Neisseria Meningitidis Group Research Articles

NHBA antibodies elicited by 4CMenB vaccination are key for serum bactericidal activity against Neisseria gonorrhoeae

The 4CMenB (BexseroR) vaccine contains detergent-extracted outer membrane vesicles (OMVs) from a Neisseria meningitidis (Nm) group B strain NZ98/254 and three recombinant Nm protein antigens: Neisseria adhesin A (NadA), Factor H binding protein (FHbp, as the C-terminal protein in the GNA2091-FHbp fusion), and Neisserial Heparin Binding Antigen (NHBA, as the N-terminal protein in the NHBA-GNA1030 fusion). Previous work has shown that 4CMenB generates serum antibodies to Nm and Neisseria gonorrhoeae (Ng) OMV proteins and lipooligosaccharide (LOS). Mounting evidence indicates 4CMenB can partially protect against mucosal infections with Ng. The immunologic basis for Ng cross protection remains to be fully elucidated. Ten paired human sera obtained pre- and post-immunization with 4CMenB (1 month after a third vaccine dose) were used in ELISAs and in Western blots to determine IgG and IgA serum responses to OMVs from Nm strain NZ98/254 (OMVNm) and two Ng strains, 1291 and CNG20 (OMVNg), and gonococcal recombinant NHBA (rNHBANg) proteins. Post 4CMenB sera, but not pre-sera, showed strong IgG and variable IgA responses to the OMVNm but lower (2–11-fold difference in signal intensity) recognition of OMVNg. All post (not pre) 4CMenB sera showed strong IgG, but variable IgA, recognition of rNHBANg by ELISAs and Western blots. Three post 4CMenB sera at 10% (v/v) concentration had serum bactericidal activity (SBA) against Ng strains 1291 and CNG20 (~30–40% killing), not seen in paired pre-sera. These data confirmed 4CMenB-induced cross-reactive functional antibody responses to Ng. In competitive SBA assays, in which sera were pre-incubated with rNHBA, minimal SBA against Nm strain NZ98/254 was titrated away. However, most of the SBA against Ng strains 1291 and CNG20 required NHBA-specific antibodies, and the Δnhba mutants were resistant to killing by post 4CMenB sera. Removing NHBA-specific and LOS-specific OMV antibodies simultaneously decreased SBA significantly more than the sum of removing individual antibodies alone, suggesting synergy between anti-NHBA and anti-OMV antibodies. Anti- NHBANm antibodies induced by 4CMenB vaccination cross react with NHBANg and substantially contribute to the bactericidal response toward Ng induced by the vaccine.

Investigation on the carriage prevalence of Neisseria meningitidis in healthy population in Jiangsu Province in 2019 and 2020

In order to investigate carriage status and serum groups distribution of Neisseria meningitidis(Nm) among healthy population in Jiangsu Province, four counties were selected as monitoring sites by random sampling method for cross-sectional study. Throat swab specimens were collected from four monitoring sites in October to November 2019 and November to December 2020 for bacterial culture and Real-time PCR detection. Chi-square test was used to compare the positive rate of Neisseria meningitidis, and multiple logistic regression model was used to analyze the influencing factors of Neisseria meningitidis carrier rate. The results showed that among 1 512 samples, 57 strains of Nm were isolated, and the carrier rate was 3.77%. Serogroup B was the dominant group (36.84%), followed by un-known serogroups (33.33%), serogroup C was the third (17.54%), serogroup W135 and serogroup A were 7.02% and 5.26%, respectively. The carriage rate (8.60%) of 15-20 years old was significantly higher than others (1.77%-3.74%)(Pearson χ2=18.211, P<0.05). Region, age and immunization history were risk factors for Neisseria meningitidis carrier rate. In summary, the Nm carrier rate of healthy population in Jiangsu Province is relatively low, which indicates that the epidemic cases will continue to be sporadic in the future. However, the prevention and control of meningococcal epidemics on campus should be strengthened, and the monitoring of neisseria meningitidis group B should be maintained as it has become the dominant epidemic strain.

