Vaccine potential and diversity of the putative cell binding factor (CBF, NMB0345/NEIS1825) protein of Neisseria meningitidis

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a ?nmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on ?nmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testin

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

The susceptibility of mast cells for rhinovirus infection: implications for asthma exacerbations

Mast cells (MCs) are classically involved in the pathogenesis of allergic asthma, however MCs also have a key role in innate immunity with an emerging role in viral immunity. During allergic asthma MCs localise in greater numbers to the bronchial epithelium which is the principal site of human rhinovirus (HRV) infection. HRVs are a major viral trigger of asthma exacerbations via mechanisms that are not completely understood. MCs are susceptible to HRV infection but their role in antiHRV responses is unknown. HRV infection of the bronchial epithelium triggers the release of the epithelial derived cytokine IL-33, which induces Th2 cytokine release from target cells with MCs being the major target of IL-33 in allergic asthma. I hypothesised that HRV infection induces MC anti-viral responses and also modulates IL-33-dependent Th2 responses in MCs.The LAD2 human MC line and/or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV (control infection) with or without IFNβ, IFN-γ, IFN-λ or IL-33. Twenty-four hours following HRV infection, anti-viral and Th2 immune responses were assessed by RT-qPCR, MSD, ELISA and flow cytometry. Viral replication and release were determined by RT-qPCR and TCID50 assay respectively.HRV infection induced the expression of IFN-β and IFN-λ and the induction of IFN stimulated genes (ISGs). Despite this MCs were permissive for HRV replication and the release of infectious HRV particles. This was confirmed in CBMCs. To determine the contribution of endogenous anti-viral responses, CBMCs were treated with a type I IFN receptor blocking antibody. Treatment with the blocking antibody failed to significantly increase HRV replication and release suggesting endogenous type I IFN responses were insufficient to protect MCs against HRV infection. Therefore, in order to enhance anti-viral responses, MCs were treated with exogenous IFN-β, IFNγ or IFN-λ. The induction of ISGs was enhanced by IFN-β and IFN-γ but not by IFN-λ. In addition, IFN-β treatment significantly suppressed viral replication and the release of infectious virus particles. To investigate the impact of HRV on IL-33-mediated Th2 responses, MCs were treated with IL-33 during HRV infection. IL-33 treatment induced a concentration-dependent increase in the release of IL-5 and IL13 but this was not modulated by HRV. However, IL-33 treatment increased ICAM1 expression, a receptor for HRV entry, and enhanced HRV-mediated induction of IFNβ and ISGs. This resulted in a significant increase in HRV replication but prevented significant release of infectious HRV particles.These findings show for the first time that MCs mount anti-viral responses to HRV infection and that HRV-induced IFN-β production is enhanced by IL-33 treatment. In severe asthma, which is associated with impaired bronchial epithelial IFN responses and an increase in the localisation of MCs to the bronchial epithelium, MCs may aggravate HRV-induced exacerbations. However, IL-33 released from the epithelium may protect MCs against productive HRV infection. These findings may have important implications in HRV-induced asthmas exacerbations and the impact of novel asthma therapies particularly anti-IL-33

Mast cells are permissive for rhinovirus replication: potential implications for asthma exacerbations

BACKGROUND: Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations, with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity.OBJECTIVE: In view of the emerging role of MCs in innate immunity and increased localisation to the asthmatic bronchial epithelium, we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection.METHODS: The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-? or IFN-?. After 24 h, innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay.RESULTS: HRV infection of LAD2 MCs induced expression of IFN-?, IFN-? and IFN-stimulated genes. However, LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralisation of the type I IFN receptor had minimal effects on viral shedding suggesting that endogenous type I IFN signalling offered limited protection against HRV. However, augmentation of these responses by exogenous IFN-?, but not IFN-?, protected MCs against HRV infection.CONCLUSION AND CLINICAL RELEVANCE: MCs are permissive for the replication and release of HRV which is prevented by exogenous IFN-? treatment. Taken together these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbation

IL-33 Induces an Antiviral Signature in Mast Cells but Enhances Their Permissiveness for Human Rhinovirus Infection

