Wolbachia depletion blocks transmission of lymphatic filariasis by preventing chitinase-dependent parasite exsheathment.

Lymphatic filariasis is a vector-borne neglected tropical disease prioritized for global elimination. The filarial nematodes that cause the disease host a symbiotic bacterium, Wolbachia, which has been targeted using antibiotics, leading to cessation of parasite embryogenesis, waning of circulating larvae (microfilariae [mf]), and gradual cure of adult infection. One of the benefits of the anti-Wolbachia mode of action is that it avoids the rapid killing of mf, which can drive inflammatory adverse events. However, mf depleted of Wolbachia persist for several months in circulation, and thus patients treated with antibiotics are assumed to remain at risk for transmitting infections. Here, we show that Wolbachia-depleted mf rapidly lose the capacity to develop in the mosquito vector through a defect in exsheathment and inability to migrate through the gut wall. Transcriptomic and Western blotting analyses demonstrate that chitinase, an enzyme essential for mf exsheathment, is down-regulated in Wolbachia-depleted mf and correlates with their inability to exsheath and escape the mosquito midgut. Supplementation of in vitro cultures of Wolbachia-depleted mf with chitinase enzymes restores their ability to exsheath to a similar level to that observed in untreated mf. Our findings elucidate a mechanism of rapid transmission-blocking activity of filariasis after depletion of Wolbachia and adds to the broad range of biological processes of filarial nematodes that are dependent on Wolbachia symbiosis.

Development of Pyrazolopyrimidine Anti-Wolbachia Agents for the Treatment of Filariasis.

Anti-Wolbachia therapy has been identified as a viable treatment for combating filarial diseases. Phenotypic screening revealed a series of pyrazolopyrimidine hits with potent anti-Wolbachia activity. This paper focuses on the exploration of the SAR for this chemotype, with improvement of metabolic stability and solubility profiles using medicinal chemistry approaches. Organic synthesis has enabled functionalization of the pyrazolopyrimidine core at multiple positions, generating a library of compounds of which many analogues possess nanomolar activity against Wolbachia in vitro with improved DMPK parameters. A lead compound, 15f, was selected for in vivo pharmacokinetics (PK) profiling in mice. The combination of potent anti-Wolbachia activity in two in vitro assessments plus the exceptional oral PK profiles in mice puts this lead compound in a strong position for in vivo proof-of-concept pharmacodynamics studies and demonstrates the strong potential for further optimization and development of this series for treatment of filariasis in the future.

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis.

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Laboratory evaluation of molecular xenomonitoring using mosquito and tsetse fly excreta/feces to amplify Plasmodium, Brugia, and Trypanosoma DNA.

Background:  Results from an increasing number of studies suggest that mosquito excreta/feces (E/F) testing has considerable potential to serve as a supplement for traditional molecular xenomonitoring techniques. However, as the catalogue of possible use-cases for this methodology expands, and the list of amenable pathogens grows, a number of fundamental methods-based questions remain. Answering these questions is critical to maximizing the utility of this approach and to facilitating its successful implementation as an effective tool for molecular xenomonitoring. Methods:  Utilizing E/F produced by mosquitoes or tsetse flies experimentally exposed to Brugia malayi, Plasmodium falciparum, or Trypanosoma brucei brucei, factors such as limits of detection, throughput of testing, adaptability to use with competent- and incompetent-vector species, and effects of additional blood feedings post parasite-exposure were evaluated.  Two platforms for the detection of pathogen signal (quantitative real-time PCR and digital PCR [dPCR]) were also compared, with strengths and weaknesses examined for each.       Results:  Experimental results indicated that high throughput testing is possible when evaluating mosquito E/F for the presence of either B. malayi or P. falciparum from both competent- and incompetent-vector mosquito species.  Furthermore, following exposure to pathogen, providing mosquitoes with a second, uninfected bloodmeal did not expand the temporal window for E/F collection during which pathogen detection was possible.  However, this collection window did appear longer in E/F collected from tsetse flies following exposure to T. b. brucei.  Testing also suggested that dPCR may facilitate detection through its increased sensitivity.  Unfortunately, logistical obstacles will likely make the large-scale use of dPCR impractical for this purpose. Conclusions:  By examining many E/F testing variables, expansion of this technology to a field-ready platform has become increasingly feasible.  However, translation of this methodology from the lab to the field will first require the completion of field-based pilot studies aimed at assessing the efficacy of E/F screening.

Interleukin-4 activated macrophages mediate immunity to filarial helminth infection by sustaining CCR3-dependent eosinophilia.

Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (Mϕ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal Mϕ populations proliferated and became alternatively-activated (AAMϕ). Filarial AAMϕ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of Mϕ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMϕ in Mϕ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMϕ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMϕ expansions, sustained eosinophilia and mediated immunological resistance in Mϕ-intact SCID mice. Co-culturing Brugia with filarial AAMϕ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMϕ orchestrate eosinophil immunity to filarial tissue helminth infection.

Corrigendum: Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

This corrects the article DOI: 10.1038/srep23458.

Author Correction: Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

Identification and prioritization of novel anti-Wolbachia chemotypes from screening a 10,000-compound diversity library.

Lymphatic filariasis and onchocerciasis are two important neglected tropical diseases (NTDs) that cause severe disability. Control efforts are hindered by the lack of a safe macrofilaricidal drug. Targeting the Wolbachia bacterial endosymbionts in these parasites with doxycycline leads to a macrofilaricidal outcome, but protracted treatment regimens and contraindications restrict its widespread implementation. The Anti-Wolbachia consortium aims to develop improved anti-Wolbachia drugs to overcome these barriers. We describe the first screening of a large, diverse compound library against Wolbachia. This whole-organism screen, streamlined to reduce bottlenecks, produced a hit rate of 0.5%. Chemoinformatic analysis of the top 50 hits led to the identification of six structurally diverse chemotypes, the disclosure of which could offer interesting avenues of investigation to other researchers active in this field. An example of hit-to-lead optimization is described to further demonstrate the potential of developing these high-quality hit series as safe, efficacious, and selective anti-Wolbachia macrofilaricides.

Brugia malayi microfilariae adhere to human vascular endothelial cells in a C3-dependent manner.

Brugia malayi causes the human tropical disease, lymphatic filariasis. Microfilariae (Mf) of this nematode live in the bloodstream and are ingested by a feeding mosquito vector. Interestingly, in a remarkable co-evolutionary adaptation, Mf appearance in the peripheral blood follows a circadian periodicity and reaches a peak when the mosquito is most likely to feed. For the remaining hours, the majority of Mf sequester in the lung capillaries. This circadian phenomenon has been widely reported and is likely to maximise parasite fitness and optimise transmission potential. However, the mechanism of Mf sequestration in the lungs remains largely unresolved. In this study, we demonstrate that B. malayi Mf can, directly adhere to vascular endothelial cells under static conditions and under flow conditions, they can bind at high (but not low) flow rates. High flow rates are more likely to be experienced diurnally. Furthermore, a non-periodic nematode adheres less efficiently to endothelial cells. Strikingly C3, the central component of complement, plays a crucial role in the adherence interaction. These novel results show that microfilariae have the ability to bind to endothelial cells, which may explain their sequestration in the lungs, and this binding is increased in the presence of inflammatory mediators.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.

A superhydrophobic cone to facilitate the xenomonitoring of filarial parasites, malaria, and trypanosomes using mosquito excreta/feces.

Background: Molecular xenomonitoring (MX), the testing of insect vectors for the presence of human pathogens, has the potential to provide a non-invasive and cost-effective method for monitoring the prevalence of disease within a community. Current MX methods require the capture and processing of large numbers of mosquitoes, particularly in areas of low endemicity, increasing the time, cost and labour required. Screening the excreta/feces (E/F) released from mosquitoes, rather than whole carcasses, improves the throughput by removing the need to discriminate vector species since non-vectors release ingested pathogens in E/F. It also enables larger numbers of mosquitoes to be processed per pool. However, this new screening approach requires a method of efficiently collecting E/F. Methods: We developed a cone with a superhydrophobic surface to allow for the efficient collection of E/F. Using mosquitoes exposed to either Plasmodium falciparum, Brugia malayi or Trypanosoma brucei brucei, we tested the performance of the superhydrophobic cone alongside two other collection methods. Results: All collection methods enabled the detection of DNA from the three parasites. Using the superhydrophobic cone to deposit E/F into a small tube provided the highest number of positive samples (16 out of 18) and facilitated detection of parasite DNA in E/F from individual mosquitoes. Further tests showed that following a simple washing step, the cone can be reused multiple times, further improving its cost-effectiveness. Conclusions: Incorporating the superhydrophobic cone into mosquito traps or holding containers could provide a simple and efficient method for collecting E/F. Where this is not possible, swabbing the container or using the washing method facilitates the detection of the three parasites used in this study.

Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

Lymphatic filariasis and onchocerciasis are parasitic helminth diseases, which cause severe morbidities such as elephantiasis, skin disease and blindness, presenting a major public health burden in endemic communities. The anti-Wolbachia consortium (A·WOL: http://www.a-wol.com/) has identified a number of registered antibiotics that target the endosymbiotic bacterium, Wolbachia, delivering macrofilaricidal activity. Here we use pharmacokinetics/pharmacodynamics (PK/PD) analysis to rationally develop an anti-Wolbachia chemotherapy by linking drug exposure to pharmacological effect. We compare the pharmacokinetics and anti-Wolbachia efficacy in a murine Brugia malayi model of minocycline versus doxycycline. Doxycycline exhibits superior PK in comparison to minocycline resulting in a 3-fold greater exposure in SCID mice. Monte-Carlo simulations confirmed that a bi-daily 25-40 mg/Kg regimen is bioequivalent to a clinically effective 100-200 mg/day dose for these tetracyclines. Pharmacodynamic studies showed that minocycline depletes Wolbachia more effectively than doxycycline (99.51% vs. 90.35%) after 28 day 25 mg/Kg bid regimens with a more potent block in microfilarial production. PK/PD analysis predicts that minocycline would be expected to be 1.7 fold more effective than doxycycline in man despite lower exposure in our infection models. Our findings warrant onward clinical investigations to examine the clinical efficacy of minocycline treatment regimens against lymphatic filariasis and onchocerciasis.

Development and validation of a high-throughput anti-Wolbachia whole-cell screen: a route to macrofilaricidal drugs against onchocerciasis and lymphatic filariasis.

There is an urgent need to develop new, safe, and affordable macrofilaricidal drugs for onchocerciasis and lymphatic filariasis treatment and control. The Anti-Wolbachia Consortium (A·WOL) aims to provide a novel treatment with macrofilaricidal activity by targeting the essential bacterial symbiont Wolbachia. The consortium is currently screening a diverse range of compounds to find new chemical space to drive this drug discovery initiative and address this unmet demand. To increase the throughput and capacity of the A·WOL cell-based screen, we have developed a 384-well format assay using a high-content imaging system (Operetta) in conjunction with optimized Wolbachia growth dynamics in the C6/36 Aedes albopictus mosquito cell line. This assay uses texture analysis of cells stained with SYTO 11 as a direct measure of bacterial load. This validated assay has dramatically increased the capacity and throughput of the A·WOL compound library screening program 25-fold, enriching the number of new anti-Wolbachia hits identified for further development as potential macrofilaricides for onchocerciasis and lymphatic filariasis.

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.

Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.

Autophagy regulates Wolbachia populations across diverse symbiotic associations.

Wolbachia are widespread and abundant intracellular symbionts of arthropods and filarial nematodes. Their symbiotic relationships encompass obligate mutualism, commensalism, parasitism, and pathogenicity. A consequence of these diverse associations is that Wolbachia encounter a wide range of host cells and intracellular immune defense mechanisms of invertebrates, which they must evade to maintain their populations and spread to new hosts. Here we show that autophagy, a conserved intracellular defense mechanism and regulator of cell homeostasis, is a major immune recognition and regulatory process that determines the size of Wolbachia populations. The regulation of Wolbachia populations by autophagy occurs across all distinct symbiotic relationships and can be manipulated either chemically or genetically to modulate the Wolbachia population load. The recognition and activation of host autophagy is particularly apparent in rapidly replicating strains of Wolbachia found in somatic tissues of Drosophila and filarial nematodes. In filarial nematodes, which host a mutualistic association with Wolbachia, the use of antibiotics such as doxycycline to eliminate Wolbachia has emerged as a promising approach to their treatment and control. Here we show that the activation of host nematode autophagy reduces bacterial loads to the same magnitude as antibiotic therapy; thus we identify a bactericidal mode of action targeting Wolbachia that can be exploited for the development of chemotherapeutic agents against onchocerciasis, lymphatic filariasis, and heartworm.

Research strategies to improve snakebite treatment: challenges and progress.

Antivenom is an effective treatment of snakebite but, because of the complex interplay of fiscal, epidemiological, therapeutic efficacy and safety issues, the mortality of snakebite remains unacceptably high. Efficiently combating this high level of preventable death amongst the world's most disadvantaged communities requires the globally-coordinated action of multiple intervention programmes. This is the overall objective of the Global Snakebite Initiative. This paper describes the challenges facing the research community to develop snakebite treatments that are more efficacious, safe and affordable than current therapy.