Invasive Meningococcal Isolates Research Articles

Genomic diversity and antimicrobial susceptibility of invasive Neisseria meningitidis in South Africa, 2016-2021.

Invasive meningococcal isolates in South Africa have in previous years (<2008) been characterized by serogroup B, C, W and Y lineages over time, with penicillin intermediate resistance (peni) at 6%. We describe the population structure and genomic markers of peni among invasive meningococcal isolates in South Africa, 2016-2021. Meningococcal isolates were collected through national, laboratory-based invasive meningococcal disease (IMD) surveillance. Phenotypic antimicrobial susceptibility testing and whole-genome sequencing were performed, and the mechanism of reduced penicillin susceptibility was assessed in silico. Of 585 IMD cases reported during the study period, culture and PCR-based capsular group was determined for 477/585 (82%); and 241/477 (51%) were sequenced. Predominant serogroups included NmB (210/477; 44%), NmW (116/477; 24%), NmY (96/477; 20%) and NmC (48/477; 10%). Predominant clonal complexes (CC) were CC41/44 in NmB (27/113; 24%), CC11 in NmW (46/56; 82%), CC167 in NmY (23/44; 53%), and CC865 in NmC (9/24; 38%). Peni was detected in 16% (42/262) of isolates, and was due to the presence of a penA mosaic, with the majority harboring penA7, penA9 or penA14. IMD lineages circulating in South Africa were consistent with those circulating prior to 2008, however peni was higher than previously reported, and occurred in a variety of lineages.

Molecular characterization of invasive Neisseria meningitidis isolates collected in Lithuania (2009-2019) and estimation of serogroup B meningococcal vaccine 4CMenB and MenB-Fhbp coverage.

Neisseria meningitidis causes invasive meningococcal disease (IMD), which is associated with significant mortality and long-term consequences, especially among young children. The incidence of IMD in Lithuania was among the highest in European Union/European Economic Area countries during the past two decades; however, the characterization of meningococcal isolates by molecular typing methods has not yet been performed. In this study, we characterized invasive meningococcal isolates (n=294) recovered in Lithuania from 2009 to 2019 by multilocus sequence typing (MLST) and typing of antigens FetA and PorA. The more recent (2017-2019) serogroup B isolates (n=60) were genotyped by analyzing vaccine-related antigens to evaluate their coverage by four-component (4CMenB) and two-component (MenB-Fhbp) vaccines using the genetic Meningococcal Antigen Typing System (gMATS) and Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index methods, respectively. The vast majority (90.5%) of isolates belonged to serogroup B. MLST revealed a predominance of clonal complex 32 (74.02%). Serogroup B strain P1.19,15: F4-28: ST-34 (cc32) accounted for 64.1% of IMD isolates. The overall level of strain coverage by the 4MenB vaccine was 94.8% (CI 85.9-98.2%). Most serogroup B isolates (87.9%) were covered by a single vaccine antigen, most commonly Fhbp peptide variant 1 (84.5% of isolates). The Fhbp peptides included in the MenB-Fhbp vaccine were not detected among the analyzed invasive isolates; however, the identified predominant variant 1 was considered cross-reactive. In total, 88.1% (CI 77.5-94.1) of isolates were predicted to be covered by the MenB-Fhbp vaccine. In conclusion, both serogroup B vaccines demonstrate potential to protect against IMD in Lithuania.

Risk Factors for Invasive Meningococcal Disease Belonging to a Novel Urethritis Clade of Neisseria meningitidis—United States, 2013–2017

Abstract We describe cases of invasive meningococcal disease caused by nongroupable Neisseria meningitidis belonging to a novel phylogenetic clade associated with urethritis. Seven cases were identified, comprising 0.6% of sequenced invasive meningococcal disease isolates from 2013 to 2017. Five patients had a known or likely immunocompromising condition, including 2 with a complement deficiency.

Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study

Neisseria meningitidis is the causative agent of invasive meningococcal disease and the polysaccharide capsule is one of its major virulence factors. Biosynthesis of the meningococcal capsule is controlled by an RNA thermosensor (RNAT) in the 5'-untranslated region (5'-UTR) of the cssA gene. The function of the RNAT depends on an 8-bp tandem repeat configuration. We aimed to identify and characterise novel RNATs in meningococcal isolates responsible for regulating capsule production. We investigated the allele igr_up_NEIS0055, containing the 5'-UTR of the cssA gene, in clinical meningococcal isolates for which whole-genome sequences are available on the Neisseria PubMLST database and that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018. Eight isolates with different RNAT tandem repeat configurations were selected for genetic and phenotypic studies. The thermosensing capability of the RNAT and capsule production was tested with immunoblots. Bacterial survival by capsule protection was assessed with a human serum stress assay and capsule interference with bacterial cell adhesion was evaluated with a bacterial adhesion assay. The dataset of RNAT configurations was analysed for an association with invasive meningococcal disease, and was stratified to visualise the distribution of RNAT configurations within the meningococcal population. Our search of PubMLST identified 112 alleles for the igr_up_NEIS0055 locus and 7013 N meningitidis isolates. Five novel RNAT tandem repeat configurations were identified and eight RNAT tandem repeat configurations, ranging from 1 × 8-bp up to 8 × 8-bp, were characterised. The disrupted RNATs (1 × 8-bp and 3 × 8-bp to 8 × 8-bp) confer upregulated CssA expression and increased capsule production compared with the native 2 × 8-bp configuration, resulting in a hypercapsulation phenotype. Increased capsule production was associated with higher survival rates in up to 25% human serum. The prevalence of a disrupted RNAT resulting in hypercapsulation was almost twice as high in invasive meningococcal disease isolates compared with carrier isolates. Disrupted RNATs were especially attributed to isolates of capsule group B and C, and clonal complexes 23, 32, 213, and 269. Hypercapsulation in one isolate led to lower adhesion onto pharyngeal cells compared with a similar isolate with low capsule production. Six non-canonical RNAT tandem repeat variants (3 × 8-bp to 8 × 8-bp) were identified in the igr_up_NEIS0055 locus of N meningitidis that induce a hypercapsulation phenotype, thus providing the meningococci with better protection against host complement-mediated killing than does the native RNAT (2 × 8-bp). Further research is warranted to strengthen the association between hypercapsulation and the progression of invasive meningococcal disease, and to investigate the role of regulatory RNAs in meningococcal virulence and as potential markers for disease progression. Swedish Foundation for Strategic Research, Knut and Alice Wallenberg Foundation, and Swedish Research Council.

Predicted coverage by 4CMenB vaccine against invasive meningococcal disease cases in the Netherlands

Neisseria meningitidis serogroup B is a major cause of invasive meningococcal disease in Europe. In the absence of a conjugate serogroup B vaccine, a subcapsular 4CMenB vaccine was developed. Data on 4CMenB vaccine efficacy is still limited. Recently, genomic MATS (Meningococcal Antigen Typing System) was developed as a tool to predict strain coverage, using vaccine antigens sequence data. We characterized all invasive meningococcal isolates received by the Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM) in two epidemiological years 2017–2019 using whole-genome sequencing and determined serogroup, clonal complex (cc) and estimated 4CMenB vaccine coverage by gMATS. Of 396 cases of invasive meningococcal disease, corresponding to an incidence of 1.22 cases/105 inhabitants, 180 (45%) were serogroup W, 155 (39%) serogroup B, 46 (12%) serogroup Y, 10 (3%) serogroup C, 2 non-groupable (0.5%) and 3 (0.7%) unknown. The incidence was the highest among 0–4 years olds (4 cases/105 inhabitants), and 57/72 (79%) of these cases were serogroup B. Serogroup W predominated among persons 45 years of age or older with 110/187 (59%) cases. Serogroup B isolates comprised 11 different clonal complexes, with 103/122 (84%) isolates belonging to 4 clonal complexes: cc32, cc41/44, cc269 and cc213. In contrast, serogroup W isolates were genetically similar with 95% belonging to cc11. Of 122 serogroup B isolates, 89 (73%; 95% CI: 64–80%) were estimated to be covered by 4CMenB and the degree of coverage varied largely by clonal complex and age. Among the 0–4 year olds, 25 of 43 (58%; 95% CI: 43–72%) MenB isolates were estimated to be covered. Since the coverage of the 4CMenB vaccine is dependent on circulating clonal complexes, our findings emphasize the need for surveillance of circulating meningococcal strains. In addition, estimation of age specific coverage is relevant to determine the right target age group for vaccination.

