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Sequence analysis of a 7.5 kb DNA plasmid isolated from Chlamydia trachomatis shows 8 open reading frames (ORFs) regularly spaced along most of the sequence. One of these ORFs encodes a 451-aminoacid polypeptide highly homologous to the DnaB protein of Escherichia coli. A region between ORFs 6 and 7 contains a cluster of alternating ATs and a 22 bp sequence tandemly repeated 4 times, suggesting a replication control region. Several ORFs correspond to plasmid-specific polypeptides that have been described. Codons ending with A or T are more frequent, as might be expected from the high A/T content (64%) of the plasmid, and codon usage is similar to that of the C. trachomatis chromosomal gene, omp1L2. [ABSTRACT FROM AUTHOR]

Background: The multicomponent meningococcal serogroup B vaccine (4CMenB) is currently indicated for active immunization against invasive meningococcal disease caused by Neisseria meningitidis serogroup B (MenB). However, genes encoding the 4CMenB antigens are also variably present and expressed in strains belonging to other meningococcal serogroups. In this study, we evaluated the ability of antibodies raised by 4CMenB immunisation to induce complement-mediated bactericidal killing of non-MenB strains., Methods: A total of 227 invasive non-MenB disease isolates were collected between 1 July 2007 and 30 June 2008 from England and Wales, France, and Germany; 41 isolates were collected during 2012 from Brazil. The isolates were subjected to genotypic analyses. A subset of 147 isolates (MenC, MenW and MenY) representative of the meningococcal genetic diversity of the total sample were tested in the human complement serum bactericidal antibody assay (hSBA) using sera from infants immunised with 4CMenB., Results: Serogroup and clonal complex repertoires of non-MenB isolates were different for each country. For the European panel, MenC, MenW and MenY isolates belonged mainly to ST-11, ST-22 and ST-23 complexes, respectively. For the Brazilian panel, most MenC and MenW isolates belonged to the ST-103 and ST-11 complexes, respectively, and most MenY isolates were not assigned to clonal complexes. Of the 147 non-MenB isolates, 109 were killed in hSBA, resulting in an overall coverage of 74%., Conclusion: This is the first study in which 147 non-MenB serogroup isolates have been analysed in hSBA to evaluate the potential of a MenB vaccine to cover strains belonging to other serogroups. These data demonstrate that antibodies raised by 4CMenB are able to induce bactericidal killing of 109 non-MenB isolates, representative of non-MenB genetic and geographic diversity. These findings support previous evidence that 4CMenB immunisation can provide cross-protection against non-MenB strains in infants, which represents an added benefit of 4CMenB vaccination., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MM and MC were, AB, GDA, ST, BB, MG, SB, SoB, MMG, RR and MP are employees of the GSK groups of companies. SoB has a patent pending (WO 2015014922 A2). PB was an employee of PRA Health Sciences c/o GSK at the time of study. MKT received grants from Pfizer and the GSK group of companies and has a patent issued (630133). JL and RB have performed contract research on behalf of Public Health England for the GSK group of companies, Pfizer, and Sanofi Pasteur. APSdL reports personal fees from Pfizer and Sanofi-Pasteur. HC and UV's institution received research grants from Novartis. MCOG and EH have nothing to disclose., (Copyright © 2020 GlaxoSmithKline Biologicals S.A. Published by Elsevier Ltd.. All rights reserved.)

Background: The 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., Methods: From 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., Results: Overall, 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%., Conclusions: gMATS 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., (Copyright © 2019 GlaxoSmithKline Biologicals SA. Published by Elsevier Ltd.. All rights reserved.)

Background: A novel meningococcal multicomponent vaccine, 4CMenB (Bexsero®), has been approved in Europe, Canada, Australia and US. The potential impact of 4CMenB on strain coverage is being estimated by using Meningococcal Antigen Typing System (MATS), an ELISA assay which measures vaccine antigen expression and diversity in each strain. Here we show the genetic characterization and the 4CMenB potential coverage of Spanish invasive strains (collected during one epidemiological year) compared to other European countries and discuss the potential reasons for the lower estimate of coverage in Spain., Material and Methods: A panel of 300 strains, a representative sample of all serogroup B Neisseria meningitidis notified cases in Spain from 2009 to 2010, was characterized by multilocus sequence typing (MLST) and FetA variable region determination. 4CMenB vaccine antigens, PorA, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were molecularly typed by sequencing. PorA coverage was assigned to strain with VR2 = 4. The levels of expression and cross-reactivity of fHbp, NHBA and NadA were analyzed using MATS ELISA., Findings: Global estimated strain coverage by MATS was 68.67% (95% CI: 47.77-84.59%), with 51.33%, 15.33% and 2% of strains covered by one, two and three vaccine antigens, respectively. The predicted strain coverage by individual antigens was: 42% NHBA, 36.33% fHbp, 8.33% PorA and 1.33% NadA. Coverage within the most prevalent clonal complexes (cc) was 70.37% for cc 269, 30.19% for cc 213 and 95.83% for cc 32., Conclusions: Clonal complexes (cc) distribution accounts for variations in strain coverage, so that country-by-country investigations of strain coverage and cc prevalence are important. Because the cc distribution could also vary over time, which in turn could lead to changes in strain coverage, continuous detailed surveillance and monitoring of vaccine antigens expression is needed in those countries where the multicomponent vaccine is introduced. This is really important in countries like Spain where most of the strains are predicted to be covered by only one vaccine antigen and the chance for escape mutants to emerge with vaccine use is higher. Based on the observed data, cc213 should receive special attention as it is associated with low predicted strain coverage, and has recently emerged in Spain.

Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The first broad-spectrum multicomponent vaccine against serogroup B meningococcus (MenB), 4CMenB (Bexsero(®)), was approved by the EMA in 2013, for prevention of MenB disease in all age groups, and by the US FDA in January 2015 for use in adolescents. A second protein-based MenB vaccine has also been approved in the USA for adolescents (rLP2086, Trumenba(®)). Both vaccines contain the lipoprotein factor H-binding protein (fHbp). Preclinical studies demonstrated that fHbp elicits a robust bactericidal antibody response that correlates with the amount of fHbp expressed on the bacterial surface. fHbp is able to selectively bind human factor H, the key regulator of the alternative complement pathway, and this has important implications both for meningococcal pathogenesis and for vaccine design. Here, we review the functional and structural properties of fHbp, the strategies that led to the design of the two fHbp-based vaccines and the data generated during clinical studies.

Neisseria adhesin A (NadA), involved in the adhesion and invasion of Neisseria meningitidis into host tissues, is one of the major components of Bexsero, a novel multicomponent vaccine licensed for protection against meningococcal serogroup B in Europe, Australia, and Canada. NadA has been identified in approximately 30% of clinical isolates and in a much lower proportion of carrier isolates. Three protein variants were originally identified in invasive meningococci and named NadA-1, NadA-2, and NadA-3, whereas most carrier isolates either lacked the gene or harbored a different variant, NadA-4. Further analysis of isolates belonging to the sequence type 213 (ST-213) clonal complex identified NadA-5, which was structurally similar to NadA-4, but more distantly related to NadA-1, -2, and -3. At the time of this writing, more than 89 distinct nadA allele sequences and 43 distinct peptides have been described. Here, we present a revised nomenclature system, taking into account the complete data set, which is compatible with previous classification schemes and is expandable. The main features of this new scheme include (i) the grouping of the previously named NadA-2 and NadA-3 variants into a single NadA-2/3 variant, (ii) the grouping of the previously assigned NadA-4 and NadA-5 variants into a single NadA-4/5 variant, (iii) the introduction of an additional variant (NadA-6), and (iv) the classification of the variants into two main groups, named groups I and II. To facilitate querying of the sequences and submission of new allele sequences, the nucleotide and amino acid sequences are available at http://pubmlst.org/neisseria/NadA/., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Surface-expressed protein antigens such as factor H-binding protein (fHbp), Neisserial adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and Porin protein A (PorA); all express sequence variability that can affect their function as protective immunogens when used in meningococcal serogroup B vaccines like the recently-approved 4CMenB (Bexsero(®)). We assessed the sequence variation of genes coding for these proteins and two additional proteins ("fusion partners" to fHbp and NHBA) in pathogenic isolates from a recent low incidence period (endemic situation; 2005-2006) in Norway. Findings among strains from this panel were contrasted to what was found among isolates from a historic outbreak (epidemic situation; 1985-1990). Multilocus sequence typing revealed 14 clonal complexes (cc) among the 66 endemic strains, while cc32 vastly predominated in the 38-strain epidemic panel. Serogroup B isolates accounted for 50/66 among endemic strains and 28/38 among epidemic strains. Potential strain-coverage ("sequence match") for the 4CMenB vaccine was identified among the majority (>70%) of the endemic serogroup B isolates and all of the epidemic serogroup B isolates evaluated. Further information about the degree of expression, surface availability and the true cross-reactivity for the vaccine antigens will be needed to fully characterize the clinical strain-coverage of 4CMenB in various geographic and epidemiological situations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Serogroup B meningococcal (MenB) isolates currently account for approximately 90% of invasive meningococcal disease (IMD) in Greece with ST-162 clonal complex predominating. The potential of a multicomponent meningococcal B vaccine (4CMenB) recently licensed in Europe was investigated in order to find whether the aforementioned vaccine will cover the MenB strains circulating in Greece. A panel of 148 serogroup B invasive meningococcal strains was characterized by multilocus sequence typing (MLST) and PorA subtyping. Vaccine components were typed by sequencing for factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA) and Neisseria adhesin A (NadA). Their expression was explored by Meningococcal Antigen Typing System (MATS)., Results: Global strain coverage predicted by MATS was 89.2% (95% CI 63.5%-98.6%) with 44.6%, 38.5% and 6.1% of strains covered by one, two and three vaccine antigens respectively. NHBA was the antigen responsible for the highest coverage (78.4%), followed by fHbp (52.7%), PorA (8.1%) and NadA (0.7%). The coverage of the major genotypes did not differ significantly. The most prevalent MLST genotype was the ST-162 clonal complex , accounting for 44.6% of the strains in the panel and with a predicted coverage of 86.4%, mainly due to NHBA and fHbp., Conclusions: 4CMenB has the potential to protect against a significant proportion of Greek invasive MenB strains.

