A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies

The presence of serum bactericidal antibodies is a proven correlate of protection against systemic infection with the important human pathogen Neisseria meningitidis. We have identified three serogroup C N. meningitidis (MenC) isolates recovered from patients with invasive meningococcal disease that resist killing by bactericidal antibodies induced by the MenC conjugate vaccine. None of the patients had received the vaccine, which has been successfully introduced in countries in North America and Europe. The increased resistance was not caused by changes either in lipopolysaccharide sialylation or acetylation of the α2-9–linked polysialic acid capsule. Instead, the resistance of the isolates resulted from the presence of an insertion sequence, IS1301, in the intergenic region (IGR) between the sia and ctr operons, which are necessary for capsule biosynthesis and export, respectively. The insertion sequence led to an increase in the transcript levels of surrounding genes and the amount of capsule expressed by the strains. The increased amount of capsule was associated with down-regulation of the alternative pathway of complement activation, providing a generic mechanism by which the bacterium protects itself against bactericidal antibodies. The strains with IS1301 in the IGR avoided complement-mediated lysis in the presence of bactericidal antibodies directed at the outer membrane protein, PorA, or raised against whole cells

Species Status of Neisseria gonorrhoeae: Evolutionary and Epidemiological Inferences from MLST

Abstract Background Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. Results A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. Conclusion The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present

Typing complex meningococcal vaccines to understand diversity and population structure of key vaccine antigens [version 1; referees: 2 approved]

Background: Protein-conjugate capsular polysaccharide vaccines can potentially control invasive meningococcal disease (IMD) caused by five (A, C, W, X, Y) of the six IMD-associated serogroups.  Concerns raised by immunological similarity of the serogroup B capsule, to human neural cell carbohydrates, has meant that ‘serogroup B substitute’ vaccines target more variable subcapsular protein antigens.  A successful approach using outer membrane vesicles (OMVs) as major vaccine components had limited strain coverage. In 4CMenB (Bexsero®), recombinant proteins have been added to ameliorate this problem.  Methods: Here, scalable, portable, genomic techniques were used to investigate the Bexsero® OMV protein diversity in meningococcal populations. Shotgun proteomics identified 461 proteins in the OMV, defining a complex proteome. Amino acid sequences for the 24 proteins most likely to be involved in cross-protective immune responses were catalogued within the PubMLST.org/neisseria database using a novel OMV peptide Typing (OMVT) scheme. Results: Among these proteins there was variation in the extent of diversity and association with meningococcal lineages, identified as clonal complexes (ccs), ranging from the most conserved peptides (FbpA, NEISp0578, and putative periplasmic protein, NEISp1063) to the most diverse (TbpA, NEISp1690).  There were 1752 unique OMVTs identified amongst 2492/3506 isolates examined by whole-genome sequencing (WGS). These OMVTs were grouped into clusters (sharing ≥18 identical OMVT peptides), with 45.3% of isolates assigned to one of 27 OMVT clusters. OMVTs and OMVT clusters were strongly associated with cc, genogroup, and Bexsero® antigen variants, demonstrating that combinations of OMV proteins exist in discrete, non-overlapping combinations associated with genogroup and Bexsero® Antigen Sequence Type. This highly structured population of IMD-associated meningococci is consistent with strain structure models invoking host immune selection. Conclusions: The OMVT scheme facilitates region-specific WGS investigation of meningococcal diversity and is an open-access, portable tool with applications for vaccine development, especially in the choice of antigen combinations, assessment and implementation

Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin–haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains

Molecular epidemiology of meningococcal disease in England and Wales 1975–1995, before the introduction of serogroup C conjugate vaccines

A comprehensive meningococcal vaccine is yet to be developed. In the absence of a vaccine that immunizes against the serogroup B capsular polysaccharide, this can only be achieved by targeting subcapsular antigens, and a number of outer-membrane proteins (OMPs) are under consideration as candidates. A major obstacle to the development of such a vaccine is the antigenic diversity of these OMPs, and obtaining population data that accurately identify and catalogue these variants is an important component of vaccine design. The recently proposed meningococcal molecular strain-typing scheme indexes the diversity of two OMPs, PorA and FetA, that are vaccine candidates, as well as the capsule and multilocus sequence type. This scheme was employed to survey 323 meningococci isolated from invasive disease in England and Wales from 1975 to 1995, before the introduction of meningococcal conjugated serogroup C polysaccharide vaccines in 1999. The eight-locus typing scheme provided high typeability (99.4 %) and discrimination (Simpson's diversity index 0.94–0.99). The data showed cycling of meningococcal genotypes and antigenic types in the absence of planned interventions. Notwithstanding high genetic and antigenic diversity, most of the isolates belonged to one of seven clonal complexes, with 11 predominant strain types. Combinations of PorA and FetA, chosen on the basis of their prevalence over time, generated vaccine recipes that included protein variants found in 80 % or more of the disease isolates for this time period. If adequate immune responses can be generated, these results suggest that control of meningococcal disease with relatively simple protein component vaccines may be possible