High genomic-based predicted strain coverage among invasive meningococcal isolates when combining Bexsero and Trumenba vaccines

Two protein-based vaccines (Bexsero® and Trumenba®) are licensed for invasive meningococcal disease (IMD) caused by Neisseria meningitidis serogroup B (MenB). The aim of this study was to evaluate the possible protection of these vaccines, based on the genomic profiles of IMD isolates. All invasive meningococcal isolates in Sweden during 2014–2018 (n = 242) were analyzed with the vaccine coverage scheme available at the PubMLST database. The overall estimated genomic strain coverage among the Swedish invasive meningococcal isolates was 55% for Bexsero and 57% for Trumenba (p = 0.714). The estimated serogroup-specific coverage for Bexsero respectively Trumenba was: MenB; 67% and 90% (p < 0.05), MenW; 93% and 4% (p < 0.05), MenC; 87% and 30% (p < 0.05) and MenY; 1% and 96% (p < 0.05). With the combination of the two vaccines, the potential genomic-based strain coverage was 95%, indicating a possible additive effect of combining Bexsero and Trumenba, which, however, needs to be confirmed by analysis of phenotypic antigen expression.

Susceptibility of invasive Neisseria meningitidis strains isolated in Germany to azithromycin, an alternative agent for post-exposure prophylaxis.

Post-exposure prophylaxis (PEP) for close contacts of invasive meningococcal disease (IMD) cases is recommended in most countries to avoid secondary cases by eradicating supposed meningococcal colonization. Currently, rifampicin, ciprofloxacin and ceftriaxone are recommended in many countries including Germany. Azithromycin has been shown to eradicate meningococcal colonization. To assess the azithromycin susceptibility of invasive Neisseria meningitidis isolates. A subset of German invasive meningococcal isolates from 2006-18 was selected for this study. Azithromycin MIC was determined using broth microdilution and agar gradient diffusion. Azithromycin MICs as determined by broth microdilution ranged from <0.003 to 2 mg/L (median 0.50 mg/L, Q75 1 mg/L). All isolates were susceptible to azithromycin according to the CLSI breakpoint (95% CI 0.0%-1.5%). There was no significant correlation between MICs determined by broth microdilution and agar gradient diffusion. Nevertheless, the two methods were consistent regarding the categorization of all isolates as susceptible. Azithromycin is an eligible antibiotic for PEP of IMD close contacts. It is approved for adults as well as children and may even be used in pregnancy. Because of easier application and lower toxicity, it might be an alternative to rifampicin and ciprofloxacin, as we found no resistant isolates. Since a gonococcal gene associated with elevated azithromycin MICs has been reported in N. meningitidis, careful monitoring of the emergence of resistant strains is nevertheless necessary for meningococci. Lack of concordance of MICs between broth microdilution and agar gradient diffusion needs to be considered.

Phylogenetic relationships and regional spread of meningococcal strains in the meningitis belt, 2011-2016.

BackgroundHistorically, the major cause of meningococcal epidemics in the meningitis belt of sub-Saharan Africa has been Neisseria meningitidis serogroup A (NmA), but the incidence has been substantially reduced since the introduction of a serogroup A conjugate vaccine starting in 2010. We performed whole-genome sequencing on isolates collected post-2010 to assess their phylogenetic relationships and inter-country transmission.MethodsA total of 716 invasive meningococcal isolates collected between 2011 and 2016 from 11 meningitis belt countries were whole-genome sequenced for molecular characterization by the three WHO Collaborating Centers for Meningitis.FindingsWe identified three previously-reported clonal complexes (CC): CC11 (n = 434), CC181 (n = 62) and CC5 (n = 90) primarily associated with NmW, NmX, and NmA, respectively, and an emerging CC10217 (n = 126) associated with NmC. CC11 expanded throughout the meningitis belt independent of the 2000 Hajj outbreak strain, with isolates from Central African countries forming a distinct sub-lineage within this expansion. Two major sub-lineages were identified for CC181 isolates, one mainly expanding in West African countries and the other found in Chad. CC10217 isolates from the large outbreaks in Nigeria and Niger were more closely related than those from the few cases in Mali and Burkina Faso.InterpretationsWhole-genome based phylogenies revealed geographically distinct strain circulation as well as inter-country transmission events. Our results stress the importance of continued meningococcal molecular surveillance in the region, as well as the development of an affordable vaccine targeting these strains.FundMeningitis Research Foundation; CDC's Office of Advanced Molecular Detection; GAVI, the Vaccine Alliance.

