porA data were 97% concordant with Sanger nucleotide sequencing. These results suggest that that microarray resequencing may be a useful tool for the characterization of meningococci, particularly for those isolates that cannot be phenotyped, offering an alternative to conventional sequencing methods.

Genetic polymorphism of the binding domain of surfactant protein-A2 increases susceptibility to meningococcal disease.

BACKGROUND: Meningococcal disease occurs after colonization of the nasopharynx with Neisseria meningitidis. Surfactant protein (SP)-A and SP-D are pattern-recognition molecules of the respiratory tract that activate inflammatory and phagocytic defences after binding to microbial sugars. Variation in the genes of the surfactant proteins affects the expression and function of these molecules. METHODS: Allele frequencies of SP-A1, SP-A2, and SP-D were determined by polymerase chain reaction in 303 patients with microbiologically proven meningococcal disease, including 18 patients who died, and 222 healthy control subjects. RESULTS: Homozygosity of allele 1A1 of SP-A2 increased the risk of meningococcal disease (odds ratio [OR], 7.4; 95% confidence interval [CI], 1.3-42.4); carriage of 1A5 reduced the risk (OR, 0.3; 95% CI, 0.1-0.97). An analysis of the multiple single-nucleotide polymorphisms in SP-A demonstrated that homozygosity for alleles encoding lysine (in 1A1) rather than glutamine (in 1A5) at amino acid 223 in the carbohydrate recognition domain was associated with an increased risk of meningococcal disease (OR, 6.7; 95% CI, 1.4-31.5). Carriage of alleles encoding lysine at residue 223 was found in 61% of patients who died, compared with 35% of those who survived (OR adjusted for age, 2.9; 95% CI, 1.1-7.7). Genetic variation of SP-A1 and SP-D was not associated with meningococcal disease. CONCLUSIONS: Gene polymorphism resulting in the substitution of glutamine with lysine at residue 223 in the carbohydrate recognition domain of SP-A2 increases susceptibility to meningococcal disease, as well as the risk of death.

Epidemiology of meningococcal disease in England and Wales 1993/94 to 2003/04: contribution and experiences of the Meningococcal Reference Unit.

The laboratory confirmation of meningococcal disease and characterization of Neisseria meningitidis isolates was improved considerably in England and Wales by the Meningococcal Reference Unit between epidemiological years 1993/94 and 2003/04 to meet the challenge of increasing numbers of cases of clinical disease and the requirement for enhanced surveillance. Improved case ascertainment was made possible by the rapid introduction of an innovative centralized reference service for non-culture PCR-based DNA detection of meningococci utilizing the ctrA and siaD PCR assays, complemented by consistent phenotypic characterization of submitted isolates from culture-proven cases. This allowed the increased prevalence of serogroup C disease in specific age groups and the apparent associated increase in mortality from 1995/96 to 1999/00 to be defined, thereby prompting accelerated intervention with the newly licensed meningococcal serogroup C conjugate (MCC) vaccines into the under-25-year UK population (in November 1999). The continued increase in and predominance of serogroup B cases (1993/94 to 2000/01) were observed in conjunction with their diverse and changing phenotypic characteristics. Trends observed to be associated with the predominant phenotypic combinations of serogroup, serotype and sero-subtype were: a decline of both C : 2b and B : 2b meningococci, and a decline of B : 15 : P1.7,16 with a concomitant increase of B : 4 : P1.4 over the 11-year period. Detailed routine surveillance rapidly confirmed the introduction of W135 : 2a : P1.5,2 meningococci into the UK during 2000 and 2001. The importance of continued detailed surveillance of this important pathogen cannot be overestimated, both to monitor the effectiveness of the MCC vaccine and to identify changes within the meningococcal population that can inform the design of anti-serogroup B vaccines.

Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation.

Serogroup-specific antibody has been shown to be present in the sera of patients recovering from meningococcal disease, and thus the detection of such antibodies may aid in the confirmation of disease. There are currently no standard methods for measuring meningococcal serogroup B-specific antibody in sera. Here, we report the development of a microsphere-based immunoassay which utilizes colominic acid from Escherichia coli 07:K1 (L):NM to detect immunoglobulin M directed against serogroup B polysaccharide. The serogroup B assay was incorporated into a multiplex assay which also detects serogroup-specific immunoglobulin M for meningococcal serogroups A, C, Y and W-135. Using the method of cross-standardization, serogroup B-specific immunoglobulin M concentrations were assigned to the standard serum CDC 1992. The assay is able to detect increases in specific immunoglobulin M concentrations from acute to convalescent phase serum from serogroup B cases, and can be utilized in conjunction with the previously developed tetraplex immunoglobulin G detection assay for serogroups A, C, Y and W-135.

