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.

Eosinophil-Mediated Immune Control of Adult Filarial Nematode Infection Can Proceed in the Absence of IL-4 Receptor Signaling.

Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα-/-) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα-/- mice. By treating IL-4Rα-/- mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα-/- mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα-/-/IL-5-/- double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα-/- mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.

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.

Onchocerca ochengi male worms implanted in SCID mice and Gerbil: Relationship between microfilaridermia status of cows, nodular worm viability and fertility and worm survival in the rodents.

BACKGROUND: Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. METHODS: Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. RESULTS: Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf-. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf- cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf- cows, respectively. Female worms from nodules of mf- cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. CONCLUSION: Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents.

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.

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.

Blood flukes exploit Peyer's Patch lymphoid tissue to facilitate transmission from the mammalian host.

Schistosomes are blood-dwelling parasitic helminths which produce eggs in order to facilitate transmission. Intestinal schistosomes lay eggs in the mesenteries, however, it is unclear how their eggs escape the vasculature to exit the host. Using a murine model of infection, we reveal that Schistosoma mansoni exploits Peyer's Patches (PP) gut lymphoid tissue as a preferential route of egress for their eggs. Egg deposition is favoured within PP as a result of their more abundant vasculature. Moreover, the presence of eggs causes significant vascular remodeling leading to an expanded venule network. Egg deposition results in a decrease in stromal integrity and lymphoid cellularity, including secretory IgA producing lymphocytes, and the focal recruitment of macrophages. In mice lacking PP, egg excretion is significantly impaired, leading to greater numbers of ova being entrapped in tissues and consequently, exacerbated morbidity. Thus, we demonstrate how schistosomes directly facilitate transmission from the host by targeting lymphoid tissue. For the host, PP-dependency of egg egress represents a trade-off, as limiting potentially life-threatening morbidity is balanced by loss of PP structure and perturbed PP IgA production.

The national distribution of lymphatic filariasis cases in Malawi using patient mapping and geostatistical modelling.

BACKGROUND: In 2020 the World Health Organization (WHO) declared that Malawi had successfully eliminated lymphatic filariasis (LF) as a public health problem. Understanding clinical case distributions at a national and sub-national level is important, so essential care packages can be provided to individuals living with LF symptoms. This study aimed to develop a national database and map of LF clinical cases across Malawi using geostatistical modelling approaches, programme-identified clinical cases, antigenaemia prevalence and climate information. METHODOLOGY: LF clinical cases identified through programme house-to-house surveys across 90 sub-district administrative boundaries (Traditional Authority (TA)) and antigenaemia prevalence from 57 sampled villages in Malawi were used in a two-step geostatistical modelling process to predict LF clinical cases across all TAs of the country. First, we modelled antigenaemia prevalence in relation to climate covariates to predict nationwide antigenaemia prevalence. Second, we modelled clinical cases for unmapped TAs based on our antigenaemia prevalence spatial estimates. PRINCIPLE FINDINGS: The models estimated 20,938 (95% CrI 18,091 to 24,071) clinical cases in unmapped TAs (70.3%) in addition to the 8,856 (29.7%), programme-identified cases in mapped TAs. In total, the overall national number of LF clinical cases was estimated to be 29,794 (95% CrI 26,957 to 32,927). The antigenaemia prevalence and clinical case mapping and modelling found the highest burden of disease in Chikwawa and Nsanje districts in the Southern Region and Karonga district in the Northern Region of the country. CONCLUSIONS: The models presented in this study have facilitated the development of the first national LF clinical case database and map in Malawi, the first endemic country in sub-Saharan Africa. It highlights the value of using existing LF antigenaemia prevalence and clinical case data together with modelling approaches to produce estimates that may be used for the WHO dossier requirements, to help target limited resources and implement long-term health strategies.

A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Trial of AWZ1066S, an Anti-Wolbachia Candidate Macrofilaricide.