Variation and molecular evolution of HmbR, the Neisseria meningitidis haemoglobin receptor

Meningococcal disease caused by serogroup B Neisseria meningitidis remains an important health problem in many parts of the world, and there are currently no comprehensive vaccines. Poor immunogenicity, combined with immunological identity to human sialic acids, have hindered the development of a serogroup B conjugate vaccine, resulting in the development of alternative vaccine candidates, including many outer-membrane protein (OMP)-based formulations. However, the design of protein-based meningococcal vaccines is complicated by the high level of genetic and antigenic diversity of the meningococcus. Knowledge of the extent and structuring of this diversity can have implications for the use of particular proteins as potential vaccine candidates. With this in mind, the diversity of the meningococcal OMP HmbR was investigated among N. meningitidis isolates representative of major hyper-invasive lineages. In common with other meningococcal antigens, the genetic diversity of hmbR resulted from a combination of intraspecies horizontal genetic exchange and de novo mutation. Furthermore, genealogical analysis showed an association of hmbR genes with clonal complexes and the occurrence of two hmbR families, A and B. Three variable regions (VR1–VR3), located in loops 2, 3 and 4, were observed with clonal complex structuring of VR types. A minority of codons (3.9 %), located within putative surface-exposed loop regions of a 2D model, were under diversifying selection, indicating regions of the protein likely to be subject to immune attack

Restructuring and repositioning of private accomodation in Primorsko-Goranska county: problems and solutions

The paper presents the results of a research conducted on private accommodation as most common form of accommodation within the tourism offer of Primorsko-Goranska County. The study was conducted in 2008, and the obtained results of the research are analysed in the paper, conclusions made, key problems identified and appropriate measures proposed, with the aim of enhancing the quality of this segment of the tourism industry. The goal of the above mentioned measures’ implementation are quality improvement, repositioning and restructuring of private accommodation and its transformation into small family hotels, boarding houses or apart-hotels

Evaluation of Critical Quality Attributes of a Pentavalent (A, C, Y, W, X) Meningococcal Conjugate Vaccine for Global Use

Towards achieving the goal of eliminating epidemic outbreaks of meningococcal disease in the African meningitis belt, a pentavalent glycoconjugate vaccine (NmCV-5) has been developed to protect against Neisseria meningitidis serogroups A, C, Y, W and X. MenA and X polysaccharides are conjugated to tetanus toxoid (TT) while MenC, Y and W polysaccharides are conjugated to recombinant cross reactive material 197 (rCRM197), a non-toxic genetic variant of diphtheria toxin. This study describes quality control testing performed by the manufacturer, Serum Institute of India Private Limited (SIIPL), and the independent control laboratory of the U.K. (NIBSC) on seven clinical lots of the vaccine to ensure its potency, purity, safety and consistency of its manufacturing. In addition to monitoring upstream-manufactured components, samples of drug substance, final drug product and stability samples were evaluated. This paper focuses on the comparison of the vaccine’s critical quality attributes and reviews key indicators of its stability and immunogenicity. Comparable results were obtained by the two laboratories demonstrating sufficient levels of polysaccharide O-acetylation, consistency in size of the bulk conjugate molecules, integrity of the conjugated saccharides in the drug substance and drug product, and acceptable endotoxin content in the final drug product. The freeze-dried vaccine in 5-dose vials was stable based on molecular sizing and free saccharide assays. Lot-to-lot manufacturing consistency was also demonstrated in preclinical studies for polysaccharide-specific IgG and complement-dependent serum bactericidal activity for each serogroup. This study demonstrates the high quality and stability of NmCV-5, which is now undergoing Phase 3 clinical trials in Africa and India

Investigation of the potential of PorA and FetA as meningococcal vaccine components

In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.This thesis is not currently available via ORA