Aberrant stromal tissue factor localisation and mycolactone-driven vascular dysfunction, exacerbated by IL-1ß, are linked to fibrin formation in Buruli ulcer lesions.

Buruli ulcer (BU) is a neglected tropical disease caused by subcutaneous infection with Mycobacterium ulcerans and its exotoxin mycolactone. BU displays coagulative necrosis and widespread fibrin deposition in affected skin tissues. Despite this, the role of the vasculature in BU pathogenesis remains almost completely unexplored. We hypothesise that fibrin-driven ischemia can be an 'indirect' route to mycolactone-dependent tissue necrosis by a mechanism involving vascular dysfunction. Here, we tracked >900 vessels within contiguous tissue sections from eight BU patient biopsies. Our aim was to evaluate their vascular and coagulation biomarker phenotype and explore potential links to fibrin deposition. We also integrated this with our understanding of mycolactone's mechanism of action at Sec61 and its impact on proteins involved in maintaining normal vascular function. Our findings showed that endothelial cell dysfunction is common in skin tissue adjacent to necrotic regions. There was little evidence of primary haemostasis, perhaps due to mycolactone-dependent depletion of endothelial von Willebrand factor. Instead, fibrin staining appeared to be linked to the extrinsic pathway activator, tissue factor (TF). There was significantly greater than expected fibrin staining around vessels that had TF staining within the stroma, and this correlated with the distance it extended from the vessel basement membrane. TF-induced fibrin deposition in these locations would require plasma proteins outside of vessels, therefore we investigated whether mycolactone could increase vascular permeability in vitro. This was indeed the case, and leakage was further exacerbated by IL-1ß. Mycolactone caused the loss of endothelial adherens and tight junctions by the depletion of VE-cadherin, TIE-1, TIE-2 and JAM-C; all Sec61-dependent proteins. Taken together, our findings suggest that both vascular and lymphatic vessels in BU lesions become "leaky" during infection, due to the unique action of mycolactone, allowing TF-containing structures and plasma proteins into skin tissue, ultimately leading to local coagulopathy and tissue ischemia.

The malaria blood stage antigen PfCyRPA formulated with the TLR-4 agonist adjuvant GLA-SE elicits parasite growth inhibitory antibodies in experimental animals.

BACKGROUND: Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. In contrast to several previously clinically tested merozoite vaccine candidate antigens, PfCyRPA is not polymorphic, making it a promising candidate antigen for blood stage vaccine development. METHODS: Mice and rabbits were immunized with vaccine formulations of recombinantly expressed PfCyRPA adjuvanted either with the glucopyranosyl lipid A (GLA) containing adjuvants GLA-LSQ, GLA-SE, GLA-Alum or with Nanoalum. ELISA and indirect immunofluorescence assays (IFA) were used to analyse elicited IgG titers and the P. falciparum growth inhibitory activity was determined with a standardized in vitro [3H]-hypoxanthine incorporation assay. RESULTS: In the mouse experiments, the GLA adjuvanted formulations were superior to the Nanoalum formulation with respect to antibody titer development, IFA sero-conversion rates and in vitro parasite growth-inhibitory activity. In rabbits, the highest titers of parasite growth inhibitory antibodies were obtained with the GLA-SE formulation. Comparable mean ELISA IgG endpoint titers were reached in rabbits after three immunizations with GLA-SE adjuvanted PfCyRPA doses of 5, 25 and 100 µg, but with 100 µg of antigen, only two immunizations were required to reach this titer. CONCLUSION: PfCyRPA formulated with the human-compatible adjuvant GLA-SE represents an attractive vaccine candidate for early clinical testing in a controlled P. falciparum blood stage challenge trial.

An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.

Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain-specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.

Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

Key Contributions by the Swiss Tropical and Public Health Institute Towards New and Better Drugs for Tropical Diseases.

Thanks to its expertise in clinical research, epidemiology, infectious diseases, microbiology, parasitology, public health, translational research and tropical medicine, coupled with deeply rooted partnerships with institutions in low- and middle-income countries (LMICs), the Swiss Tropical and Public Health Institute (Swiss TPH) has been a key contributor in many drug research and development consortia involving academia, pharma and product development partnerships. Our know-how of the maintenance of parasites and their life-cycles in the laboratory, plus our strong ties to research centres and disease control programme managers in LMICs with access to field sites and laboratories, have enabled systems for drug efficacy testing in vitro and in vivo, clinical research, and modelling to support the experimental approaches. Thus, Swiss TPH has made fundamental contributions towards the development of new drugs - and the better use of old drugs - for neglected tropical diseases and infectious diseases of poverty, such as Buruli ulcer, Chagas disease, food-borne trematodiasis (e.g. clonorchiasis, fascioliasis and opisthorchiasis), human African trypanosomiasis, leishmaniasis, malaria, schistosomiasis, soil-transmitted helminthiasis and tuberculosis. In this article, we show case the success stories of molecules to which Swiss TPH has made a substantial contribution regarding their use as anti-infective compounds with the ultimate aim to improve people's health and well-being.

