Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index: a Rapid and Accessible Tool That Exploits Genomic Data in Public Health and Clinical Microbiology Applications.

As microbial genomics makes increasingly important contributions to clinical and public health microbiology, the interpretation of whole-genome sequence data by nonspecialists becomes essential. In the absence of capsule-based vaccines, two protein-based vaccines have been used for the prevention of invasive serogroup B meningococcal disease (IMD) since their licensure in 2013 and 2014. These vaccines have different components and different levels of coverage of meningococcal variants. Hence, decisions regarding which vaccine to use in managing serogroup B IMD outbreaks require information about the index case isolate, including (i) the presence of particular vaccine antigen variants, (ii) the expression of vaccine antigens, and (iii) the likely susceptibility of its antigen variants to antibody-dependent bactericidal killing. To obtain this information requires a multitude of laboratory assays, impractical in real-time clinical settings, where the information is most urgently needed. To facilitate assessment for public health and clinical purposes, we synthesized genomic and experimental data from published sources to develop and implement the Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index, which is publicly available on PubMLST (https://pubmlst.org). Using whole-genome sequences or individual gene sequences obtained from IMD isolates or clinical specimens, the MenDeVAR Index provides rapid evidence-based information on the presence and possible immunological cross-reactivity of different meningococcal vaccine antigen variants. The MenDeVAR Index enables practitioners who are not genomics specialists to assess the likely reactivity of vaccines for individual cases, outbreak management, or the assessment of public health vaccine programs. The MenDeVAR Index has been developed in consultation with, but independently of, both the 4CMenB (Bexsero; GSK) and rLP2086 (Trumenba; Pfizer, Inc.) vaccine manufacturers.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Dissection of the function of the RmpM periplasmic protein from Neisseria meningitidis.

RmpM is a periplasmic protein from Neisseria meningitidis that comprises an N-terminal domain (residues 1-47) and a separate globular C-terminal domain (residues 65-219) responsible for binding to peptidoglycan. Here we show, through the use of size exclusion chromatography and pull-down assays, that a recombinant N-terminal fragment of RmpM binds to both the major outer membrane porins, PorA and PorB. Analysis by semi-native SDS-PAGE established that both recombinant full-length RmpM and an N-terminal fragment, but not the C-terminal peptidoglycan-binding domain, were sufficient to stabilize the PorA and PorB oligomeric complexes. Evidence from binding assays indicated that the meso-diaminopimelate moiety plays an important role in peptidoglycan recognition by RmpM. Site-directed mutagenesis showed that two highly conserved residues, Asp120 and Arg135, play an important role in peptidoglycan binding. The yield of outer membrane vesicles, which have been used extensively as a vaccine against N. meningitidis, was considerably higher in an N. meningitidis strain expressing a truncated N-terminal fragment of RmpM (ΔC-term rmpM) than in the WT strain. The native oligomeric state of the PorA/PorB complexes was maintained in this strain. We conclude that the dual functions of RmpM are independent, and that it is possible to use this knowledge to engineer a strain with higher yield of outer membrane vesicles, whilst preserving PorA and PorB, which are key protective antigens, in their native oligomeric state.

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, "phasotypes," was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.

Description and nomenclature of Neisseria meningitidis capsule locus.

Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.

The effect of iron availability on transcription of the Neisseria meningitidis fHbp gene varies among clonal complexes.

Factor H binding protein (fHbp) is a major antigenic component of novel vaccines designed to protect against meningococcal disease. Prediction of the potential coverage of these vaccines is difficult, as fHbp is antigenically variable and levels of expression differ among isolates. Transcriptional regulation of the fHbp gene is poorly understood, although evidence suggests that oxygen availability is involved. In this study iron accessibility was found to affect fHbp transcription. However, regulation differed among meningococcal clonal complexes (ccs). For the majority of isolates, increased iron concentrations upregulated transcription. This effect was enhanced by the presence of a 181 bp insertion element upstream of fHbp, associated with isolates belonging to cc4 and cc5. Conversely, meningococci belonging to cc32 showed iron-repressed control of fHbp, as regulation was dominated by cotranscription with the iron-repressed upstream gene cbbA. These results highlight the complexity of fHbp regulation and demonstrate that control of transcription can vary among genetic lineages.