Characterization of the β-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the β-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a β-KDO linkage.

Characteristics of the tree shrew humoral immune system

Preclinical studies require an immune response similar to that of humans in a small animal model that is convenient to operate. Based on genome alignment, tree shrews are small animals considered to be more similar to primates than are rodents, and many human disease models have been established with tree shrews. However, the characteristics of the humoral immune response of tree shrews remain to be elucidated. In this study, the genetic sequence of the heavy chain constant region of tree shrew immunoglobulin (Ig) was complemented, and the results of immunoglobulin domain homology and transcriptome analysis showed that the tree shrew genome encodes only four classes of antibodies and does not encode IgD. The oldest IgM antibody has the highest homology with primates. After the complete sequence of each type of antibody was obtained, the tree shrew antibody protein was further expressed and purified by in vitro recombination, and an IgG quantitative evaluation system was established. The highly effective immuno protective effect induced by HSV-1 infection and the significant bactericidal effect induced by Neisseria meningitidis group C polysaccharide immunization showed that tree shrews exhibited immune responses more similar to humans than to mice. This may provide better predictive value for vaccine preclinical research.

Meningococcal B Vaccine and Meningococcal Carriage in Adolescents in Australia

BackgroundThe meningococcal group B vaccine 4CMenB is a new, recombinant protein-based vaccine that is licensed to protect against invasive group B meningococcal disease. However, its role in preventing transmission and, therefore, inducing population (herd) protection is uncertain.MethodsWe used cluster randomization to assign, according to school, students in years 10 to 12 (age, 15 to 18 years) in South Australia to receive 4CMenB vaccination either at baseline (intervention) or at 12 months (control). The primary outcome was oropharyngeal carriage of disease-causing Neisseria meningitidis (group A, B, C, W, X, or Y) in students in years 10 and 11, as identified by polymerase-chain-reaction assays for PorA (encoding porin protein A) and N. meningitidis genogroups. Secondary outcomes included carriage prevalence and acquisition of all N. meningitidis and individual disease-causing genogroups. Risk factors for carriage were assessed at baseline.ResultsA total of 237 schools participated. During April through June 2017, a total of 24,269 students in years 10 and 11 and 10,220 students in year 12 were enrolled. At 12 months, there was no difference in the prevalence of carriage of disease-causing N. meningitidis between the vaccination group (2.55%; 326 of 12,746) and the control group (2.52%; 291 of 11,523) (adjusted odds ratio, 1.02; 95% confidence interval [CI], 0.80 to 1.31; P=0.85). There were no significant differences in the secondary carriage outcomes. At baseline, the risk factors for carriage of disease-causing N. meningitidis included later year of schooling (adjusted odds ratio for year 12 vs. year 10, 2.75; 95% CI, 2.03 to 3.73), current upper respiratory tract infection (adjusted odds ratio, 1.35; 95% CI, 1.12 to 1.63), cigarette smoking (adjusted odds ratio, 1.91; 95% CI, 1.29 to 2.83), water-pipe smoking (adjusted odds ratio, 1.82; 95% CI, 1.30 to 2.54), attending pubs or clubs (adjusted odds ratio, 1.54; 95% CI, 1.28 to 1.86), and intimate kissing (adjusted odds ratio, 1.65; 95% CI, 1.33 to 2.05). No vaccine safety concerns were identified.ConclusionsAmong Australian adolescents, the 4CMenB vaccine had no discernible effect on the carriage of disease-causing meningococci, including group B. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT03089086.)