Efficacy of an Acid-Oxidizing Solution against Mycobacterium ulcerans.

For the treatment of chronic wounds, acid-oxidizing solutions (AOSs) with broad-spectrum microbicidal activity without disturbing granulation tissue formation have been developed. We found AOSs to efficiently kill Mycobacterium ulcerans, the causative agent of Buruli ulcer, which is able to survive harsh decontamination treatments. Topical AOS treatment of Buruli ulcer lesions may support the recommended antibiotic therapy (oral rifampin and clarithromycin), prevent contamination of the environment by the mycobacteria, and control secondary infections, which are a prevalent wound management problem in resource-poor settings where Buruli ulcer is endemic.

Toward a Single-Dose Cure for Buruli Ulcer.

A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

Development of Dengue Virus Serotype-Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera.

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients.

wIRA: hyperthermia as a treatment option for intracellular bacteria, with special focus on Chlamydiae and Mycobacteria.

The emergence of antibiotic-resistant bacteria in the last century is alarming and calls for alternative, nonchemical treatment strategies. Thermal medicine uses heat for the treatment of infectious diseases but its use in facultative and obligate intracellular bacteria remains poorly studied. In this review, we summarize previous research on reducing the infectious burden of Mycobacterium ulcerans and Chlamydia trachomatis by using water-filtered infrared A-radiation (wIRA), a special form of heat radiation with high tissue penetration and low thermal load on the skin surface. Mycobacterium ulcerans is a thermosensitive bacterium causing chronic necrotizing skin disease. Therefore, previous data on wIRA-induced improvement of wound healing and reduction of wound infections is summarized first. Then, pathogenesis and treatment of infections with M. ulcerans causing Buruli ulcer and of those with C. trachomatis infecting the ocular conjunctiva and resulting in blinding trachoma are discussed. Both bacteria cause neglected tropical diseases and have similar geographical distributions. Results of previous in vitro and in vivo studies using wIRA on M. ulcerans and C. trachomatis infections are presented. Finally, technical aspects of using wIRA in patients are critically reviewed and open questions driving future research are highlighted. In conclusion, wIRA is a promising tool for reducing infectious burden due to intracellular bacteria such as M. ulcerans and C. trachomatis.

Thermal field formation during wIRA-hyperthermia: temperature measurements in skin and subcutis of piglets as a basis for thermotherapy of superficial tumors and local skin infections caused by thermosensitive microbial pathogens.

Purpose: The temporal and spatial formation of the temperature field and its changes during/upon water-filtered infrared-A (wIRA)-irradiation in porcine skin and subcutis were investigated in vivo in order to get a detailed physical basis for thermotherapy of superficial tumors and infections caused by thermosensitive microbial pathogens (e.g., Mycobacterium ulcerans causing Buruli ulcer). Methods: Local wIRA-hyperthermia was performed in 11 anesthetized piglets using 85.0 mW cm-2, 103.2 mW cm-2 and 126.5 mW cm-2, respectively. Invasive temperature measurements were carried out simultaneously in 1-min intervals using eight fiber-optical probes at different tissue depths between 2 and 20 mm, and by an IR thermometer at the skin surface. Results: Tissue temperature distribution depended on incident irradiance, exposure time, tissue depths and individual 'physiologies' of the animals. Temperature maxima were found at depths between 4 and 7 mm, exceeding skin surface temperatures by about 1-2 K. Tissue temperatures above 37 °C, necessary to eradicate M. ulcerans at depths <20 mm, were reached reliably. Conclusions: wIRA-hyperthermia may be considered as a novel therapeutic option for treatment of local skin infections caused by thermosensitive pathogens (e.g., in Buruli ulcer). To ensure temperatures required for heat treatment of superficial tumors deeper than 4 mm, the incident irradiance needed can be controlled either by (a) invasive temperature measurements or (b) control of skin surface temperature and considering possible temperature increases up to 1-2 K in underlying tissue.

Mycobacterium ulcerans DNA in Bandicoot Excreta in Buruli Ulcer-Endemic Area, Northern Queensland, Australia.