Resolution of a meningococcal disease outbreak from whole-genome sequence data with rapid Web-based analysis methods.

The increase in the capacity and reduction in cost of whole-genome sequencing methods present the imminent prospect of such data being used routinely in real time for investigations of bacterial disease outbreaks. For this to be realized, however, it is necessary that generic, portable, and robust analysis frameworks be available, which can be readily interpreted and used in real time by microbiologists, clinicians, and public health epidemiologists. We have achieved this with a set of analysis tools integrated into the PubMLST.org website, which can in principle be used for the analysis of any pathogen. The approach is demonstrated with genomic data from isolates obtained during a well-characterized meningococcal disease outbreak at the University of Southampton, United Kingdom, that occurred in 1997. Whole-genome sequence data were collected, de novo assembled, and deposited into the PubMLST Neisseria BIGSdb database, which automatically annotated the sequences. This enabled the immediate and backwards-compatible classification of the isolates with a number of schemes, including the following: conventional, extended, and ribosomal multilocus sequence typing (MLST, eMLST, and rMLST); antigen gene sequence typing (AGST); analysis based on genes conferring antibiotic susceptibility. The isolates were also compared to a reference isolate belonging to the same clonal complex (ST-11) at 1,975 loci. Visualization of the data with the NeighborNet algorithm, implemented in SplitsTree 4 within the PubMLST website, permitted complete resolution of the outbreak and related isolates, demonstrating that multiple closely related but distinct strains were simultaneously present in asymptomatic carriage and disease, with two causing disease and one responsible for the outbreak itself.

Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis.

FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, ß = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit.

WHO/Health Canada meeting on regulatory considerations for evaluation and licensing of new meningococcal Group B vaccines, Ottawa, Canada, 3-4 October 2011.

Serogroup B Neisseria meningitides (MenB) is a significant cause of endemic and epidemic outbreaks of the disease worldwide. Although polysaccharide and conjugate vaccines are available against other meningococcal serogroups, the poor immunogenicity of MenB polysaccharide has led to the development of protein-based vaccines. However, the diversity and antigenic variability of MenB strains has been a major challenge. Recently a new generation of MenB vaccines that contain conserved antigens has been developed to provide broader coverage and they are in an advanced stage of development and regulatory consideration. In October 2011, the World Health Organization and Health Canada jointly organized a consultation on regulatory considerations for the evaluation and licensing of new MenB vaccines. The aim was to seek consensus on key regulatory issues relevant to the evaluation of candidate MenB vaccines and on approaches to the standardisation of in vitro assays used in the evaluation process. Participants agreed that functional antibodies as measured in the Serum Bactericidal Activity (SBA) assay could be used to evaluate MenB vaccine efficacy and ways of improving assay standardization proposed. Approaches to bridging SBA data to large collections of strains in order to give an indication of the prospective breadth of vaccine coverage were discussed.

Application of a Neisseria meningitidis antigen microarray to identify candidate vaccine proteins from a human Phase I clinical trial.

Meningococcal meningitis is a rare but serious condition affecting mainly children and young adults. Outer membrane vesicles (OMV) from Neisseria meningitidis have been used successfully as vaccines against the disease, although they only provide protection against a limited number of the many existing variants. There have been many attempts to identify suitable protein antigens for use in defined vaccines that provide broad protection against the disease, such as that leading to the development of the four component 4CMenB vaccine. We previously reported the use of a protein antigen microarray to screen for IgG antibodies in sera derived from human recipients of an OMV-based vaccine, as part of a Phase I clinical trial. Here, we show that computational methods can be used to cluster antigens that elicit similar responses in the same individuals. Fitting of IgG antibody binding data to 4,005 linear regressions identified pairs of antigens that exhibited significant correlations. Some were from the same antigens in different quaternary states, whilst others might be correlated for functional or immunological reasons. We also conducted statistical analyses to examine correlations between individual serum bactericidal antibody (SBA) titres and IgG reactivity against specific antigens. Both Kendall's tau and Spearman's rank correlation coefficient statistics identified specific antigens that correlated with log(SBA) titre in five different isolates. The principal antigens identified were PorA and PorB, RmpM, OpcA, and the type IV pilus assembly secretin, PilQ. Other minor antigens identified included a lipoprotein, two proteins from the BAM complex and the efflux channel MtrE. Our results suggest that consideration of the entire antigen composition, and allowance for potential interaction between antigens, could be valuable in designing future meningococcal vaccines. Such an approach has the advantages that it uses data derived from human, rather than animal, immunization and that it avoids the need to screen individual antigens.

