Protective efficacy of a recombinant plague vaccine when co-administered with another sub-unit or live attenuated vaccine.

Vaccines against bioterrorism agents offer the prospect of providing high levels of protection against airborne pathogens. However, the diversity of the bioterrorism threat means that it may be necessary to use several vaccines simultaneously. In this study we have investigated whether there are changes to the protective immune response to a recombinant sub-unit plague vaccine when it is co-administered with other sub-unit or live attenuated vaccines. Our results indicate that the co-administration of these vaccines did not influence the protection afforded by the plague vaccine. However, the co-administration of the plague sub-unit vaccine with a live vaccine resulted in markedly increased levels of IgG2a subclass antibodies, and markedly reduced levels of IgG1 subclass antibodies, to the plague sub-unit vaccine. This finding might have implications when considering the co-administration of other vaccine combinations

Characterization of the Ca2+-gated and voltage-dependent k+-channel slo-1 of nematodes and its interaction with emodepside

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class

Repurposing of approved drugs from the human pharmacopoeia to target Wolbachia endosymbionts of onchocerciasis and lymphatic filariasis

AbstractLymphatic filariasis and onchocerciasis are debilitating diseases caused by parasitic filarial nematodes infecting around 150 million people throughout the tropics with more than 1.5 billion at risk. As with other neglected tropical diseases, classical drug-discovery and development is lacking and a 50year programme of macrofilaricidal discovery failed to deliver a drug which can be used as a public health tool. Recently, antibiotic targeting of filarial Wolbachia, an essential bacterial symbiont, has provided a novel drug treatment for filariasis with macrofilaricidal activity, although the current gold-standard, doxycycline, is unsuitable for use in mass drug administration (MDA). The anti-Wolbachia (A·WOL) Consortium aims to identify novel anti-Wolbachia drugs, compounds or combinations that are suitable for use in MDA. Development of a Wolbachia cell-based assay has enabled the screening of the approved human drug-pharmacopoeia (∼2600 drugs) for a potential repurposing. This screening strategy has revealed that approved drugs from various classes show significant bacterial load reduction equal to or superior to the gold-standard doxycycline, with 69 orally available hits from different drug categories being identified. Based on our defined hit criteria, 15 compounds were then selectively screened in a Litomosoides sigmodontis mouse model, 4 of which were active. These came from the tetracycline, fluoroquinolone and rifamycin classes. This strategy of repurposing approved drugs is a promising development in the goal of finding a novel treatment against filariasis and could also be a strategy applicable for other neglected tropical diseases

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 × 10(-5)M) was the most effective anti-mycobacterial agent; clofazimine (1.25 × 10(-5)M and 3.13 × 10(-6)M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 × 10(-5)M) was filaricidal, but minocycline was more effective and at a lower concentration (5 × 10(-5)M and 1.25 × 10(-5)M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode

Pilot trial and process evaluation of a multilevel smoking prevention intervention in further education settings

Background: Preventing smoking uptake among young people is a public health priority. Further education (FE) settings provide access to the majority of 16- to 18-year-olds, but few evaluations of smoking prevention interventions have been reported in this context to date. Objectives: To evaluate the feasibility and acceptability of implementing and trialling a new multilevel smoking prevention intervention in FE settings. Design: Pilot cluster randomised controlled trial and process evaluation. Setting: Six UK FE institutions. Participants: FE students aged 16–18 years. Intervention: ‘The Filter FE’ intervention. Staff working on Action on Smoking and Health Wales’ ‘The Filter’ youth project applied existing staff training, social media and youth work resources in three intervention settings, compared with three control sites with usual practice. The intervention aimed to prevent smoking uptake by restricting the sale of tobacco to under-18s in local shops, implementing tobacco-free campus policies, training FE staff to deliver smoke-free messages, publicising The Filter youth project’s online advice and support services, and providing educational youth work activities. Main outcome measures: (1) The primary outcome assessed was the feasibility and acceptability of delivering and trialling the intervention. (2) Qualitative process data were analysed to explore student, staff and intervention team experiences of implementing and trialling the intervention. (3) Primary, secondary and intermediate (process) outcomes and economic evaluation methods were piloted. Data sources: New students at participating FE settings were surveyed in September 2014 and followed up in September 2015. Qualitative process data were collected via interviews with FE college managers (n = 5) and the intervention team (n = 6); focus groups with students (n = 11) and staff (n = 5); and observations of intervention settings. Other data sources were semistructured observations of intervention delivery, intervention team records, ‘mystery shopper’ audits of local shops and college policy documents. Results: The intervention was not delivered as planned at any of the three intervention settings, with no implementation of some community- and college-level components, and low fidelity of the social media component across sites. Staff training reached 28 staff and youth work activities were attended by 190 students across the three sites (< 10% of all eligible staff and students), with low levels of acceptability reported. Implementation was limited by various factors, such as uncertainty about the value of smoking prevention activities in FE colleges, intervention management weaknesses and high turnover of intervention staff. It was feasible to recruit, randomise and retain FE settings. Prevalence of weekly smoking at baseline was 20.6% and was 17.2% at follow-up, with low levels of missing data for all pilot outcomes. Limitations: Only 17% of eligible students participated in baseline and follow-up surveys; the representativeness of student and staff focus groups is uncertain. Conclusions: In this study, FE settings were not a supportive environment for smoking prevention activities because of their non-interventionist institutional cultures promoting personal responsibility. Weaknesses in intervention management and staff turnover also limited implementation. Managers accept randomisation but methodological work is required to improve student recruitment and retention rates if trials are to be conducted in FE settings. Trial registration: Current Controlled Trials ISRCTN19563136. Funding: This project was funded by the National Institute for Health Research (NIHR) Public Health Research programme and will be published in full in Public Health Research; Vol. 5, No. 8. See the NIHR Journals Library website for further project information. It was also funded by the Big Lottery Fund

