Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high-medium-low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype.

Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a lung disease characterised by airflow-limiting inflammation and mucus production. Acute exacerbations are a major cause of COPD-related morbidity and mortality and are mostly associated with bacterial or viral infections. A vaccine targeting non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), the main bacteria associated with exacerbations, was tested in a Phase 2 trial. We assessed "ex-vivo" expression of vaccine candidate and housekeeping genes pd, pe, pilA, gapA, ompP6 of NTHi, and uspA2, parE, polA of Mcat in sputum samples of COPD patients and determined whether expression of the vaccine candidate genes pd, pe, pilA (NTHi) and uspA2 (Mcat) differed between stable and exacerbation samples. METHODS: A single-centre, prospective, observational cohort study was conducted where 123 COPD patients were seen on enrolment, followed monthly for 2 years, and reviewed after onset of acute exacerbations. We selected 69 patients with sputum samples positive for NTHi or Mcat by PCR during at least one stable and one exacerbation visit. mRNA was isolated from the sputum, and expression of NTHi and Mcat genes was analysed with RT-PCR. Statistical analyses compared mRNA concentrations between stable and exacerbation samples and in relationship to COPD severity and exacerbation frequency. RESULTS: The vaccine candidate genes were variably expressed in sputum samples, suggesting they are expressed in the lung. Absolute and relative expression of all NTHi vaccine candidate genes and Mcat uspA2 were similar between exacerbation and stable samples. Expression of pd and pilA was slightly associated with the number of exacerbations in the year before enrolment, and uspA2 with the disease severity status at enrolment. CONCLUSIONS: The NTHi-Mcat vaccine candidate genes were expressed in sputum samples, and each gene had a specific level of expression. No statistically significant differences in gene expression were detectable between stable and exacerbation samples. However, the history of COPD exacerbations was slightly associated with the expression of pd, pilA and uspA2. Trial registration NCT01360398 ( https://www. CLINICALTRIALS: gov ).

STRAIN: an R package for multi-locus sequence typing from whole genome sequencing data.

BACKGROUND: Multi-locus sequence typing (MLST) is a standard typing technique used to associate a sequence type (ST) to a bacterial isolate. When the output of whole genome sequencing (WGS) of a sample is available the ST can be assigned directly processing the read-set. Current approaches employ reads mapping (SRST2) against the MLST loci, k-mer distribution (stringMLST), selective assembly (GRAbB) or whole genome assembly (BIGSdb) followed by BLASTn sequence query. Here we present STRAIN (ST Reduced Assembly IdentificatioN), an R package that implements a hybrid strategy between assembly and mapping of the reads to assign the ST to an isolate starting from its read-sets. RESULTS: Analysis of 540 publicly accessible Illumina read sets showed STRAIN to be more accurate at correct allele assignment and new alleles identification compared to SRTS2, stringMLST and GRAbB. STRAIN assigned correctly 3666 out of 3780 alleles (capability to identify correct alleles 97%) and, when presented with samples containing new alleles, identified them in 3730 out of 3780 STs (capability to identify new alleles 98.7%) of the cases. On the same dataset the other tested tools achieved lower capability to identify correct alleles (from 28.5 to 96.9%) and lower capability to identify new alleles (from 1.1 to 97.1%). CONCLUSIONS: STRAIN is a new accurate method to assign the alleles and ST to an isolate by processing the raw reads output of WGS. STRAIN is also able to retrieve new allele sequences if present. Capability to identify correct and new STs/alleles, evaluated on a benchmark dataset, are higher than other existing methods. STRAIN is designed for single allele typing as well as MLST. Its implementation in R makes allele and ST assignment simple, direct and prompt to be integrated in wider pipeline of downstream bioinformatics analyses.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure.

One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.

Diversity of Greek meningococcal serogroup B isolates and estimated coverage of the 4CMenB meningococcal vaccine.

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.

