Genomic characterization of invasive meningococcal X isolates from Brazil, 1992-2022.

INTRODUCTION: Invasive meningococcal disease (IMD) is a major health problem. Given the post-COVID-19 pandemic scenario with the loosening of the non-pharmacological measures to control the virus transmission and considering the observed global reduction of meningococcal vaccination coverage, an increase in IMD cases can be expected. METHODOLOGY: Using whole-genome sequencing, we characterized six Neisseria meningitidis serogroup X (MenX) isolates recovered from IMD cases in Brazil in the last 30 years. RESULTS: The predominance (66.6%, 4/6) of ST2888 presenting fHbp 160, NHBA 129, NadA 21, and PorA 19,15 was found on isolates. Two novel STs, 15458 and 15477, were described. CONCLUSION: This study describes the circulation of MenX lineage ST2888 in Brazil, previously reported only in Europe. Continuous universal surveillance is crucial to implement prompt public health measures aiming to prevent and control non-vaccine preventable serogroup X IMD cases.

Invasive Meningococcal X Disease during the COVID-19 Pandemic, Brazil.

Invasive meningococcal disease persists as a fulminant disorder worldwide. Although cases caused by Neisseria meningitidis serogroup X (MenX) occur infrequently, outbreaks have been reported in countries in Africa in recent decades. We report 2 cases of MenX invasive meningococcal disease in São Paulo, Brazil, in 2021 and 2022, during the COVID-19 pandemic.

Lung abscess due to Neisseria meningitidis serogroup X-unexpected virulence of a commensal resulting from putative serogroup B capsular switching.

To report the first case of a lung abscess caused by Neisseria meningitidis (Nm) and to genetically characterize the rare underlying capsule switching event. The strain (PT NmX) was subjected to whole genome sequencing, and a comparative gene-by-gene analysis was performed based on 1605 N. meningitidis core loci that constitute the MLST core-genome scheme (cgMLST) V1.0. All ~ 9,600 genomes available on Neisseria PubMLST (until 30th November 2019) from all serogroups were used to better identify the genome make-up of the PT NmX strain. This strain was found to be highly divergent from other NmX reported worldwide and to belong to a new sequence type (ST-14273), with the finetype X: P1.19,15-1:F5-2. Moreover, it revealed a closer genetic proximity to strains from serogroup B than to other serogroups, suggesting a genome backbone associated with serogroup B, while it presents a capsule synthesis region derived from a NmX strain. We describe a new hybrid NmB/X isolate from a noninvasive meningococcal infection, causing lung abscess. Despite capsular switching events involving serogroup X are rare, it may lead to the emergence of pathogenic potential. Studies should continue to better understand the molecular basis underlying Neisseria strains' ability to spread to body compartments other than the tissues for which their tropism is already known.

Genetic Analysis of Neisseria meningitidis Sequence Type 7 Serogroup X Originating from Serogroup A.

Neisseria meningitidis causes meningococcal disease, often resulting in fulminant meningitis, sepsis, and death. Vaccination programs have been developed to prevent infection of this pathogen, but serogroup replacement is a problem. Capsular switching has been an important survival mechanism for N. meningitidis, allowing the organism to evolve in the present vaccine era. However, related mechanisms have not been completely elucidated. Genetic analysis of capsular switching between diverse serogroups would help further our understanding of this pathogen. In this study, we analyzed the genetic characteristics of the sequence type 7 (ST-7) serogroup X strain that was predicted to arise from ST-7 serogroup A at the genomic level. By comparing the genomic structures and sequences, ST-7 serogroup X was closest to ST-7 serogroup A, whereas eight probable recombination regions, including the capsular gene locus, were identified. This indicated that serogroup X originated from serogroup A by recombination leading to capsular switching. The recombination involved approximately 8,540 bp from the end of the ctrC gene to the middle of the galE gene. There were more recombination regions and strain-specific single-nucleotide polymorphisms in serogroup X than in serogroup A genomes. However, no specific gene was found for each serogroup except those in the capsule gene locus.

Meningococci of Serogroup X Clonal Complex 181 in Refugee Camps, Italy.