Whence river blindness? The domestication of mammals and host-parasite co-evolution in the nematode genus Onchocerca

The genus Onchocerca includes 34 described species and represents one of the largest genera of the filarial nematodes within the family Onchocercidae. Representative members of this genus are mainly parasites of ungulates, with some exceptions such as Onchocerca lupi and Onchocerca volvulus, infecting carnivores and/or humans. For a long time, the evolutionary relationships amongst onchocercids remained poorly studied, as the systematics of this genus was impaired by the high morphological variability of species included in the taxon. Although some molecular phylogenies were developed, these studies were mainly focused on bovine Onchocerca spp. and O. volvulus, including assessments of Wolbachia endosymbionts. In the present study, we analysed 13 Onchocerca spp. from a larger host spectrum using a panel of seven different genes. Analysis of the coxI marker supports its usefulness for the identification of species within the genus. The evolutionary history of the genus has been herein revised by multi-gene phylogenies, presenting three strongly supported clades of Onchocerca spp. Analyses of co-evolutionary scenarios between Onchocerca and their vertebrate hosts underline the effect of domestication on Onchocerca speciation. Our study indicates that a host switch event occurred between Bovidae, Canidae and humans. Cophylogenetic analyses between Onchocerca and the endosymbiotic bacterium Wolbachia indicate the strongest co-evolutionary pattern ever registered within the filarial nematodes. Finally, this dataset indicates that the clade composed by O. lupi, Onchocerca gutturosa, Onchocerca lienalis, Onchocerca ochengi and O. volvulus derived from recent speciation

"Keeping it on your radar"- assessing the barriers and facilitators to a timely diagnosis of type 1 diabetes in childhood: a qualitative study from the early detection of type 1 diabetes in youth study

Aims The aim of this study was to explore from the perspectives of key stakeholders involved in the pathway to diagnosis, the barriers and facilitators to a timely diagnosis of type 1 diabetes in childhood. Methods Qualitative interviews and free‐text analyses were undertaken in 21 parents with a child diagnosed with type 1 diabetes, 60 parents without a child diagnosed with type 1 diabetes, 9 primary healthcare professionals, 9 teachers and 3 community diabetes liaison nurses. Data were analysed thematically and 30% double coded. Results Two key themes were identified, namely the importance of widespread awareness and knowledge and seeking healthcare professional help. Parents with a child diagnosed with type 1 diabetes described seeking opinions from a number of individuals prior to seeking health professional help. Healthcare professionals recognized the rarity of the condition and the need for it to be kept on their “radar”, to ensure they considered it when examining an unwell child. The process of obtaining a primary healthcare appointment was identified as potentially playing a crucial role in the diagnostic process. However, most parents with a child diagnosed with type 1 diabetes described receiving an appointment on the day they sought it. Conclusions Knowledge and awareness of type 1 diabetes in childhood remain limited in the general population and misconceptions persist relating to how children present with this serious condition. An effective community‐based intervention to raise awareness amongst key stakeholders is required to ensure children receive a timely diagnosis once symptomatic

Development of emodepside as a possible adulticidal treatment for human onchocerciasis-The fruit of a successful industrial-academic collaboration

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer–DNDi partnership is an outstanding example of “One World Health,” in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area

Pilot trial and process evaluation of a multi-level smoking prevention intervention in further education settings

Background: Preventing smoking uptake among young people is a public health priority. Further education (FE) settings provide access to the majority of 16- to 18-year-olds, but few evaluations of smoking prevention interventions have been reported in this context to date. Objectives: To evaluate the feasibility and acceptability of implementing and trialling a new multilevel smoking prevention intervention in FE settings. Design: Pilot cluster randomised controlled trial and process evaluation. Setting: Six UK FE institutions. Participants: FE students aged 16–18 years. Intervention: ‘The Filter FE’ intervention. Staff working on Action on Smoking and Health Wales’ ‘The Filter’ youth project applied existing staff training, social media and youth work resources in three intervention settings, compared with three control sites with usual practice. The intervention aimed to prevent smoking uptake by restricting the sale of tobacco to under-18s in local shops, implementing tobacco-free campus policies, training FE staff to deliver smoke-free messages, publicising The Filter youth project’s online advice and support services, and providing educational youth work activities. Main outcome measures: (1) The primary outcome assessed was the feasibility and acceptability of delivering and trialling the intervention. (2) Qualitative process data were analysed to explore student, staff and intervention team experiences of implementing and trialling the intervention. (3) Primary, secondary and intermediate (process) outcomes and economic evaluation methods were piloted. Data sources: New students at participating FE settings were surveyed in September 2014 and followed up in September 2015. Qualitative process data were collected via interviews with FE college managers (n = 5) and the intervention team (n = 6); focus groups with students (n = 11) and staff (n = 5); and observations of intervention settings. Other data sources were semistructured observations of intervention delivery, intervention team records, ‘mystery shopper’ audits of local shops and college policy documents. Results: The intervention was not delivered as planned at any of the three intervention settings, with no implementation of some community- and college-level components, and low fidelity of the social media component across sites. Staff training reached 28 staff and youth work activities were attended by 190 students across the three sites (< 10% of all eligible staff and students), with low levels of acceptability reported. Implementation was limited by various factors, such as uncertainty about the value of smoking prevention activities in FE colleges, intervention management weaknesses and high turnover of intervention staff. It was feasible to recruit, randomise and retain FE settings. Prevalence of weekly smoking at baseline was 20.6% and was 17.2% at follow-up, with low levels of missing data for all pilot outcomes