Measurement of surface protein antigens, PorA and PorB, in Bexsero vaccine using quantitative mass spectrometry

Bexsero is a multivalent vaccine containing outer membrane vesicles (OMV) derived from Neisseria meningitidis group B strain NZ98/254 and three recombinant meningococcal proteins, Neisserial adhesin A, Heparin binding antigen and factor H binding protein. OMV production relies on the growth of large-scale cultures of N. meningitidis under controlled conditions. Changes to environmental factors, such as temperature, pH, nutrient availability and trace elements, can impact the growth rate of the meningococcus. Furthermore outer membrane expression levels vary in response to the environmental milieu, thus any changes in environmental conditions can result in changes in OMV protein content. This makes consistent production of OMVs challenging and the ability to measure the protein content of the final product is desirable to ensure product quality. The aim of this work was to develop a mass spectrometry (MS) method for measuring the porin proteins and to evaluate this approach for assessing the batch consistency of Bexsero vaccine. Using isotope dilution MS, we measured the PorA and PorB content in 75 lots of Bexsero vaccine. PorA ranged from 4.0 to 5.95 μg/dose with an average of 4.8 μg/dose. PorB ranged from 5.4 to 8.7 μg/dose with an average of 6.5 μg/dose. This is the first description of the quantitative characterisation of adjuvanted Bexsero vaccine drug product at the final stage of the production process, once the aluminium adjuvanted vaccine has been packaged into syringes, to assess manufacturing consistency. The significance of our findings to quality control in the future is discussed.

Vaccine-preventable meningitis in French children with incorrect vaccination status from 2011 to 2013

The objective of this study was to determine the number of cases of pediatric meningitis or purpura fulminans associated with an incorrect vaccination status from 2011 to 2013 in France. A total of 48 children with vaccine-preventable meningitis or purpura fulminans, including three deaths, had an incorrect vaccination status: 26 cases were due to Neisseria meningitidis group C (54.2%), 19 to Streptococcus pneumoniae (39.6%), and three to Haemophilus influenzae type b (6.3%). The majority of patients (n=35, 72.9%) had received no injection of the vaccine concerned. Over a 3-year period, 48 cases of bacterial meningitis or purpura fulminans in children could have been avoided if the French immunization schedule had been followed.

Effect of Acceptor Chain Length and Hydrophobicity on Polymerization Kinetics of the Neisseria meningitidis Group C Polysialyltransferase.

Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in virulence make the polysialyltransferases (PST) targets for therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here, we have generated recombinant bovine nucleotide monophosphate kinase to facilitate steady state kinetic assays of the PST. We have characterized the N. meningitidis group C (NmC) PST kinetically, using substrate analogues to describe the polymerization reaction. We observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased, indicating a tighter binding of longer oligomers. In addition, we observed a biphasic relationship between kcat and chain length, which can be attributed to a switch in the mechanism of transfer of sialic acid from distributive to processive as the chain length increased above six sialic acid units. Substitution of donor substrate with the analogue CMP-9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose that this decrease in kcat is caused by a destabilization of the transition state and/or an increase affinity of the product due to presence of the fluoro substituent. The acceptor's hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned over at a faster rate than the α-2,9 polysialic acid substrates lacking the hydrophobic end. This finding suggests the presence of a secondary ligand binding site that tethers the acceptor substrate to the enzyme active site.

Role of the KDO Glycosyltransferase KpsS in the Biosynthesis of the Polysialyltransferase Acceptor for Escherichia coli K1

Many pathogenic bacteria produce polysaccharide capsules to evade immune recognition and prevent lysis by the host immune response. Neisseria meningitidis groups B and C as well as Escherichia coli serogroups K1 and K92 are coated with a polysialic acid (PSA) capsule. The PSA chain is synthesized by polysialyltransferases that makes an α‐2,8 linkage for E. coli K1 and N. meningitidis serotype B, an α‐2,9 linkage for N. meningitidis serotype C, or an alternating α‐2,8, α‐2,9 linkage for E. coli K92. These transferases cannot initiate synthesis of PSA de novo, but rather, a more complex set of machinery is required to generate an appropriate acceptor. This acceptor has been proposed to be a phosphatidylglycerol lipid anchor with a short keto‐deoxyoctulosonate (KDO) linker to one or more sialic acid residues. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized KpsS, the first enzyme in the pathway for acceptor synthesis and a membrane associated beta KDO glycosyltransferase. Much of the machinery required to produced bacterial capsules consists of transmembrane or membrane‐associated enzymes. Isolation and characterization of these enzymes in an active state has proven challenging. Here, we have purified KpsS in a soluble and active form and have begun to investigate its function. We show that KpsS can transfer a KDO residue to a fluorescent labeled phosphatidylglycerol lipid substrate. The enzyme tolerates various lengths of lipid tails on the phosphatidylglycerol substrate, including fluorescent tags that facilitate the characterization of the substrate and product. We have also shown that the enzyme can transfer modified KDO residues. Furthermore, we have isolated the product of the KpsS reaction and analyzed its structure. That KpsS is the first KDO transferase in the pathway is confirmed by the further modification of this purified product by KpsC with additional KDO units.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rise in Group W Meningococcal Carriage in University Students, United Kingdom.