MF59 and Pam3CSK4 boost adaptive responses to influenza subunit vaccine through an IFN type I-independent mechanism of action.

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.

Prediction by genetic MATS of 4CMenB vaccine strain coverage of invasive meningococcal serogroup B isolates circulating in Taiwan between 2003 and 2020.

Neisseria meningitidis serogroup B (NmB) strains have diverse antigens, necessitating methods for predicting meningococcal serogroup B (MenB) vaccine strain coverage. The genetic Meningococcal Antigen Typing System (gMATS), a correlate of MATS estimates, predicts strain coverage by the 4-component MenB (4CMenB) vaccine in cultivable and non-cultivable NmB isolates. In Taiwan, 134 invasive, disease-causing NmB isolates were collected in 2003-2020 (23.1%, 4.5%, 5.2%, 29.8%, and 37.3% from individuals aged ≤11 months, 12-23 months, 2-4 years, 5-29 years, and ≥30 years, respectively). NmB isolates were characterized by whole-genome sequencing and vaccine antigen genotyping, and 4CMenB strain coverage was predicted using gMATS. Analysis of phylogenetic relationships with 502 global NmB genomes showed that most isolates belonged to three global hyperinvasive clonal complexes: ST-4821 (27.6%), ST-32 (23.9%), and ST-41/44 (14.9%). Predicted strain coverage by gMATS was 62.7%, with 27.6% isolates covered, 2.2% not covered, and 66.4% unpredictable by gMATS. Age group coverage point estimates ranged from 42.9% (2-4 years) to 66.1% (≤11 months). Antigen coverage estimates and percentages predicted as covered/not covered were highly variable, with higher estimates for isolates with one or more gMATS-positive antigens than for isolates positive for one 4CMenB antigen. In conclusion, this first study on NmB strain coverage by 4CMenB in Taiwan shows 62.7% coverage by gMATS, with predictable coverage for 29.8% of isolates. These could be underestimated since the gMATS calculation does not consider synergistic mechanisms associated with simultaneous antibody binding to multiple targets elicited by multicomponent vaccines or the contributions of minor outer membrane vesicle vaccine components.IMPORTANCEMeningococcal diseases, caused by the bacterium Neisseria meningitidis (meningococcus), include meningitis and septicemia. Although rare, invasive meningococcal disease is often severe and can be fatal. Nearly all cases are caused by six meningococcal serogroups (types), including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B, but the antigens targeted by these vaccines have highly variable genetic features and expression levels, so the effectiveness of vaccination may vary depending on the strains circulating in particular countries. It is therefore important to test meningococcal serogroup B strains isolated from specific populations to estimate the percentage of bacterial strains that a vaccine can protect against (vaccine strain coverage). Meningococcal isolates were collected in Taiwan between 2003 and 2020, of which 134 were identified as serogroup B. We did further investigations on these isolates, including using a method (called gMATS) to predict vaccine strain coverage by the 4-component meningococcal serogroup B vaccine (4CMenB).

An integrative network-based approach to identify driving gene communities in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is an etiologically complex disease characterized by acute exacerbations and stable phases. We aimed to identify biological functions modulated in specific COPD conditions, using whole blood samples collected in the AERIS clinical study (NCT01360398). Considered conditions were exacerbation onset, severity of airway obstruction, and presence of respiratory pathogens in sputum samples. With an integrative multi-network gene community detection (MNGCD) approach, we analyzed expression profiles to identify communities of correlated genes. The approach combined different layers of gene interactions for each explored condition/subset of samples: gene expression similarity, protein-protein interactions, transcription factors, and microRNAs validated regulons. Heme metabolism, interferon-alpha, and interferon-gamma pathways were modulated in patients at both exacerbation and stable-state visits, but with the involvement of distinct sets of genes. An important gene community was enriched with G2M checkpoint, E2F targets, and mitotic spindle pathways during exacerbation. Targets of TAL1 regulator and hsa-let-7b - 5p microRNA were modulated with increasing severity of airway obstruction. Bacterial infections with Moraxella catarrhalis and, particularly, Haemophilus influenzae triggered a specific cellular and inflammatory response in acute exacerbations, indicating an active reaction of the host to infections. In conclusion, COPD is a complex multifactorial disease that requires in-depth investigations of its causes and features during its evolution and whole blood transcriptome profiling can contribute to capturing some relevant regulatory mechanisms associated with this disease. In this work, we explored multi-network modeling that integrated diverse layers of regulatory gene networks and enhanced our comprehension of the biological functions implicated in the COPD pathogenesis.