Four cases of infection with serogroup X meningococci (MenX) (1 in 2015 and 3 in 2016) occurred in migrants living in refugee camps or reception centers in Italy. All MenX isolates were identified as clonal complex 181. Our report suggests that serogroup X represents an emerging health threat for persons arriving from African countries.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Neisseria meningitidis Serogroup X in Sub-Saharan Africa.

The epidemiology of meningococcal disease varies by geography and time. Whole-genome sequencing of Neisseria meningitidis serogroup X isolates from sub-Saharan Africa and Europe showed that serogroup X emergence in sub-Saharan Africa resulted from expansion of particular variants within clonal complex 181. Virulence of these isolates in experimental mouse models was high.

Functional expression of the capsule polymerase of Neisseria meningitidis serogroup X: a new perspective for vaccine development.

Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis and sepsis. A key feature in pathogenicity is the capsular polysaccharide (CPS) that prevents complement activation and thus supports bacterial survival in the host. Twelve serogroups characterized by immunologically and structurally different CPSs have been identified. Meningococcal CPSs elicit bactericidal antibodies and consequently are used for the development of vaccines. Vaccination against the epidemiologically most relevant serogroups was initially carried out with purified CPS and later followed by conjugate vaccines which consist of CPS covalently linked to a carrier protein. Of increasing importance in the African meningitis belt is NmX for which no vaccine is currently available. Here, we describe the molecular cloning, recombinant expression and purification of the capsule polymerase (CP) of NmX called CsxA. The protein expressed with N- and/or C-terminal epitope tags was soluble and could be purified to near homogeneity. With short oligosaccharide primers derived from the NmX capsular polysaccharide (CPSX), recombinant CsxA produced long polymer chains in vitro that in immunoblots were detected with NmX-specific antibodies. Moreover, the chemical identity of in vitro produced NmX polysaccharides was confirmed by NMR. Besides the demonstration that the previously identified gene csxA encodes the NmX CP CsxA, the data presented in this study pave the way for the use of the recombinant CP as a safe and economic way to generate the CPSX in vaccine developmental programs.

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines.

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.

An integrated in silico approach for the identification of novel potential drug target and chimeric vaccine against Neisseria meningitides strain 331401 serogroup X by subtractive genomics and reverse vaccinology.

Neisseria meningitidis, commonly known as the meningococcus, leads to substantial illness and death among children and young adults globally, revealing as either epidemic or sporadic meningitis and/or septicemia. In this study, we have designed a novel peptide-based chimeric vaccine candidate against the N. meningitidis strain 331,401 serogroup X. Through rigorous analysis of subtractive genomics, two essential cytoplasmic proteins, namely UPI000012E8E0(UDP-3-O-acyl-GlcNAc deacetylase) and UPI0000ECF4A9(UDP-N-acetylglucosamine acyltransferase) emerged as potential drug targets. Additionally, using reverse vaccinology, the outer membrane protein UPI0001F4D537 (Membrane fusion protein MtrC) identified by subcellular localization and recognized for its known indispensable role in bacterial survival was identified as a novel chimeric vaccine target. Following a careful comparison of MHC-I, MHC-II, T-cell, and B-cell epitopes, three epitopes derived from UPI0001F4D537 were linked with three types of linkers-GGGS, EAAAK, and the essential PADRE-for vaccine construction. This resulted in eight distinct vaccine models (V1-V8). Among them V1 model was selected as the final vaccine construct. It exhibits exceptional immunogenicity, safety, and enhanced antigenicity, with 97.7 % of its residues in the Ramachandran plot's most favored region. Subsequently, the vaccine structure was docked with the TLR4/MD2 complex and six different HLA allele receptors using the HADDOCK server. The docking resulted in the lowest HADDOCK score of 39.3 ± 9.0 for TLR/MD2. Immune stimulation showed a strong immune response, including antibodies creation and the activation of B-cells, T Cytotoxic cells, T Helper cells, Natural Killer cells, and interleukins. Furthermore, the vaccine construct was successfully expressed in the Escherichia coli system by reverse transcription, optimization, and ligation in the pET-28a (+) vector for the expression study. The current study proposes V1 construct has the potential to elicit both cellular and humoral responses, crucial for the developing an epitope-based vaccine against N. meningitidis strain 331,401 serogroup X.