Mast cells (MCs) are classically associated with allergic asthma but their role in antiviral immunity is unclear. Human rhinoviruses (HRVs) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of HRV infection is the airway epithelium and MCs localise to this site with increasing asthma severity. The asthma susceptibility gene, IL-33, encodes an epithelial-derived cytokine released following HRV infection but its impact on MC antiviral responses has yet to be determined. In this study we investigated the global response of LAD2 MCs to IL-33 stimulation using RNA sequencing and identified genes involved in antiviral immunity. In spite of this, IL-33 treatment increased permissiveness of MCs to HRV16 infection which, from the RNA-Seq data, we attributed to upregulation of ICAM1. Flow cytometric analysis confirmed an IL-33-dependent increase in ICAM1 surface expression as well as LDLR, the receptors used by major and minor group HRVs for cellular entry. Neutralisation of ICAM1 reduced the IL-33-dependent enhancement in HRV16 replication and release in both LAD2 MCs and cord blood derived MCs. These findings demonstrate that although IL-33 induces an antiviral signature in MCs, it also upregulates the receptors for HRV entry to enhance infection. This highlights the potential for a gene-environment interaction involving IL33 and HRV in MCs to contribute to virus-induced asthma exacerbations

IL-33 induces an antiviral signature in mast cells but enhances their permissiveness for human rhinovirus infection

Mast cells (MCs) are classically associated with allergic asthma but their role in antiviral immunity is unclear. Human rhinoviruses (HRVs) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of HRV infection is the airway epithelium and MCs localise to this site with increasing asthma severity. The asthma susceptibility gene, IL-33, encodes an epithelial-derived cytokine released following HRV infection but its impact on MC antiviral responses has yet to be determined. In this study we investigated the global response of LAD2 MCs to IL-33 stimulation using RNA sequencing and identified genes involved in antiviral immunity. In spite of this, IL-33 treatment increased permissiveness of MCs to HRV16 infection which, from the RNA-Seq data, we attributed to upregulation of ICAM1. Flow cytometric analysis confirmed an IL-33-dependent increase in ICAM1 surface expression as well as LDLR, the receptors used by major and minor group HRVs for cellular entry. Neutralisation of ICAM1 reduced the IL-33-dependent enhancement in HRV16 replication and release in both LAD2 MCs and cord blood derived MCs. These findings demonstrate that although IL-33 induces an antiviral signature in MCs, it also upregulates the receptors for HRV entry to enhance infection. This highlights the potential for a gene-environment interaction involving IL33 and HRV in MCs to contribute to virus-induced asthma exacerbations

Serum bactericidal activity of murine anti-rCBF sera.

<p>Serum bactericidal activity of murine anti-rCBF sera.</p

ELISA reactivity of antisera raised to different rCBF formulations against pure rCBF protein, outer membranes (OM) and whole meningococcal cells.

<p>Antisera from individual animals immunized with rCBF in various adjuvant and delivery formulations and from animals immunised with OM, were reacted in ELISA against <b>A) purified rCBF protein, B) MC58 OM and C) whole MC58 meningococcal cells</b>. The columns represent the geometric mean reciprocal ELISA titres (n = 4 or 5 animals per group) and the error bars represent the 95% confidence limits. No significant reactivity with rCBF, OM or whole cells in ELISA was observed with sera from sham-immunized animals or with normal mouse serum (absorbance values λ₄₅₀nm <0.1 for serum dilutions of 1/10). The * denotes that the responses were statistically higher than the corresponding sham-immunized animals (p<0.05), using an independent t-Test to compare differences between the mean values.</p

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of <i>Neisseria meningitidis</i> - Fig 1

<p><b>A. Amino acid sequence alignment of NMB0345 (NEIS1825, Cell Binding Factor, CBF) from <i>N</i>. <i>meningitidis</i> MC58, <i>N</i>.<i>gonorrhoeae</i> FA1090 and the <i>Campylobacter jejuni</i> PEB3/CBF2 protein. *</b> denotes identical amino acids between all three proteins. Yellow-shaded amino acid sequences denote the putative signal leader peptides. The blue-shaded amino acid sequence denotes the presence of a putative conserved PPIC-type peptidyl prolyl <i>cis/trans</i> isomerase domain (interval 133–259) belonging to the Rotamase_2 superfamily. <b>B) Putative structure of meningococcal CBF based on <i>C</i>.<i>jejuni</i> CBF</b>. The structure of meningococcal CBF was putatively modelled in SWISS-model, using as a template the crystallized structure of 3rfw.1.A protein (PEB4), which is the most closely related structure to <i>C</i>. <i>jejuni</i> PEB3/CBF2 that we have available in Protein Data Bank.</p