To identify potential reservoirs/vectors of Mycobacterium ulcerans in northern Queensland, Australia, we analyzed environmental samples collected from the Daintree River catchment area, to which Buruli ulcer is endemic, and adjacent coastal lowlands by species-specific PCR. We detected M. ulcerans DNA in soil, mosquitoes, and excreta of bandicoots, which are small terrestrial marsupials.

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions.

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2 ng/ml and as early as 8 hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Evaluating decontamination protocols for the isolation of Mycobacterium ulcerans from swabs.

BACKGROUND: Mycobacterium ulcerans (M. ulcerans) is the causative agent of Buruli Ulcer (BU) disease. In order to inhibit the growth of the microbial contaminants during culture of M. ulcerans, it is necessary to decontaminate BU samples with effective chemical agents. This study aimed at investigating some selected chemicals as potential decontamination agents for the isolation of M. ulcerans from swabs. RESULTS: Povidone iodine at 0.5 and 1% exhibited the lowest contamination and recovery rate for microbial contaminants and M. ulcerans. The most effective decontamination method was the protocol using 2% cetylpyridinium chloride/4% sodium chloride (recovery rate = 53%, contamination rate = 14%). The observed difference between the recovery rate of 2% CPC/4% NaC and the other protocols was however not statistically significant (p = 0.76). CONCLUSIONS: Two percent (2%) cetylpyridinium chloride/4% sodium chloride can be conveniently used as an alternative decontamination method for the isolation of M. ulcerans from swabs.

Exudate collection using wound sponges-An easy, non-invasive and reliable method to explore protease activities in ulcers.

Proteases are important for wound healing, but in excessive amounts or left uncontrolled, they may cause healing impairment or other severe wound complications. Point-of-care testing for protease activities in wounds may be useful for monitoring the effectiveness of treatment, and for early identification of wounds that potentially fail to heal. Here we describe an easy, noninvasive method to collect wound fluid for evaluating the protease milieu of wounds. Wound fluids were collected using sterile sponges applied between wound surface and normal wound dressing. Wound fluid could be easily squeezed or centrifuged out of the sponges and was tested for gelatinase (MMP-2 and MMP-9) activities by gel zymography. In addition, we measured polymorphonuclear granulocyte elastase levels by ELISA. Both gelatinases were remarkably stable in sponge derived fluids, as no significant loss was observed even when samples were stored for 3 days at room temperature. Protease levels were highly diverse amongst patients and, in some cases, showed substantial variations in the course of the treatment. The here described wound sponge approach represents a patient-friendly and reliable method to collect wound fluid for evaluating wound healing relevant biomarkers, such as matrix metalloproteinases.

Local Heat Application for the Treatment of Buruli Ulcer: Results of a Phase II Open Label Single Center Non Comparative Clinical Trial.

BACKGROUND: Buruli ulcer (BU) is a necrotizing skin disease most prevalent among West African children. The causative organism, Mycobacterium ulcerans, is sensitive to temperatures above 37°C. We investigated the safety and efficacy of a local heat application device based on phase change material. METHODS: In a phase II open label single center noncomparative clinical trial (ISRCTN 72102977) under GCP standards in Cameroon, laboratory confirmed BU patients received up to 8 weeks of heat treatment. We assessed efficacy based on the endpoints 'absence of clinical BU specific features' or 'wound closure' within 6 months ("primary cure"), and 'absence of clinical recurrence within 24 month' ("definite cure"). RESULTS: Of 53 patients 51 (96%) had ulcerative disease. 62% were classified as World Health Organization category II, 19% each as category I and III. The average lesion size was 45 cm(2). Within 6 months after completion of heat treatment 92.4% (49 of 53, 95% confidence interval [CI], 81.8% to 98.0%) achieved cure of their primary lesion. At 24 months follow-up 83.7% (41 of 49, 95% CI, 70.3% to 92.7%) of patients with primary cure remained free of recurrence. Heat treatment was well tolerated; adverse effects were occasional mild local skin reactions. CONCLUSIONS: Local thermotherapy is a highly effective, simple, cheap and safe treatment for M. ulcerans disease. It has in particular potential as home-based remedy for BU suspicious lesions at community level where laboratory confirmation is not available. CLINICAL TRIALS REGISTRATION: ISRCT 72102977.

Comparative Study of Activities of a Diverse Set of Antimycobacterial Agents against Mycobacterium tuberculosis and Mycobacterium ulcerans.