MenACWY conjugate vaccination was recently introduced in the United Kingdom for adolescents and young adults to reduce disease from infection by Neisseria meningitidis group W. We conducted a cross-sectional meningococcal carriage study in first-year UK university students. Despite 71% MenACWY vaccine coverage, carriage of group W increased substantially.

Complement factor P is a ligand for the natural killer cell-activating receptor NKp46.

Innate lymphoid cells (ILCs) are involved in immune responses to microbes and various stressed cells, such as tumor cells. They include group 1 [such as natural killer (NK) cells and ILC1], group 2, and group 3 ILCs. Besides their capacity to respond to cytokines, ILCs detect their targets through a series of cell surface-activating receptors recognizing microbial and nonmicrobial ligands. The nature of some of these ligands remains unclear, limiting our understanding of ILC biology. We focused on NKp46, which is highly conserved in mammals and expressed by all mature NK cells and subsets of ILC1 and ILC3. We show here that NKp46 binds to a soluble plasma glycoprotein, the complement factor P (CFP; properdin), the only known positive regulator of the alternative complement pathway. Consistent with the selective predisposition of patients lacking CFP to lethal Neisseria meningitidis (Nm) infections, NKp46 and group 1 ILCs bearing this receptor were found to be required for mice to survive Nm infection. Moreover, the beneficial effects of CFP treatment for Nm infection were dependent on NKp46 and group 1 NKp46+ ILCs. Thus, group 1 NKp46+ ILCs interact with the complement pathway, via NKp46, revealing a cross-talk between two partners of innate immunity in the response to an invasive bacterial infection.

Analysis for protection rate and antibody levels of epidemic cerebrospinal meningitis among children aged between 3-23 months in Liuzhou, in 2012

Objective: To understand the level of bactericidal antibodies against Neisseria meningitidis and their rate of protection in children aged between 3 and 23 months, in Liuzhou, in 2012. Methods: Convenience sampling was performed in Liujiang, Rong'an and Sanjiang Counties from May through August, 2012. The inclusion criteria for 603 subjects were: Children aged between 3 and 23 months; no history of meningococcal meningitis; no vaccination against Neisseria meningitidis serogroup C; more than 30 days from the last vaccination against Neisseria meningitidis serogroup A. Demographic information and immunization history of the subjects were obtained using questionnaires. Venous blood samples (2.0 ml each) were collected and levels of Neisseria meningitides antibodies determined using a Serum Bactericidal Assay (SBA). The geometric mean titer (GMT) of serum bacterial antibodies was positive when it was greater than or equal to 1∶2 and protective when greater than 1∶8. Chi-square and Fisher's exact tests were used to compare differences in the positive and protective rates of serum antibodies of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup C, among children with different demographic characteristics. Kruskal-Wallis H test was used to compare differences in the GMT of serum antibodies of Neisseria meningitidis serogroup A and Neisseria meningitidis serogroup C, among children with different demographic characteristics. Results: Of 603 subjects, 325 (53.9%) were female and 278 (46.1%) were male; 276 (45.8%), 143 (23.7%) and 184 (30.5%) subjects were administered, respectively, no treatment, 1 dose vaccine and 2 doses vaccine. The GMT of serum antibodies against group A Neisseria meningitidis was 1∶1.11, the positive rate was 7.6% (46) and the protective rate was 2.0% (12). The GMT of antibodies in children receiving 1 vaccine dose was 1∶1.16 and the protective rate was 3.5% (5), both values higher than those in children receiving 2 vaccine doses (GMT: 1∶1.2, protective rate: 3.5% (5)). However, these differences were not statistically significant (GMT: H=0.64, P=0.728; protective rate: Fisher's exact test, P= 0.080). The GMT of antibodies in children receiving 1 and 2 doses of meningococcal polysaccharide vaccines were 1∶1.12 and 1∶2.30, respectively (≤180 d). The GMT of serum antibodies for group C meningococcal vaccine was 1∶1.18 and its positive and protective rates were 14.6% (88) and 2.2% (13), respectively. Conclusion: Children aged between 3 and 23 months are susceptible to Neisseria meningitidis groups A and C. The immune effectiveness of group A meningococcal polysaccharide vaccine is relatively poor in this age group and their antibody levels decreased rapidly.