Effectiveness of meningococcal serogroup C conjugate vaccine 4 years after introduction.

The meningococcal serogroup C conjugate (MCC) vaccine programme in England has successfully controlled the incidence of serogroup C disease, as a result of high short-term vaccine effectiveness and substantial herd immunity. However, the long-term effectiveness of the vaccine remains unknown. We assessed surveillance data from the 4 years since introduction of the programme. Vaccine effectiveness remained high in children vaccinated in the catch-up campaign (aged 5 months to 18 years). However, for children vaccinated in the routine infant immunisation programme, the effectiveness of the MCC vaccine fell to low levels after only 1 year. The number of individuals in these cohorts remains low, but alternative routine immunisation schedules should be considered to ensure high levels of protection are sustained.

Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease.

BACKGROUND: Genetically determined variation in proinflammatory cytokine release influences severity of meningococcal disease and other serious infections. OBJECTIVE: To ascertain the relative frequencies of single nucleotide polymorphisms within the interleukin-1 gene locus among patients who survived and those who died of meningococcal disease and a control population of blood donors. DESIGN: Association study. SETTING: England and Wales. PATIENTS: 1106 consecutively received blood samples from persons with microbiologically confirmed meningococcal disease and 839 samples from blood donors. MEASUREMENTS: Patient demographic and outcome data, infecting meningococcal serogroups, and genotype at the IL1B(-511) and IL1RN(+2018) loci of patients and blood donor controls. RESULTS: Genotype frequency did not differ between patients with meningococcal disease and blood donor controls. Logistic regression analysis revealed that the likelihood of death was significantly influenced by age but not socioeconomic status and was higher in patients who were infected with serogroup C (odds ratio for survival, 0.50 [95% CI, 0.33 to 0.78]). Patients carrying the common allele at IL1B(-511) were more likely to survive (odds ratio, 2.01 [CI, 1.11 to 3.79]). Patients with this allele were less likely to survive if they also carried the rare allele at IL1RN(+2018) (odds ratio, 0.61 [CI, 0.38 to 0.993]). CONCLUSION: Genotype at the interleukin-1 gene locus influences likelihood of survival of meningococcal disease but has no effect on susceptibility to the infection. Increasing age and infection with serogroup C also influence the likelihood of death.

Reduced turnaround time and improved diagnosis of invasive serogroup B Neisseria meningitidis and Streptococcus pneumoniae infections using a lyophilized quadruplex quantitative PCR.

Since 1996 the Meningococcal Reference Unit (MRU) in Manchester has provided a national service for PCR confirmation of meningococcal and pneumococcal disease. Neisseria meningitidis serogroup B is predominant in the UK, accounting for >60% of cases. In response to this, the MRU has developed a quadruplex quantitative PCR that detects N. meningitidis capsule transporter (ctrA), serogroup B sialyltransferase (siaDB), Streptococcus pneumoniae pneumolysin (ply) and an internal control. The assay was prepared in a ready-to-use lyophilized format by Applied Biosystems. Laboratory validation showed excellent performance in a specificity panel of 52 isolates and improved detection in comparison with the routine assay. Testing of 244 patient samples showed sensitivity of 93% [95% confidence interval (CI): 88-98%] for the ctrA assay, 95% (95% CI: 91-100%) for the siaDB assay and 100% (95% CI: 95-100%) for the ply assay. Specificity was 100% (95% CI: 98-100%) for both meningococcal targets and 95% (95% CI: 92-98%) for ply. The quadruplex also retained high performance in mixed samples and had acceptable reproducibility. After introduction of the quadruplex into routine use the turnaround time for N. meningitidis group B PCR confirmation reduced from 37 to 29 h and the internal control has proved useful for detecting inhibitory samples. The quadruplex assay provides rapid group B confirmation of meningococcal positive samples, enabling timely public health interventions for the most common disease-causing meningococcal serogroup in the UK.

Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

Fatal outcome from meningococcal disease--an association with meningococcal phenotype but not with reduced susceptibility to benzylpenicillin.