Studies of meningococcal evolution and genetic population structure, including the long-term stability of non-random associations between variants of surface proteins, are essential for vaccine development. We analyzed the sequence variability of factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisseria adhesin A (NadA), three major antigens in the multicomponent meningococcal serogroup B vaccine 4CMenB. A panel of invasive isolates collected in the Netherlands over a period of 50 years was used. To our knowledge, this strain collection covers the longest time period of any collection available worldwide. Long-term persistence of several antigen sub/variants and of non-overlapping antigen sub/variant combinations was observed. Our data suggest that certain antigen sub/variants including those used in 4CMenB are conserved over time and promoted by selection.

Background: A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe., Methods: We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain., Findings: 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries., Interpretation: MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future., Funding: Novartis Vaccines and Diagnostics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A new vaccine, 4CMenB, is composed of surface proteins of Neisseria meningitidis and is aimed to target serogroup B (MenB) isolates. The vaccine components are present in meningococcal isolates of other serogroups allowing potential use against meningococcal isolates belonging to non-B serogroups. Isolates of serogroup X (MenX) have been emerged in countries of the African meningitis belt. 4CMenB may offer a vaccine strategy against these isolates as there is no available capsule-based vaccine against MenX. We used the Meningococcal Antigen Typing System (MATS) to determine presence, diversity and levels of expression of 4CMenB antigens among 9 MenX isolates from several African countries in order to estimate the potential coverage of MenX by the 4CMenB vaccine. We performed bactericidal assays against these isolates, using pooled sera from 4CMenB-vaccinated infants, adolescents and adults. The African MenX isolates belonged to the same genotype but showed variation in the vaccine antigens. MATS data and bactericidal assays suggest coverage of the 9 African MenX isolates by 4CMenB but not of two unrelated MenX isolates from France. 4CMenB vaccine can be considered for further investigation to control MenX outbreaks in Africa., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

Neisseria meningitidis (Nm) serogroups B, C and Y are the major causes of meningococcal diseases in the United States. NmB accounts for ∼1/3 of the disease but no licensed vaccine is yet available. Two candidate vaccines are being developed specifically to target NmB, but may also provide protection against other serogroups. To assess the potential impact of these vaccines on NmB and other serogroups causing disease in the US, we determined the prevalence, genetic diversity and epidemiological characteristics of three candidate antigen genes in Nm isolates collected through Active Bacterial Core surveillance (ABCs), a population-based active surveillance program. fHbp was detected in all NmB, NmY and NmW135 isolates. Eleven NmC isolates contain fHbp with a single base-pair deletion creating a frame shift in the C-terminal region. Among NmB, 59% were FHbp subfamily/variant B/v1 and 41% A/v2-3. Among NmC and NmY, 39% and 3% were B/v1, respectively. nadA was detected in 39% of NmB, 61% of NmC and 4% of NmY. Among isolates tested, nhbA was present in all NmB and 96% of non-B. For the subset of strains sequenced for NadA and NhbA, pairwise identity was greater than 93% and 78%, respectively. The proportion of FHbp subfamily/variant was different between ABCs site and year, but no linear temporal trend was observed. Although assessment of the vaccine coverage also requires understanding of the antigen expression and the ability to induce bactericidal activity, our finding that all isolates contain one or more antigen genes suggests these candidate vaccines may protect against multiple Nm serogroups., (Published by Elsevier Ltd.)

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling downregulation of complement activation on the bacterial surface. fHbp is a component of two serogroup B meningococcal vaccines currently in clinical development. Here we characterize 12 fHbp subvariants for their level of surface exposure and ability to bind fH, to mediate serum resistance, and to induce bactericidal antibodies. Flow cytometry and Western analysis revealed that all strains examined expressed fHbp on their surface to different extents and bound fH in an fHbp-dependent manner. However, differences in fH binding did not always correlate with the level of fHbp expression, indicating that this is not the only factor affecting the amount of fH bound. To overcome the issue of strain variability in fHbp expression, the MC58ΔfHbp strain was genetically engineered to express different subvariants from a constitutive heterologous promoter. These recombinant strains were characterized for fH binding, and the data confirmed that each subvariant binds different levels of fH. Surface plasmon resonance revealed differences in the stability of the fHbp-fH complexes that ranged over 2 orders of magnitude, indicating that differences in residues between and within variant groups can influence fH binding. Interestingly, the level of survival in human sera of recombinant MC58 strains expressing diverse subvariants did not correlate with the level of fH binding, suggesting that the interaction of fHbp with fH is not the only function of fHbp that influences serum resistance. Furthermore, cross-reactive bactericidal activity was seen within each variant group, although the degree of activity varied, suggesting that amino acid differences within each variant group influence the bactericidal antibody response.