Prevalence of meningococcal carriage in children and adolescents aged 10-19 years in Chile in 2013.

In 2011, Chile experienced an increase in the number of cases of IMD caused by Neisseria meningitidis group W. This epidemiological scenario prompted authorities to implement prevention strategies. As part of these strategies, the Institute of Public Heath of Chile conducted a cross-sectional study to determine the prevalence of pharyngeal carriage of N. meningitidis in a representative sample of healthy children and adolescents aged 10-19 years. The identification of presumptive N. meningitidis strains was performed by testing carbohydrate utilization in the National Reference Laboratory at the ISP. Association of meningococcal carriage with risk factors was analyzed by calculating the Odds Ratio. Selected variables were included in a logistic model for risk analyses. The prevalence of carriage of N. meningitidis was 6.5% (CI: 5.7-7.3%). Older age (carriers: 14.2±0.29 vs. non-carriers: 13.8±0.08 years old; p=0.009), cohabitation with children (carriers: 0.9±0.13 vs. non-carriers: 0.7±0.03; p=0.028), number of smoking cohabitants (carriers: 0.55±0.13 vs. non-carriers: 0.44±0.03) and frequent attendance to crowded social venues (carriers: 49% vs. non-carriers: 37%; p=0.008) were determined to favor carriage. Statistical modeling showed that meningococcal carriage was associated with older age (OR: 1.077, p-value: 0.002) and cohabitation with children (OR: 1.182, p-value: 0.02).

Population-Level Persistence of Immunity 2 Years After the PsA-TT Mass-Vaccination Campaign in Mali.

Background. In 2010, Africa's first preventive meningococcal mass vaccination campaign was launched using a newly developed Neisseria meningitidis group A (NmA) polysaccharide–tetanus toxoid conjugate vaccine, PsA-TT (MenAfriVac), designed specifically for the meningitis belt. Given PsA-TT's recent introduction, the duration of protection against meningococcal group A is unknown.Methods. We conducted a household-based, age-stratified seroprevalence survey in Bamako, Mali, in 2012, 2 years after the vaccination campaign targeted all 1- to 29-year-olds. Randomly selected participants who had been eligible for PsA-TT provided a blood sample and responded to a questionnaire. Sera were analyzed to assess NmA-specific serum bactericidal antibody titers using rabbit complement (rSBA) and NmA-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. The proportion of participants putatively protected and the age group- and sex-specific rSBA geometric mean titers (GMTs) and IgG geometric mean concentrations (GMCs) were determined.Results. Two years postvaccination, nearly all of the 800 participants (99.0%; 95% confidence interval [CI], 98.3%–99.7%) maintained NmA-specific rSBA titers ≥8, the accepted threshold for protection; 98.6% (95% CI, 97.8%–99.4%) had titers ≥128, and 89.5% (95% CI, 87.4%–91.6%) had titers ≥1024. The rSBA GMTs were significantly higher in females than in males aged <18 years at vaccination (P < .0001). NmA-specific IgG levels ≥2 µg/mL were found in 88.5% (95% CI, 86.3%–90.7%) of participants.Conclusions. Two years after PsA-TT introduction, a very high proportion of the population targeted for vaccination maintains high antibody titers against NmA. Assessing the duration of protection provided by PsA-TT is a priority for implementing evidence-based vaccination strategies. Representative, population-based seroprevalence studies complement clinical trials and provide this key evidence.