4CMenB journey to the 10-year anniversary and beyond.

The 4-component meningococcal serogroup B (MenB) vaccine, 4CMenB, the first broadly protective, protein-based MenB vaccine to be licensed, is now registered in more than 50 countries worldwide. Real-world evidence (RWE) from the last decade confirms its effectiveness and impact, with infant immunization programs showing vaccine effectiveness of 71-95% against invasive MenB disease and cross-protection against non-B serogroups, including a 69% decrease in serogroup W cases in 4CMenB-eligible cohorts in England. RWE from different countries also demonstrates the potential for additional moderate protection against gonorrhea in adolescents. The real-world safety profile of 4CMenB is consistent with prelicensure reports. Use of the endogenous complement human serum bactericidal antibody (enc-hSBA) assay against 110 MenB strains may enable assessment of the immunological effectiveness of multicomponent MenB vaccines in clinical trial settings. Equitable access to 4CMenB vaccination is required to better protect all age groups, including older adults, and vulnerable groups through comprehensive immunization policies.

Invasive meningococcal disease, caused by the bacterium Neisseria meningitidis(meningococcus), is rare but often devastating and can be deadly. Effective vaccines are available, including vaccines against meningococcal serogroup B disease. In 2013, the 4-component meningococcal serogroup B vaccine, 4CMenB, became the first broadly protective, protein-based vaccine against serogroup B to be licensed, with the second (bivalent vaccine, MenB-FHbp) licensed the following year. 4CMenB is now registered in more than 50 countries, in the majority, for infants and all age groups. In the US, it is approved for individuals aged 10­25 years. Evidence from immunization programs in the last decade, comparing vaccinated and unvaccinated individuals and the same population before and after vaccination, confirms the effectiveness and positive impact of 4CMenB against serogroup B disease. This also demonstrates that 4CMenB can provide protection against invasive diseases caused by other meningococcal serogroups. Furthermore, N. meningitidis is closely related to the bacterium that causes gonorrhea, N. gonorrhoeae, and emerging real-world evidence suggests that 4CMenB provides additional moderate protection against gonococcal disease. The safety of 4CMenB when given to large numbers of infants, children, adolescents, and adults is consistent with the 4CMenB safety profile reported before licensure.For the future, it would be beneficial to address differences among national guidelines for the recommended administration of 4CMenB, particularly where there is supportive epidemiological evidence but no equitable access to vaccination. New assays for assessing the potential effectiveness of meningococcal serogroup B vaccines in clinical trials are also required because serogroup B strains circulating in the population are extremely diverse across different countries.

Classification of Neisseria meningitidis genomes with a bag-of-words approach and machine learning.

Whole genome sequencing of bacteria is important to enable strain classification. Using entire genomes as an input to machine learning (ML) models would allow rapid classification of strains while using information from multiple genetic elements. We developed a "bag-of-words" approach to encode, using SentencePiece or k-mer tokenization, entire bacterial genomes and analyze these with ML. Initial model selection identified SentencePiece with 8,000 and 32,000 words as the best approach for genome tokenization. We then classified in Neisseria meningitidis genomes the capsule B group genotype with 99.6% accuracy and the multifactor invasive phenotype with 90.2% accuracy, in an independent test set. Subsequently, in silico knockouts of 2,808 genes confirmed that the ML model predictions aligned with our current understanding of the underlying biology. To our knowledge, this is the first ML method using entire bacterial genomes to classify strains and identify genes considered relevant by the classifier.