Genetic Meningococcal Antigen Typing System (gMATS): A genotyping tool that predicts 4CMenB strain coverage worldwide

BackgroundThe Meningococcal Antigen Typing System (MATS) was developed to identify meningococcus group B strains with a high likelihood of being covered by the 4CMenB vaccine, but is limited by the requirement for viable isolates from culture-confirmed cases. We examined if antigen genotyping could complement MATS in predicting strain coverage by the 4CMenB vaccine. MethodsFrom a panel of 3912 MATS-typed invasive meningococcal disease isolates collected in England and Wales in 2007–2008, 2014–2015 and 2015–2016, and in 16 other countries in 2000–2015, 3481 isolates were also characterized by antigen genotyping. Individual associations between antigen genotypes and MATS coverage for each 4CMenB component were used to define a genetic MATS (gMATS). gMATS estimates were compared with England and Wales human complement serum bactericidal assay (hSBA) data and vaccine effectiveness (VE) data from England. ResultsOverall, 81% of the strain panel had genetically predictable MATS coverage, with 92% accuracy and highly concordant results across national panels (Lin’s accuracy coefficient, 0.98; root-mean-square deviation, 6%). England and Wales strain coverage estimates were 72–73% by genotyping (66–73% by MATS), underestimating hSBA values after four vaccine doses (88%) and VE after two doses (83%). The gMATS predicted strain coverage in other countries was 58–88%. ConclusionsgMATS can replace MATS in predicting 4CMenB strain coverage in four out of five cases, without requiring a cultivable isolate, and is open to further improvement. Both methods underestimated VE in England. Strain coverage predictions in other countries matched or exceeded England and Wales estimates.An Audio Summary linked to this article that can be found on Figshare https://figshare.com/articles/gMATS_video_Vaccine-_2019-_37_7_991-1000_mp4/9326687

Molecular characterization of invasive meningococcal isolates in Burkina Faso as the relative importance of serogroups X and W increases, 2008\u20132012

BackgroundNeisseria meningitidis serogroup A disease in Burkina Faso has greatly decreased following introduction of a meningococcal A conjugate vaccine in 2010, yet other serogroups continue to pose a risk of life-threatening disease. Capsule switching among epidemic-associated serogroup A N. meningitidis strains could allow these lineages to persist despite vaccination. The introduction of new strains at the national or sub-national levels could affect the epidemiology of disease.MethodsIsolates collected from invasive meningococcal disease in Burkina Faso between 2008 and 2012 were characterized by serogrouping and molecular typing. Genome sequences from a subset of isolates were used to infer phylogenetic relationships.ResultsThe ST-5 clonal complex (CC5) was identified only among serogroup A isolates, which were rare after 2010. CC181 and CC11 were the most common clonal complexes after 2010, having serogroup X and W isolates, respectively. Whole-genome phylogenetic analysis showed that the CC181 isolates collected during and after the epidemic of 2010 formed a single clade that was closely related to isolates collected in Niger during 2005 and Burkina Faso during 2007. Geographic population structure was identified among the CC181 isolates, where pairs of isolates collected from the same region of Burkina Faso within a single year had less phylogenetic diversity than the CC181 isolate collection as a whole. However, the reduction of phylogenetic diversity within a region did not extend across multiple years. Instead, CC181 isolates collected during the same year had lower than average diversity, even when collected from different regions, indicating geographic mixing of strains across years. The CC11 isolates were primarily collected during the epidemic of 2012, with sparse sampling during 2011. These isolates belong to a clade that includes previously described isolates collected in Burkina Faso, Mali, and Niger from 2011 to 2015. Similar to CC181, reduced phylogenetic diversity was observed among CC11 isolate pairs collected from the same regions during a single year.ConclusionsThe population of disease-associated N. meningitidis strains within Burkina Faso was highly dynamic between 2008 and 2012, reflecting both vaccine-imposed selection against serogroup A strains and potentially complex clonal waves of serogroup X and serogroup W strains.