Modeling Long-term Vaccination Strategies With MenAfriVac in the African Meningitis Belt.

Background. The introduction of MenAfriVac in campaigns targeting people aged 1–29 years across the African meningitis belt has successfully reduced meningitis incidence and carriage due to Neisseria meningitidis group A (MenA). It is important to consider how best to sustain population protection in the long term.Methods. We created a mathematical model of MenA transmission and disease to investigate the potential impact of a range of immunization strategies. The model is age structured; includes classes of susceptible, carrier, ill, and immune people (who may be vaccinated or unvaccinated); and incorporates seasonal transmission and a stochastic forcing term that models between year variation in rates of transmission. Model parameters were primarily derived from African sources. The model can describe the typical annual incidence of meningitis in the prevaccine era, with irregular epidemics of varying size. Parameter and structural uncertainty were explored in sensitivity analyses.Results. Following MenAfriVac introduction at high uptake, the model predicts excellent short-term disease control. With no subsequent immunization, strong resurgences in disease incidence were predicted after approximately 15 years (assuming 10 years’ average vaccine protection). Routine immunization at 9 months of age resulted in lower average annual incidence than regular mass campaigns of 1- to 4-year-olds, provided coverage was above approximately 60%. The strategy with the lowest overall average annual incidence and longest time to resurgence was achieved using a combination strategy of introduction into the Expanded Programme on Immunization at 9 months, 5 years after the initial mass campaigns, with a catch-up targeting unvaccinated 1- to 4-year-olds.Conclusions. These results can be used to inform policy recommendations for long-term vaccination strategies with MenAfriVac.

Meningococcal cellulitis: An unusual presentation in the elderly

An 87-year-old Caucasian woman was referred to the medical team with a 2-day history of pain and swelling on the left side of her neck associated with odynophagia resulting in minimal oral intake. Her past medical history included ischaemic heart disease and a previous stroke which had left no residual neurological deficit. She was not diabetic and was not known to have any immunodeficiency disorders. On admission, her temperature was 39 8C, oxygen saturation of 99% on 10 litres oxygen, blood pressure of 108/58 mmHg and a respiratory rate of 24 breaths per minute. There was significant erythema and swelling over the lower left side of her face and neck with supraclavicular region involvement which was moderately tender. There was no visible rash and the remainder of the examination was unremarkable. Blood tests done on admission showed a significantly raised CRP (Table 1). Chest X-ray was normal. Blood cultures were drawn and she was given intravenous flucloxacillin 2 g four times a day based on a presumptive diagnosis of cellulitis. Her Laboratory risk indicator for necotising fascitis (LRINEC) score was calculated to be 7 [1] and she was thus referred for a surgical review to rule out the condition. The surgical team felt it was unlikely to be necrotising fasciitis but recommended a CT scan. This showed bilateral lymphadenopathy with no evidence of cervical abscess. Nasoendoscopy, performed by the Ear, nose and throat (ENT) team, was normal. The day after admission, her blood cultures showed possible growth of meningococcus and her antibiotics were changed to ceftriaxone 2 g once a day given intravenously for a total of 7 days based on microbiologist advice. Neisseria meningitidis group W135

Brazilian meningococcal C conjugate vaccine: Scaling up studies

Several outbreaks caused by Neisseria meningitidis group C have been occurred in different regions of Brazil. A conjugate vaccine for Neisseria meningitidis was produced by chemical linkage between periodate-oxidized meningococcal C polysaccharide and hydrazide-activated monomeric tetanus toxoid via a modified reductive amination conjugation method. Vaccine safety and immunogenicity tested in Phase I and II trials showed satisfactory results. Before starting Phase III trials, vaccine production was scaled up to obtain industrial lots under Good Manufacture Practices (GMP). Comparative analysis between data obtained from industrial and pilot scales of the meningococcal C conjugate bulk showed similar execution times in the scaling up production process without significant losses or alterations in the quality attributes of purified compounds. In conclusion, scale up was considered satisfactory and the Brazilian meningococcal conjugate vaccine production aiming to perform Phase III trials is feasible.