Penicillin has been the mainstay of treatment for meningococcal disease. Isolates of Neisseria meningitidis that are less susceptible to penicillin have been reported in several countries and in recent years have become more common. The clinical significance of this reduced susceptibility has not been investigated on a large scale. Hence, N. meningitidis isolates from culture-confirmed cases of meningococcal disease in England and Wales, between 1993 and 2000, were routinely serogrouped, serotyped and tested for susceptibility to penicillin. These data were linked to death registrations and analysed retrospectively. The changing trends in susceptibility were described and multivariate logistic regression was used to examine associations between strain characteristics and fatal outcome. The frequency of N. meningitidis isolates less susceptible to penicillin increased from < 6% in 1993 to > 18% in 2000. In particular, isolates expressing serogroup C with serotype 2b and serogroup W135 had a higher frequency of reduced penicillin susceptibility (49% and 55%, respectively). There was no evidence of an association between fatal outcome and infection with a less penicillin-susceptible isolate. Fatal outcome was associated with serogroup and serotype, with the odds of death for cases infected with C:2a and B:2a strains three-fold higher when compared with the baseline. For this large dataset the serogroup and serotype of the infecting strain influenced mortality from meningococcal disease and may be markers for hypervirulence. No association was found between reduced penicillin susceptibility and fatal outcome, but the increasing frequency of isolates less susceptible to penicillin highlights the need for continued surveillance.

O-Acetylation status of the capsular polysaccharides of serogroup Y and W135 meningococci isolated in the UK.

At a time when tetravalent conjugate vaccines for meningococcal serogroups A/C/Y/W135 are being formulated the O-acetylation status of their respective capsular polysaccharides has not previously been studied in the UK for all components. Although this has been elucidated for serogroup C, little is known about the O-acetylation status of serogroups W135 and Y. Meningococcal serogroup W135 (n=181) and Y (n=90) isolates submitted to the PHLS Meningococcal Reference Unit in 1996, 2000 and 2001 were investigated for O-acetylation capsular status by dot blot assay. Eight per cent of W135 and 79% of Y isolates respectively were found to be O-acetylated with a similar distribution found in both carrier and case isolates. An increase in O-acetylated W135 isolates was noted between 2000 (0%) and 2001 (21%) which was not due to the introduction of the Hajj associated W135 (ET 37 complex; serosubtype P1.5,2) isolates, all of which were de-O-acetylated. Although the biological relevance of O-acetylation status is unknown for these serogroups, an understanding of O-acetylation status of the respective polysaccharides may provide useful insights into the optimal vaccine formulation.

Genetic distribution of noncapsular meningococcal group B vaccine antigens in Neisseria lactamica.

The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genus Neisseria that also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal, Neisseria lactamica. All the isolates possessed nhba but were devoid of fhbp and nadA. The nhba alleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.

The distribution and 'in vivo' phase variation status of haemoglobin receptors in invasive meningococcal serogroup B disease: genotypic and phenotypic analysis.

Two haemoglobin-binding proteins, HmbR and HpuAB, contribute to iron acquisition by Neisseria meningitidis. These receptors are subject to high frequency, reversible switches in gene expression--phase variation (PV)--due to mutations in homopolymeric (poly-G) repeats present in the open reading frame. The distribution and PV state of these receptors was assessed for a representative collection of isolates from invasive meningococcal disease patients of England, Wales and Northern Ireland. Most of the major clonal complexes had only the HmbR receptor whilst the recently expanding ST-275-centred cluster of the ST-269 clonal complex had both receptors. At least one of the receptors was in an 'ON' configuration in 76.3% of the isolates, a finding that was largely consistent with phenotypic analyses. As PV status may change during isolation and culture of meningococci, a PCR-based protocol was utilised to confirm the expression status of the receptors within contemporaneously acquired clinical specimens (blood/cerebrospinal fluid) from the respective patients. The expression state was confirmed for all isolate/specimen pairs with <15 tract repeats indicating that the PV status of these receptors is stable during isolation. This study therefore establishes a protocol for determining in vivo PV status to aid in determining the contributions of phase variable genes to invasive meningococcal disease. Furthermore, the results of the study support a putative but non-essential role of the meningococcal haemoglobin receptors as virulence factors whilst further highlighting their vaccine candidacy.