Genetic characterization and estimated 4CMenB vaccine strain coverage of 284 Neisseria meningitidis isolates causing invasive meningococcal disease in Argentina in 2010-2014.

Meningococcal (Neisseria meningitidis) serogroup B (MenB) strain antigens are diverse and a limited number of strains can be evaluated using the human serum bactericidal antibody (hSBA) assay. The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict the likelihood of coverage for large numbers of isolates by the 4CMenB vaccine, which includes antigens Neisseria adhesin A (NadA), Neisserial Heparin-Binding Antigen (NHBA), factor H-binding protein (fHbp), and Porin A (PorA). In this study, we characterized by whole-genome analyses 284 invasive MenB isolates collected from 2010 to 2014 by the Argentinian National Laboratories Network (52-61 isolates per year). Strain coverage was estimated by gMATS on all isolates and by hSBA assay on 74 randomly selected isolates, representative of the whole panel. The four most common clonal complexes (CCs), accounting for 81.3% of isolates, were CC-865 (75 isolates, 26.4%), CC-32 (59, 20.8%), CC-35 (59, 20.8%), and CC-41/44 (38, 13.4%). Vaccine antigen genotyping showed diversity. The most prevalent variants/peptides were fHbp variant 2, NHBA peptides 24, 21, and 2, and PorA variable region 2 profiles 16-36 and 14. The nadA gene was present in 66 (23.2%) isolates. Estimated strain coverage by hSBA assay showed 78.4% of isolates were killed by pooled adolescent sera, and 51.4% and 64.9% (based on two different thresholds) were killed by pooled infant sera. Estimated coverage by gMATS (61.3%; prediction interval: 55.5%, 66.7%) was consistent with the infant hSBA assay results. Continued genomic surveillance is needed to evaluate the persistence of major MenB CCs in Argentina.

The most common clinical manifestations of invasive meningococcal disease include meningitis and septicemia, which can be deadly, and many survivors suffer long-term serious after-effects. Most cases of invasive meningococcal disease are caused by six meningococcal serogroups (types), including serogroup B. Although vaccines are available against meningococcal serogroup B infection, these vaccines target antigens that are highly diverse. Consequently, the effectiveness of vaccination may vary from country to country because the meningococcal serogroup B strains circulating in particular regions carry different forms of the target vaccine antigens. This means it is important to test serogroup B strains isolated from specific populations to estimate the percentage of strains that a vaccine is likely to be effective against (known as 'vaccine strain coverage'). The genetic Meningococcal Antigen Typing System (gMATS) was developed to predict strain coverage by the four-component meningococcal serogroup B vaccine, 4CMenB, against large numbers of serogroup B strains. In this study, we analyzed 284 invasive meningococcal serogroup B isolates collected between 2010 and 2014 in Argentina. Genetic analyses showed that the vaccine antigens of the isolates were diverse and some genetic characteristics had not been found in isolates from other countries. However, vaccine strain coverage estimated by gMATS was consistent with that reported in other parts of the world and with strain coverage results obtained for a subset via another method, the human serum bactericidal antibody (hSBA) assay. These results highlight the need for continued monitoring of circulating bacterial strains to assess the estimated strain coverage of meningococcal serogroup B vaccines.

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.

Qualitative and quantitative assessment of meningococcal antigens to evaluate the potential strain coverage of protein-based vaccines.