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×106g/mol to larger than 20×106g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.

Characterization of Escherichia coli K1 colominic acid-specific murine antibodies that are cross-protective against Neisseria meningitidis groups B, C, and Y

The capsular polysaccharide (PS) of Neisseria meningitidis serogroup B (NMGB) is α(2-8)-linked N-acetylneuraminic acid (Neu5Ac), which is almost identical to the O-acetylated colominic acid (CA) of Escherichia coli K1 Although E. coli K1 has long been known to elicit cross-protective antibodies against NMGB, limited information on these highly cross-reactive antibodies is available.In the present study, six new monoclonal antibodies (mAbs) specific to both E. coli K1 CA and NMGB PS were produced by immunizing Balb/c mice with E. coli K1, and their serological and molecular properties were characterized, together with 12 previously reported hybridoma mAbs.Among the bactericidal mAbs against NMGB, both HmenB5 and HmenB18, which are genetically identical though of different mouse origins, were able to kill serogroup C and Y meningococci.Based on SPR sensograms, the binding affinity of HmenB18 for PS was suggested to be associated with at least two different binding forces: the polyanionicity of Neu5Ac and an interaction with the O-acetyl groups of Neu5Ac.Molecular analysis showed that similar to most mAbs presenting a few restricted V region germline genes, the V region genes of HmenB18 were 979% and 986% identical to the closest IGHV1-1401 and IGLV15-10301 germline gene alleles, respectively, and V-D-J editing in this mAb generated an unusually long VH-CDR3 sequence (17 amino acid residues), containing one basic arginine, two hydrophobic isoleucine residues and a ‘YAMDY’ motif.Models of the mAb combining sites demonstrate that most of the mAbs exhibited a wide, shallow groove with a high overall positive charge, as seen in mAb735, which is specific for a polyanionic helical epitope. In contrast, the combining site of HmenB18 was shown to be wide but to present a relatively weak positive charge, consistent with the extensive recognition by HmenB18 of the various structural epitopes formed with the Neu5Ac residue and its O-acetylation.

Latex Agglutination Test for Early Detection of Causative Organism in Acute Bacterial Meningitis

Introduction: Acute bacterial meningitis is one of the leading causes of mortality and morbidity in children. Identification of the causative organism is crucial to its management and outcome. The objective of this study was to see the usefulness of latex agglutination test in the early diagnosis of acute bacterial meningitis. Materials and Methods: A hospital based prospective cross-sectional study was conducted at Kanti Children’s Hospital during December 2004 to August 2005. Cerebrospinal fluid from 150 consecutive clinically suspected cases of acute bacterial meningitis between the age group of 2 months to 14 years were analyzed. Bacterial culture and latex agglutination test was done on cerebrospinal fluid obtained from all 150 suspected cases of acute bacterial meningitis. Latex agglutination test was done using the BD DirectigenTM Meningitis Combo test kit (Becton, Dickinson and company, USA) for Streptococcus pneumoniae, group B Streptococcus, Escherichia coli, Neisseria meningitidis group A,C and Y/ W135, and Hemophilus influenzae type b. Data was analysed by using SPSS Version 11.5. Results: Of the 150 Cerebrospinal fluid samples analysed bacterial culture identified only 4 meningitis cases giving an isolation rate of 1.3% whereas latex agglutination test identified 29 cases giving an isolation rate of 19.3% from 150 samples. Streptococcus pneumoniae, Hemophilus influenzae type b and Group B Streptococcus were the most common causative organism. Conclusion: Latex agglutination test has a better yield, higher sensitivity, provides microbiological diagnosis earlier than the traditional cerebrospinal fluid culture and is easy to perform. DOI: http://dx.doi.org/10.3126/jnps.v33i1.7047 J Nepal Paediatr Soc. 2013;33(1):34-38