Viral vectors expressing group B meningococcal outer membrane proteins induce strong antibody responses but fail to induce functional bactericidal activity.

OBJECTIVE: Adenoviral vectored vaccines, with the appropriate gene insert, induce cellular and antibody responses against viruses, parasites and intracellular pathogens such as Mycobacterium tuberculosis. Here we explored their capacity to induce functional antibody responses to meningococcal transmembrane outer membrane proteins. METHODS: Vectors expressing porin A and ferric enterobactin receptor A antigens were generated, and their immunogenicity assessed in mice using binding and bactericidal assays. RESULTS: The viral vectors expressed the bacterial proteins in an in vitro cell-infection assay and, after immunisation of mice, induced higher titres (>105 end-point titre) and longer lasting (>32 weeks) transgene-specific antibody responses in vivo than did outer membrane vesicles containing the same antigens. However, bactericidal antibodies, which are the primary surrogate of protection against meningococcus, were undetectable, despite different designs to support the presentation of the protective B-cell epitopes. CONCLUSION: These results demonstrate that, while the transmembrane bacterial proteins expressed by the viral vector induced strong and persistent antigen-specific antibodies, this platform failed to induce bactericidal antibodies. The results suggest that conformation or post-translational modifications of bacterial outer membrane antigens produced in eukaryote cells might not result in presentation of the necessary epitopes for induction of functional antibodies.

Human B Cell Responses to Dominant and Subdominant Antigens Induced by a Meningococcal Outer Membrane Vesicle Vaccine in a Phase I Trial.

Neisseria meningitidis outer membrane vesicle (OMV) vaccines are safe and provide strain-specific protection against invasive meningococcal disease (IMD) primarily by inducing serum bactericidal antibodies against the outer membrane proteins (OMP). To design broader coverage vaccines, knowledge of the immunogenicity of all the antigens contained in OMVs is needed. In a Phase I clinical trial, an investigational meningococcal OMV vaccine, MenPF1, made from a meningococcus genetically modified to constitutively express the iron-regulated FetA induced bactericidal responses to both the PorA and the FetA antigen present in the OMP. Using peripheral blood mononuclear cells collected from this trial, we analyzed the kinetics of and relationships between IgG, IgA, and IgM B cell responses against recombinant PorA and FetA, including (i) antibody-secreting cells, (ii) memory B cells, and (iii) functional antibody responses (opsonophagocytic and bactericidal activities). Following MenPF1vaccination, PorA-specific IgG secreting cell responses were detected in up to 77% of participants and FetA-specific responses in up to 36%. Memory B cell responses to the vaccine were low or absent and mainly detected in participants who had evidence of preexisting immunity (P = 0.0069). Similarly, FetA-specific antibody titers and bactericidal activity increased in participants with preexisting immunity and is consistent with the idea that immune responses are elicited to minor antigens during asymptomatic Neisseria carriage, which can be boosted by OMV vaccines. IMPORTANCE Neisseria meningitidis outer membrane vesicles (OMV) are a component of the capsular group B meningococcal vaccine 4CMenB (Bexsero) and have been shown to induce 30% efficacy against gonococcal infection. They are composed of multiple antigens and are considered an interesting delivery platform for vaccines against several bacterial diseases. However, the protective antibody response after two or three doses of OMV-based meningococcal vaccines appears short-lived. We explored the B cell response induced to a dominant and a subdominant antigen in a meningococcal OMV vaccine in a clinical trial and showed that immune responses are elicited to minor antigens. However, memory B cell responses to the OMV were low or absent and mainly detected in participants who had evidence of preexisting immunity against the antigens. Failure to induce a strong B cell response may be linked with the low persistence of protective responses.

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.