Molecular Characterization of Invasive Isolates of Neisseria meningitidis in Casablanca, Morocco.

Meningococcal epidemiology may change unpredictably, and typing of Neisseria meningitidis isolates is crucial for the surveillance of invasive meningococcal disease (IMD). Few data are available regarding the meningococcal epidemiology in countries of North Africa. We aimed to explore invasive meningococcal isolates from the Casablanca region in Morocco. We used whole-genome sequencing (WGS) to characterize 105 isolates from this region during the period of 2011 to 2016. Our data showed that the majority (n = 100) of the isolates belonged to serogroup B. Genotyping indicated that most of the isolates (n = 62) belonged to sequence type 33 of clonal complex 32. The isolates also showed the same PorA and FetA markers and clustered together on the basis of WGS phylogenetic analysis; they seemed to correspond to an expansion of local isolates in the Casablanca region, as reported for similar isolates in several other countries. These data suggest that serogroup B isolates may predominate in Morocco, which may have an important impact in the design of vaccination strategies.

Clonal replacement and expansion among invasive meningococcal isolates of serogroup W in France

Neisseria meningitidis group W (NmW) belonging to the clonal complex ST-11 (NmW/cc11) spread in Europe and in France in 2000 and declined thereafter. In France, invasive meningococcal disease (IMD) due to NmW increased again in 2012 and thereafter since 2015. Several sub-lineages of NmW/cc11 are circulating worldwide with successive epidemic waves. We aimed to describe recent epidemiological trends of NmW in France and to explore the microbiological and epidemiological characteristics associated with different NmW/cc11 sub-lineages. The epidemiology of NmW was described based on data collected through mandatory notification of IMD and strain typing data for culture-confirmed and PCR-confirmed cases for the period 2000-2016. All culture-confirmed cases due to NmW from the period 2010-2016 were characterised by whole genome sequencing (WGS). A detailed epidemiological analysis was performed for culture-confirmed cases on the basis of WGS data. During the period 2010-2016, genotyping was obtained for 148 cases including all the 132 culture-confirmed cases, among which 127 were matched with epidemiological data, and 16 PCR-confirmed cases (out of a total of 47 PCR-confirmed cases). An increase in IMD was observed in 2012 and was linked to isolates belonging to the "Anglo-French-Hajj" sub-lineage. These isolates have decreased significantly since 2013 and have been replaced by NmW/cc11 isolates related to the "South American - UK" sub-lineage which caused a marked increase in the number of cases of NmW in 2016. In this sub-lineage, the "original UK strain" was first detected in 2012 and increased thereafter, followed by the recently described "UK 2013-strain". Isolates related to the "South American-UK" sub-lineage represented 45% of all NmW cultured isolates from the whole period 2010-2016 but were the most frequent isolates in 2016, representing 76% of the total NmW typed isolates and 94% of the typed NmW/cc11 isolates. A changing pattern in the epidemiology of NmW has been observed in 2015-2016 in relation to the spread of the "UK 2013-strain" with a sharp increase in the number of cases among persons aged 15 years and over and a high case fatality rate (CFR). Among cases due to the "UK 2013-strain", 94% of cases were aged 15 years and over and the CFR was 28%. Our data suggest a recent clonal replacement among NmW/cc11 isolates with the expansion of the "South American-UK" sub-lineage in France and particularly the "UK 2013-strain" which was predominant in 2015 and 2016. A shift in the age-distribution of IMD due to NmW to older ages and the high CFR are consistent with the expansion of a new virulent clone in a naive population. These data may have an impact on tailoring vaccination strategies against NmW.