A library of compounds covering a broad chemical space was selected from a tuberculosis drug development program and was screened in a whole-cell assay against Mycobacterium ulcerans, the causative agent of the necrotizing skin disease Buruli ulcer. While a number of potent antitubercular agents were only weakly active or inactive against M. ulcerans, five compounds showed high activity (90% inhibitory concentration [IC90], ≤1 µM), making screening of focused antitubercular libraries a good starting point for lead generation against M. ulcerans.

Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization.

BACKGROUND: Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need. RESULTS: Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples. CONCLUSION: The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies.

Generation of Plasmodium falciparum parasite-inhibitory antibodies by immunization with recombinantly-expressed CyRPA.

BACKGROUND: The pathogenesis of malaria is primarily associated with blood-stage infection and there is strong evidence that antibodies specific for parasite blood-stage antigens can control parasitaemia. This provides a strong rationale for incorporation of asexual blood-stage antigen components into an effective multivalent malaria subunit vaccine. On the basis of available genome-wide transcriptomic and proteomic data, previously uncharacterized Plasmodium falciparum open reading frames were screened for new blood stage vaccine candidates. This has led to the identification of the cysteine-rich protective antigen (PfCyRPA), which forms together with PfRH5 and PfRipr a multiprotein complex that is crucial for erythrocyte invasion. METHODS: Glycosylated and non-glycosylated variants of recombinant PfCyRPA were expressed and produced as secreted protein in mammalian cells. Adjuvanted formulations of purified PfCyRPA were tested to assess whether they can effectively elicit parasite inhibitory antibodies, and to investigate whether or not the glycosylation status affects antibody binding. For this purpose, two sets of PfCyRPA-specific mouse monoclonal antibodies (mAbs) have been raised and evaluated for functional activity. RESULTS: Generated PfCyRPA-specific mAbs, irrespective of the immunogen's glycosylation status, showed substantial parasite in vitro growth-inhibitory activity due to inhibition of erythrocyte invasion by merozoites. Furthermore, passive immunization experiments in P. falciparum infected NOD-scid IL2Rγ (null) mice engrafted with human erythrocytes demonstrated potent in vivo growth-inhibitory activity of generated mAbs. CONCLUSIONS: Recombinantly expressed PfCyRPA tested as adjuvanted vaccine formulations in mice elicited antibodies that significantly inhibit P. falciparum asexual blood stage parasite growth both in vitro and in vivo. These findings render PfCyRPA a promising blood-stage candidate antigen for inclusion into a multicomponent malaria subunit vaccine.

Understanding pneumococcal serotype 1 biology through population genomic analysis.

BACKGROUND: Pneumococcus kills over one million children annually and over 90 % of these deaths occur in low-income countries especially in Sub-Saharan Africa (SSA) where HIV exacerbates the disease burden. In SSA, serotype 1 pneumococci particularly the endemic ST217 clone, causes majority of the pneumococcal disease burden. To understand the evolution of the virulent ST217 clone, we analysed ST217 whole genomes from isolates sampled from African and Asian countries. METHODS: We analysed 226 whole genome sequences from the ST217 lineage sampled from 9 African and 4 Asian countries. We constructed a whole genome alignment and used it for phylogenetic and coalescent analyses. We also screened the genomes to determine presence of antibiotic resistance conferring genes. RESULTS: Population structure analysis grouped the ST217 isolates into five sequence clusters (SCs), which were highly associated with different geographical regions and showed limited intracontinental and intercontinental spread. The SCs showed lower than expected genomic sequence, which suggested strong purifying selection and small population sizes caused by bottlenecks. Recombination rates varied between the SCs but were lower than in other successful clones such as PMEN1. African isolates showed higher prevalence of antibiotic resistance genes than Asian isolates. Interestingly, certain West African isolates harbored a defective chloramphenicol and tetracycline resistance-conferring element (Tn5253) with a deletion in the loci encoding the chloramphenicol resistance gene (cat pC194), which caused lower chloramphenicol than tetracycline resistance. Furthermore, certain genes that promote colonisation were absent in the isolates, which may contribute to serotype 1's rarity in carriage and consequently its lower recombination rates. CONCLUSIONS: The high phylogeographic diversity of the ST217 clone shows that this clone has been in circulation globally for a long time, which allowed its diversification and adaptation in different geographical regions. Such geographic adaptation reflects local variations in selection pressures in different locales. Further studies will be required to fully understand the biological mechanisms which makes the ST217 clone highly invasive but unable to successfully colonise the human nasopharynx for long durations which results in lower recombination rates.

Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X.

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.