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease.

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student ('Case'), who died of IMD, and a close contact ('Carrier') who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

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.

Molecular diagnostic assays for the detection of common bacterial meningitis pathogens: A narrative review.

Bacterial meningitis is a major global cause of morbidity and mortality. Rapid identification of the aetiological agent of meningitis is essential for clinical and public health management and disease prevention given the wide range of pathogens that cause the clinical syndrome and the availability of vaccines that protect against some, but not all, of these. Since microbiological culture is complex, slow, and often impacted by prior antimicrobial treatment of the patient, molecular diagnostic assays have been developed for bacterial detection. Distinguishing between meningitis caused by Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, and Streptococcus agalactiae and identifying their polysaccharide capsules is especially important. Here, we review methods used in the identification of these bacteria, providing an up-to-date account of available assays, allowing clinicians and diagnostic laboratories to make informed decisions about which assays to use.

Variation in the Neisseria meningitidis FadL-like protein: an evolutionary model for a relatively low-abundance surface antigen.

The molecular diversity of a novel Neisseria meningitidis antigen, encoded by the ORF NMB0088 of MC58 (FadL-like protein), was assessed in a panel of 64 diverse meningococcal strains. The panel consisted of strains belonging to different serogroups, serotypes, serosubtypes and MLST sequence types, of different clinical sources, years and countries of isolation. Based on the sequence variability of the protein, the FadL-like protein has been divided into four variant groups in this species. Antigen variants were associated with specific serogroups and MLST clonal complexes. Maximum-likelihood analyses were used to determine the relationships among sequences and to compare the selection pressures acting on the encoded protein. Furthermore, a model of population genetics and molecular evolution was used to detect natural selection in DNA sequences using the non-synonymous : synonymous substitution (d(N) : d(S)) ratio. The meningococcal sequences were also compared with those of the related surface protein in non-pathogenic commensal Neisseria species to investigate potential horizontal gene transfer. The N. meningitidis fadL gene was subject to only weak positive selection pressure and was less diverse than meningococcal major outer-membrane proteins. The majority of the variability in fadL was due to recombination among existing alleles from the same or related species that resulted in a discrete mosaic structure in the meningococcal population. In general, the population structuring observed based on the FadL-like membrane protein indicates that it is under intermediate immune selection. However, the emergence of a new subvariant within the hyperinvasive lineages demonstrates the phenotypic adaptability of N. meningitidis, probably in response to selective pressure.

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.

An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine against serogroup B meningococci have focused on the use of surface-exposed outer membrane proteins. Here we report the use of virus-like particles derived from the core protein of Hepatitis B Virus, HBc, to incorporate antigen domains derived from Factor H binding protein (FHbp) and the adhesin NadA. The extracellular domain of NadA was inserted into the major immunodominant region of HBc, and the C-terminal domain of FHbp at the C-terminus (CFHbp), creating a single polypeptide chain 3.7-fold larger than native HBc. Remarkably, cryoelectron microscopy revealed that the construct formed assemblies that were able to incorporate both antigens with minimal structural changes to native HBc. Electron density was weak for NadA and absent for CFHbp, partly attributable to domain flexibility. Following immunization of mice, three HBc fusions (CFHbp or NadA alone, NadA + CFHbp) were able to induce production of IgG1, IgG2a and IgG2b antibodies reactive against their respective antigens at dilutions in excess of 1:18,000. However, only HBc fusions containing NadA elicited the production of antibodies with serum bactericidal activity. It is hypothesized that this improved immune response is attributable to the adoption of a more native-like folding of crucial conformational epitopes of NadA within the chimeric VLP. This work demonstrates that HBc can incorporate insertions of large antigen domains but that maintenance of their three-dimensional structure is likely to be critical in obtaining a protective response.

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild-type and specific knock-out mutant strains.

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1-D blue native (BN) electrophoresis, 2-D BN/SDS-PAGE and 2-D diagonal electrophoresis, followed by LC/MS-MS (for 1-D gels) or MALDI-TOF (for 2-D gels) revealed at least six porin complexes in the wild-type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild-type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (approximately 800 kDa). Our results suggest that PorA and PorB could form a 'basic' template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.