Differences in the population structure of Neisseria meningitidis in two Australian states: Victoria and Western Australia.

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). A recombinant vaccine called Bexsero® incorporates four subcapsular antigens (fHbp, NHBA, NadA and PorA) which are used to assign a Bexsero® antigen sequence type (BAST) to each meningococcal strain. The vaccine elicits an immune response against combinations of variants of these antigens which have been grouped into specific BAST profiles that have been shown to have different distributions within geographical locations thus potentially affecting the efficacy of the vaccine. In this study, invasive meningococcal disease isolates from the western seaboard of Australia (Western Australia; WA) were compared to those from the south-eastern seaboard (Victoria; VIC) from 2008 to 2012. Whole-genome sequencing (WGS) of 131 meningococci from VIC and 70 meningococci from WA were analysed for MLST, FetA and BAST profiling. Serogroup B predominated in both jurisdictions and a total of 10 MLST clonal complexes (cc) were shared by both states. Isolates belonging to cc22, cc103 and cc1157 were unique to VIC whilst isolates from cc60 and cc212 were unique to WA. Clonal complex 41/44 represented one-third of the meningococcal population in each state but the predominant ST was locally different: ST-6058 in VIC and ST-146 in WA. Of the 108 BAST profiles identified in this collection, only 9 BASTs were simultaneously observed in both states. A significantly larger proportion of isolates in VIC harboured alleles for the NHBA-2 peptide and fHbp-1, antigenic variants predicted to be covered by the Bexsero® vaccine. The estimate for vaccine coverage in WA (47.1% [95% CI: 41.1–53.1%]) was significantly lower than that in VIC (66.4% [95% CI: 62.3–70.5%]). In conclusion, the antigenic structure of meningococci causing invasive disease in two geographically distinct states of Australia differed significantly during the study period which may affect vaccine effectiveness and highlights the need for representative surveillance when predicting potential impact of meningococcal B vaccines.

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1.

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV.

Hierarchical genomic analysis of carried and invasive serogroup A Neisseria meningitidis during the 2011 epidemic in Chad

BackgroundSerogroup A Neisseria meningitidis (NmA) was the cause of the 2011 meningitis epidemics in Chad. This bacterium, often carried asymptomatically, is considered to be an “accidental pathogen”; however, the transition from carriage to disease phenotype remains poorly understood. This study examined the role genetic diversity might play in this transition by comparing genomes from geographically and temporally matched invasive and carried NmA isolates.ResultsAll 23 NmA isolates belonged to the ST-5 clonal complex (cc5). Ribosomal MLST comparison with other publically available NmA:cc5 showed that isolates were closely related, although those from Chad formed two distinct branches and did not cluster with other NmA, based on their MLST profile, geographical and temporal location. Whole genome MLST (wgMLST) comparison identified 242 variable genes among all Chadian isolates and clustered them into three distinct phylogenetic groups (Clusters 1, 2, and 3): no systematic clustering by disease or carriage source was observed. There was a significant difference (p = 0.0070) between the mean age of the individuals from which isolates from Cluster 1 and Cluster 2 were obtained, irrespective of whether the person was a case or a carrier.ConclusionsWhole genome sequencing provided high-resolution characterization of the genetic diversity of these closely related NmA isolates. The invasive meningococcal isolates obtained during the epidemic were not homogeneous; rather, a variety of closely related but distinct clones were circulating in the human population with some clones preferentially colonizing specific age groups, reflecting a potential age-related niche adaptation. Systematic genetic differences were not identified between carriage and disease isolates consistent with invasive meningococcal disease being a multi-factorial event resulting from changes in host-pathogen interactions along with the bacterium.