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard.

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX.

Method development and validation of unbound saccharide content (serogroup A, C, Y, W, X) in novel pentavalent meningococcal polysaccharide conjugate vaccine with two different carrier proteins.

Exclusive DOC-HCl formulations were developed for free polysaccharide content estimation in Meningococcal serogroup A, C, Y, W and X from pentavalent meningococcal vaccine (A, C, Y, W, X). The DOC precipitation method reported herein stands as an alternative to the ultra-filtration method for free polysaccharide estimation. DOC content was optimized for all the serogroups at a single concentration, where as effective acid concentration was altered as per serogroup. Briefly, two DOC-HCl formulations were developed for intended purpose, one for TT conjugated serogroups Men A & Men X where as other for CRM conjugated serogroups Men C, Men Y and Men W with effective HCl concentration of 23 mM and 193 mM for precipitation of Protein-DOC complex respectively. Furthermore, an exclusive buffer/DOC-HCl formulation for estimation of Men X free polysaccharide in fill finished product was developed. Accuracy of the method was proven at 12.5 %, 25 %, 50 % and 100 % of test specification where recoveries were found in the range of 70-130 %. In case of repeatability, intra assay variation ranged from 2 % to 7 % whereas inter assay variation was noted to be 2-14 %. Specificity studied revealed no interference of assay components such as sample excipients, DOC, acids. Critical quality and stability-indicating characteristics were measured. Monovalent polysaccharide standards of Men A, C, Y, W and X were developed and assigned the unitage concentration 1.01, 1.10, 1.09, 1.08 and 1.00 mg/mL respectively. Linearity curve was optimized from 0.17 to 27 µg/mL for Men A and C whereas from 0.33 to 27 µg/mL for Men Y and W considering free polysaccharide content estimation. The study suggests that DOC-HCl method meets all the criteria for free polysaccharide estimation in multivalent vaccines with additional advantages of high throughput and sized independent separation hence can be used for quality control testing.

Validation and use of a serum bactericidal antibody assay for Neisseria meningitidis serogroup X in a seroprevalence study in Niger, West Africa.

Invasive meningococcal disease (IMD) affects approximately 1.2 million people worldwide annually. Prevention of IMD is mostly provided through vaccination; however, no licensed vaccine is currently available to protect against meningococcal serogroup X associated infection. Limited data are available on the natural immunity to Neisseria meningitidis serogroup X within the African sub-Saharan meningitis belt. The objective of the study was to provide an overview of natural immunity to serogroup X within a community in the African meningitis belt prior to the introduction of a pentavalent conjugate vaccine (NmCV-5). Prior to its introduction, a validated assay to assess vaccine efficacy was also required. This study therefore incorporated two objectives: a seroprevalence study to assess natural immunity in serum samples (n = 377) collected from Niger, West Africa in 2012, and the validation of a serogroup X serum bactericidal antibody (SBA) assay. Seroprevalence data obtained found that natural immunity to N. meningitidis serogroup X were present in 52.3% of study participants. The highest putative protective titres (≥8) to serogroup X were seen in age group 5-14 years-old (73.9%) and lowest in ages < 1 year old (0%). The SBA assay was successfully validated for selectivity/specificity, precision/reproducibility, linearity, and stability. This study demonstrated the suitability of the serogroup X SBA assay in clinical trials for future meningococcal conjugate vaccines containing serogroup X polysaccharides.

Quantitation of novel pentavalent meningococcal polysaccharide conjugate vaccine (Men A-TT, Men C-CRM, Men Y-CRM, Men W-CRM, Men X-TT) using sandwich ELISA.