A unique multicomponent vaccine against serogroup B meningococci incorporates the novel genome-derived proteins fHbp, NHBA, and NadA that may vary in sequence and level of expression. Measuring the effectiveness of such vaccines, using the accepted correlate of protection against invasive meningococcal disease, could require performing the serum bactericidal assay (SBA) against many diverse strains for each geographic region. This approach is impractical, especially for infants, where serum volumes are very limited. To address this, we developed the meningococcal antigen typing system (MATS) by combining a unique vaccine antigen-specific ELISA, which detects qualitative and quantitative differences in antigens, with PorA genotyping information. The ELISA correlates with killing of strains by SBA and measures both immunologic cross-reactivity and quantity of the antigens NHBA, NadA, and fHbp. We found that strains exceeding a threshold value in the ELISA for any of the three vaccine antigens had ≥80% probability of being killed by immune serum in the SBA. Strains positive for two or more antigens had a 96% probability of being killed. Inclusion of multiple different antigens in the vaccine improves breadth of coverage and prevents loss of coverage if one antigen mutates or is lost. The finding that a simple and high-throughput assay correlates with bactericidal activity is a milestone in meningococcal vaccine development. This assay allows typing of large panels of strains and prediction of coverage of protein-based meningococcal vaccines. Similar assays may be used for protein-based vaccines against other bacteria.

Use of expanded Neisseria meningitidis serogroup B panels with the serum bactericidal antibody assay for the evaluation of meningococcal B vaccine effectiveness.

INTRODUCTION: Neisseria meningitidis serogroup B (NmB) antigens are inherently diverse with variable expression among strains. Prediction of meningococcal B (MenB) vaccine effectiveness therefore requires an assay suitable for use against large panels of epidemiologically representative disease-causing NmB strains. Traditional serum bactericidal antibody assay using exogenous human complement (hSBA) is limited to the quantification of MenB vaccine immunogenicity on a small number of indicator strains. AREAS COVERED: Additional and complementary methods for assessing strain coverage developed previously include the Meningococcal Antigen Typing System (MATS), Meningococcal Antigen Surface Expression (MEASURE) assay, and genotyping approaches, but these do not estimate vaccine effectiveness. We provide a narrative review of these methods, highlighting a more recent approach involving the hSBA assay in conjunction with expanded NmB strain panels: hSBA assay using endogenous complement in each vaccinated person's serum (enc-hSBA) against a 110-strain NmB panel and the traditional hSBA assay against 14 (4 + 10) NmB strains. EXPERT OPINION: The enc-hSBA is a highly standardized, robust method that can be used in clinical trials to measure the immunological effectiveness of MenB vaccines under conditions that mimic real-world settings as closely as possible, through the use of endogenous complement and a diverse, epidemiologically representative panel of NmB strains.

Meningococcal disease refers to illnesses caused by the bacterium Neisseria meningitidis (meningococcus), including infections of the brain lining and spinal cord (meningitis) and bloodstream (septicemia). It is rare but often severe and can be deadly. Invasive meningococcal disease can be prevented through vaccination. Nearly all cases are caused by six serogroups (types) of meningococci, including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B but, because of the uncommonness of the disease, standard clinical trials could not be performed to prove these vaccines are effective. Instead, an indirect measure, called the 'hSBA assay' (serum bactericidal antibody assay using human complement), is used to measure the ability of vaccines to provide protection against specific N. meningitidis strains that have antigens (substances that cause the immune system to react) sharing characteristics with components of the vaccines. However, meningococcal serogroup B strains are diverse in the genetic composition and expression of vaccine antigens. Hence, a large number of N. meningitidis serogroup B strains would have to be tested to make sure that the vaccine is effective against these strains. This is not feasible using the traditional hSBA assay, which requires a human complement (a protein system, which is part of the immune system) that has not come from the vaccinated person and is difficult and time-consuming to source. Recently, an alternative hSBA assay was developed that uses the complement present in each vaccinated person's blood (endogenous complement) and which overcomes these challenges. By allowing testing against a broad panel of N. meningitidis serogroup B strains, this new assay may enable a more accurate estimation of the effectiveness of vaccines against serogroup B meningococci.

OpcA and PorB are novel bactericidal antigens of the 4CMenB vaccine in mice and humans.