Genomic surveillance and meningococcal group B vaccine coverage estimates after introduction of the vaccine into the national immunisation programme in the UK

BackgroundIn September, 2015, the meningococcal group B vaccine (Bexsero, GlaxoSmithKline) was introduced into the UK childhood immunisation schedule. Prelicensure coverage estimates used Meningococcal Antigen Typing System (MATS) to determine phenotype and function of circulating meningococci. Using whole-genome sequencing, we aimed to perform rapid, scalable, high-resolution analysis of peptide vaccine antigens in disease-causing meningococci before and after vaccine implementation. MethodsAll culture-confirmed invasive meningococcal disease isolates from July 1, 2010, to June 30, 2016, from the UK underwent whole-genome sequencing (n=2994), representing 56·7% of cases reported to Public Health England. Genomes were hosted on the PubMLST database. The Bexsero Antigen Sequence Type (BAST) scheme was used to catalogue antigenic variants for comparison with the Bexsero type, BAST-1 (fHbp:1, NHBA:2, NadA:8, PorA VR1:7-2, PorA VR2:4). Statistical analysis was performed with R software (v3.2.4). FindingsComplete BASTs were obtained for 2915 isolates (97·4%). Within the fHbp family 1, variant 1 was only present in 105 (3·5%) of 2994 isolates. Variant 4 dominated since 2010–11 in BASTs 220, 226, and 229. From 2013–14 onwards, family 2 variants were more frequent than family 1 variants. NHBA variant 2 decreased from 373 (15·3%) of 2441 prevaccine isolates to 45 (9·4%) of 479 postvaccine isolates (p=0·001). The most common variant was 29 (557/2994). NadA was absent in 2049 (70·1%) of 2921 isolates. PorA VR2 had 106 variants, most frequently variants 2 (n=542), 9 (350), and 4 (333). Genotypic matches to BAST-1 (≥1 antigen) decreased from 30·4% (167/550) to 15·5% (83/536) over a 6 year period. Coverage including cross-reactive antigens was 60·2–69·0%. Genotype–phenotype modelling estimated coverage of 62·1% (95% CI 60·9–63·3), with no significant change before or after vaccine implementation. InterpretationRecent clonal expansion of South American/UK serogroup W strain accounts for many antigenic changes, such as the increase in family 2 fHbp and NHBA variant 29. Cross-reactivity within the fHbp family 1 is a key determinant of vaccine immunogenicity. MATS coverage estimates of 73% (95% CI 57–87) were key to policy decisions for introduction of the vaccine in the UK. Analysis of recent disease isolates with genomic and genotype–phenotype correlations suggests that coverage estimates are lower but within the limits of previous MATS studies. FundingWellcome Trust.

Emergence of meningococci with reduced susceptibility to third-generation cephalosporins.

Reduced susceptibility to penicillin G in Neisseria meningitidis is mainly due to alterations in PBP2 encoded by the penA gene. However, this phenotype was not associated with reduced susceptibility to third-generation cephalosporins (C3Gs). We aimed to study the emergence of meningococci with reduced susceptibility to C3Gs (MIC >0.06 mg/L) in France since 2012. Invasive meningococcal isolates were typed by MLST and penA sequencing and their antibiotic susceptibility was tested. Isolates with reduced susceptibility to C3Gs represented 2% of all invasive isolates from 2012-15, but were absent before. They harboured a new penA allele, penA327, that was also detected in isolates from urethritis cases and in gonococci. Surveillance of these isolates should be enhanced as they may jeopardize the use of C3Gs in the management of invasive meningococcal disease.

Distribution of Bexsero® Antigen Sequence Types (BASTs) in invasive meningococcal disease isolates: Implications for immunisation

Serogroup B is the only major disease-associated capsular group of Neisseria meningitidis for which no protein-polysaccharide conjugate vaccine is available. This has led to the development of multi-component protein-based vaccines that target serogroup B invasive meningococcal disease (IMD), including Bexsero®, which was implemented for UK infants in 2015, and Trumenba®. Given the diversity of meningococcal protein antigens, post-implementation surveillance of IMD isolates, including characterisation of vaccine antigens, is essential for assessing the effectiveness of such vaccines. Whole genome sequencing (WGS), as realised in the Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL), provides a rapid, comprehensive, and cost-effective approach to this. To facilitate the surveillance of the antigen targets included in Bexsero® (fHbp, PorA, NHBA and NadA) for protective immunity, a Bexsero® Antigen Sequence Type (BAST) scheme, based on deduced peptide sequence variants, was implemented in the PubMLST.org/neisseria database, which includes the MRF-MGL and other isolate collections. This scheme enabled the characterisation of vaccine antigen variants and here the invasive meningococci isolated in Great Britain and Ireland in the epidemiological years 2010/11 to 2013/14 are analysed. Many unique BASTs (647) were present, but nine of these accounted for 39% (775/1966) of isolates, with some temporal and geographic differences in BAST distribution. BASTs were strongly associated with other characteristics, such as serogroup and clonal complex (cc), and a significant increase in BAST-2 was associated with increased prevalence of serogroup W clonal complex 11 meningococci. Potential coverage was assessed by the examination of the antigen peptide sequences present in the vaccine and epidemiological dataset. There were 22.8–30.8% exact peptide matches to Bexsero® components and predicted coverage of 66.1%, based on genotype-phenotype modelling for 63.7% of serogroup B isolates from 2010/14 in UK and Ireland. While there are many caveats to this estimate, it lies within the range of other published estimates.