Meningococcal vaccines have been developed over the years, to control outbreaks of meningitis. There are 12 immunologically distinct serogroups of which, A, B, C, W, Y and X are predominant and invasive in nature. Meningococcal vaccines can be sorted out as, polysaccharide vaccines, polysaccharide protein conjugate vaccines or protein based (independent of polysaccharides) vaccines. Stringent quality control analysis as per the regulatory guidelines is a requirement for any vaccine manufacturer for commercial release of vaccines. Quantitation becomes challenging when it comes to analysis of multivalent vaccines. We describe the quantitation of novel pentavalent meningococcal polysaccharide conjugate vaccine manufactured at Serum Institute of India Pvt Ltd (SIIPL). Briefly, sandwich ELISA assay was developed to quantitate five different serogroups (Men A-TT, Men C-CRM, Men Y-CRM, Men W-CRM, Men X-TT) in pentavalent meningococcal polysaccharide conjugate vaccine containing two different carrier proteins (TT and CRM). ELISA reported herein showed significant reproducibility and repeatability over a range of developed standard curve with acceptable coefficient of variation (<15%) for both intra and inter assay. Furthermore analysis showed that, polysaccharide conjugate (PC) contents were within the accepted range (±30%) of the stated label claim. The immunoassay was verified for its sensitivity, accuracy and precision. Based on the relevant experimental data; it is proposed that, reported sandwich ELISA is well suited for quantitation of individual polysaccharide conjugate from pentavalent formulation. Furthermore, the ELISA was explored for forced degradation and polysaccharide spiking studies. This leads to open up an area of research for sandwich ELISA as a tool to assess the integrity of vaccine as well as for lot to lot consistency monitoring.

Genomic characterization of invasive meningococcal X isolates from Brazil, 1992–2022 / Caracterización genómica de aislamientos invasivos de meningococo X de Brasil, 1992–2022

Introduction: Invasive meningococcal disease (IMD) is a major health problem. Given the post-COVID-19 pandemic scenario with the loosening of the non-pharmacological measures to control the virus transmission and considering the observed global reduction of meningococcal vaccination coverage, an increase in IMD cases can be expected. Methodology: Using whole-genome sequencing, we characterized six Neisseria meningitidis serogroup X (MenX) isolates recovered from IMD cases in Brazil in the last 30 years. Results: The predominance (66.6%, 4/6) of ST2888 presenting fHbp 160, NHBA 129, NadA 21, and PorA 19,15 was found on isolates. Two novel STs, 15458 and 15477, were described. Conclusion: This study describes the circulation of MenX lineage ST2888 in Brazil, previously reported only in Europe. Continuous universal surveillance is crucial to implement prompt public health measures aiming to prevent and control non-vaccine preventable serogroup X IMD cases.(AU)

Capsule switching of Neisseria meningitidis sequence type 7 serogroup A to serogroup X.

OBJECTIVES: The bacterial pathogen Neisseria meningitidis is able to escape the currently available capsule-based vaccines by undergoing capsule switching. In this study, we investigated whether capsule switching has occurred in a recently emerged sequence type (ST) 7 serogroup X isolate in China, for which currently no vaccine is available. METHODS: To identify capsule switching breakpoints, the capsule locus and flanking regions of the ST-7 serogroup X isolate and three endemic ST-7 serogroup A isolates were sequenced and compared. To obtain further insight into capsule switching frequency and length of DNA fragments involved, capsule switching assays were performed using genomic DNA containing combinations of antibiotic selection markers at various locations in the capsule locus and flanking regions. RESULTS: Sequence analyses showed that capsule switching has occurred and involved a 8450 bp serogroup X DNA fragment spanning the region from galE to ctrC. Capsule switching assays indicate that capsule switching occurs at a frequency of 6.3 × 10-6 per bacterium per µg of DNA and predominantly involved DNA fragments of about 8.1-9.6 kb in length. CONCLUSIONS: Our results show that capsule switching in N. meningitidis occurs at high frequency and involves recombination in the flanking regions of the capsule biosynthesis genes.

The case of a new sequence type 7 serogroup X Neisseria meningitidis infection in China: may capsular switching change serogroup profile?

Neisseria meningitidis remains the leading cause of bacterial meningitis and septicemia worldwide. We describe a case of invasive meningococcal infection caused by serogroup X N. meningitidis with a unique sequence type (ST-7) in China. The strain may have arisen from locally prevalent hypervirulent serogroup A clones by capsular switching events.