The ability of Neisseria meningitidis Outer Membrane Vesicles (OMV) to induce protective responses in humans is well established and mainly attributed to Porin A (PorA). However, the contribution of additional protein antigens to protection remains to be elucidated. In this study we dissected the immunogenicity of antigens originating from the OMV component of the 4CMenB vaccine in mice and humans. We collected functional data on a panel of strains for which bactericidal responses to 4CMenB in infants was attributable to the OMV component and evaluated the role of 30 OMV-specific protein antigens in cross-coverage. By using tailor-made protein microarrays, the immunosignature of OMV antigens was determined. Three of these proteins, OpcA, NspA, and PorB, triggered mouse antibodies that were bactericidal against several N. meningitidis strains. Finally, by genetic deletion and/or serum depletion studies, we demonstrated the ability of OpcA and PorB to induce functional immune responses in infant sera after vaccination. In conclusion, while confirming the role of PorA in eliciting protective immunity, we identified two OMV antigens playing a key role in protection of infants vaccinated with the 4CMenB vaccine against different N. meningitidis serogroup B strains.

Analysis of the regulated transcriptome of Neisseria meningitidis in human blood using a tiling array.

Neisseria meningitidis is the major cause of septicemia and meningococcal meningitis. During the course of infection, the bacterium must adapt to different host environments as a crucial factor for survival and dissemination; in particular, one of the crucial factors in N. meningitidis pathogenesis is the ability to grow and survive in human blood. We recently showed that N. meningitidis alters the expression of 30% of the open reading frames (ORFs) of the genome during incubation in human whole blood and suggested the presence of fine regulation at the gene expression level in order to control this step of pathogenesis. In this work, we used a customized tiling oligonucleotide microarray to define the changes in the whole transcriptional profile of N. meningitidis in a time course experiment of ex vivo bacteremia by incubating bacteria in human whole blood and then recovering RNA at different time points. The application of a newly developed bioinformatic tool to the tiling array data set allowed the identification of new transcripts--small intergenic RNAs, cis-encoded antisense RNAs, mRNAs with extended 5' and 3' untranslated regions (UTRs), and operons--differentially expressed in human blood. Here, we report a panel of expressed small RNAs, some of which can potentially regulate genes involved in bacterial metabolism, and we show, for the first time in N. meningitidis, extensive antisense transcription activity. This analysis suggests the presence of a circuit of regulatory RNA elements used by N. meningitidis to adapt to proliferate in human blood that is worthy of further investigation.

A novel Hfq-dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis.

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.

A novel epigenetic regulator associated with the hypervirulent Neisseria meningitidis clonal complex 41/44.

Neisseria meningitidis is a major cause of septicemia and meningitis. The hypervirulent clonal complex 41/44 (cc41/44) has emerged as the predominant cause of serogroup B meningococcal disease, having been responsible for recent outbreaks and epidemics worldwide. However, the meningococcal factors that enable transition from asymptomatic carriage to rapidly progressing disease are poorly understood. Here we describe a novel phase-variable DNA methyltransferase, ModD, which was identified in the genome sequence of a New Zealand epidemic isolate. Investigation of the distribution of modD in the wider meningococcal population, by PCR and sequence analysis of genetically diverse N. meningitidis strains, revealed the presence of modD in 20/27 strains in cc41/44, but in only 2/47 strains from other clonal complexes, indicating a significant association of modD with cc41/44 (Fisher's exact P value=3×10(-10)). The modD gene contains 5'-ACCGA-3' repeats that mediate phase variation, leading to reversible on/off switching of modD expression. Microarray analysis of modD-on/off variants revealed that ModD regulates expression of multiple genes involved in colonization, infection, and protection against host defenses, with increased catalase expression in the modD-on variant conferring increased resistance to oxidative stress. The modulation of gene expression via the ModD phase-variable regulon (phasevarion), and its significant association with the cc41/44, suggest a role in the fitness and/or pathogenesis of strains belonging to the cc41/44.