Typing and surface charges of the variable loop regions of PorB from Neisseria meningitidis.

PorB is a pan-Neisserial major outer membrane protein with a trimeric β-barrel structure. Each monomer presents eight periplasmic turns and eight surface exposed loop regions with sequence variability. PorB induces activation of host cell responses via a TLR2-dependent mechanism likely mediated by electrostatic interactions between TLR2 and PorB surface exposed loops. Variability in the loop amino acid sequence is known to influence cell responses to PorB in vitro, particularly for the residues in L5 and L7. In this work, the sequence of the porB gene and the electrostatic surface charges of PorB from 35 invasive meningococcal isolates belonging to the main clonal complexes identified in Italy and from five carriage genomes available on the website http://pubmlst.org/neisseria/ were examined. Analysis of the porB encoding regions from the invasive meningococci has identified four new alleles and a potential association between porB alleles, serogroup, and clonal complexes. Through computer-based modeling and analysis of the electrostatic surface charges of PorB from these strains, loop charge segregation between PorB from invasive serogroups B and C was observed. Specifically, loops 1, 4, and 7 were negatively charged and L2 and L8 were mostly neutral in serogroup B isolates, while an overall homogeneous positive surface charge was present in PorB from invasive serogroup C strains. A higher PorB sequence variability was observed among carriage genomes, and a general prevalence of negative loop surface charges. The surface charge differences in PorB from serogroups B and C invasive and carriage strains may, in part, influence the outcomes of Neisseriae interactions with host cells. © 2016 IUBMB Life, 68(6):488-495, 2016.

Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis

ABSTRACTThe identification of “factor H binding protein (fHbp)-null” invasive meningococcal isolates and the realization that widespread use of fHbp-based vaccines could herald selection of such strains prompted us to characterize novel mechanisms of alternative pathway (AP) inhibition on meningococci. Of seven strains engineered to lack four known AP-inhibiting molecules, capsular polysaccharide, lipooligosaccharide sialic acid, fHbp, and neisserial surface protein A (quadruple mutants), four strains inhibited human AP-mediated C3 deposition. All four expressed the porin B2 (PorB2) molecule, and three strains belonged to the hypervirulent ST-11 lineage. Consistent with reduced C3 deposition, the rate of C3a generation by a PorB2 isolate was lower than that by a PorB3 strain. Allelic replacement of PorB3 with PorB2, in both encapsulated and unencapsulated strains, confirmed the role of PorB2 in AP inhibition. Expression of PorB2 increased resistance to complement-dependent killing relative to that seen in an isogenic PorB3-expressing strain. Adult rabbit and mouse APs were unimpeded on all mutants, and human fH inhibited nonhuman C3 deposition on PorB2-expressing strains, which provided functional evidence for human fH-dependent AP regulation by PorB2. Low-affinity binding of full-length human fH to quadruple mutants expressing PorB2 was demonstrated. fH-like protein 1 (FHL-1; contains fH domains 1 through 7) and fH domains 6 and 7 fused to IgG Fc bound to one PorB2-expressing quadruple mutant, which suggested that fH domains 6 and 7 may interact with PorB2. These results associate PorB2 expression with serum resistance and presage the appearance of fHbp-null and hypervirulent ST-11 isolates that may evade killing by fHbp-based vaccines.