AWZ1066S has been developed as a potential treatment for the neglected tropical diseases lymphatic filariasis and onchocerciasis. AWZ1066S targets the Wolbachia bacterial endosymbiont present in the causative nematode parasites. This phase 1, first-in-human study aimed to assess the safety and pharmacokinetics of AWZ1066S in healthy human participants. In a randomized double-blind, placebo-controlled, single ascending dose study, healthy adults received a single oral dose of AWZ1066S (or placebo) and were followed up for 10 days. The planned single doses of AWZ1066S ranged from 100 to 1600 mg, and each dose was administered to a cohort of 8 participants (6 AWZ1066S and 2 placebo). In total 30 people participated, 18 (60%) female, median age 30.0 years (minimum 20, maximum 61). The cohorts administered 100, 200, 300, and 400 mg of AWZ1066S progressed unremarkably. After single 700-mg doses all 4 participants developed symptoms of acute gastritis and transient increases in liver enzymes. The severity of these adverse events ranged from mild to severe, with 1 participant needing hospital admission. Pharmacokinetic analysis indicated that AWZ1066S is rapidly absorbed with predictable pharmacokinetics. In conclusion, safety concerns prevented this study from reaching the human exposures needed for AWZ1066S to be clinically effective against lymphatic filariasis and onchocerciasis.

Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Sequential Infection with Influenza A Virus Followed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads to More Severe Disease and Encephalitis in a Mouse Model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection ('twinfection') is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.

Multiple helminth infection of the skin causes lymphocyte hypo-responsiveness mediated by Th2 conditioning of dermal myeloid cells.

Infection of the mammalian host by schistosome larvae occurs via the skin, although nothing is known about the development of immune responses to multiple exposures of schistosome larvae, and/or their excretory/secretory (E/S) products. Here, we show that multiple (4x) exposures, prior to the onset of egg laying by adult worms, modulate the skin immune response and induce CD4(+) cell hypo-responsiveness in the draining lymph node, and even modulate the formation of hepatic egg-induced granulomas. Compared to mice exposed to a single infection (1x), dermal cells from multiply infected mice (4x), were less able to support lymph node cell proliferation. Analysis of dermal cells showed that the most abundant in 4x mice were eosinophils (F4/80(+)MHC-II(-)), but they did not impact the ability of antigen presenting cells (APC) to support lymphocyte proliferation to parasite antigen in vitro. However, two other cell populations from the dermal site of infection appear to have a critical role. The first comprises arginase-1(+), Ym-1(+) alternatively activated macrophage-like cells, and the second are functionally compromised MHC-II(hi) cells. Through the administration of exogenous IL-12 to multiply infected mice, we show that these suppressive myeloid cell phenotypes form as a consequence of events in the skin, most notably an enrichment of IL-4 and IL-13, likely resulting from an influx of RELMα-expressing eosinophils. We further illustrate that the development of these suppressive dermal cells is dependent upon IL-4Rα signalling. The development of immune hypo-responsiveness to schistosome larvae and their effect on the subsequent response to the immunopathogenic egg is important in appreciating how immune responses to helminth infections are modulated by repeated exposure to the infective early stages of development.

Mental health conditions in people affected by filarial lymphoedema in Malawi: prevalence, associated risk factors and the impact of an enhanced self-care intervention.

BACKGROUND: This study aimed to determine the key mental health indicators affecting people affected by lymphatic filariasis (LF) lymphoedema by assessing the prevalence of depressive symptoms and quality of life (QOL), identifying associated sociodemographic and clinical risk factors, and evaluating the impact of an enhanced self-care intervention for lymphoedema management. METHODS: A prospective cohort study of adults with filarial lymphoedema from two regions of Malawi was conducted over six months in 2021. Depressive symptoms and QOL were assessed using Patient Health Questionnaire (PHQ-9) and LF Specific QOL Questionnaire, respectively, at baseline (pre-intervention), 3- and 6-months (postintervention). Beta regression analysis identified risk factors, and assessed the impact of the intervention. RESULTS: Three hundred eleven affected individuals were surveyed with 23% (95% CI 18%-29%) reporting mild/moderate depressive symptoms and 31% (95% CI 26%-37%) reporting moderately low/low QOL. Higher depressive symptom scores were associated with high frequency of acute filarial attack episodes. Individuals with higher depressive symptoms (Adjusted Odds Ratios (AOR) 0.93, 95% CI 0.93-0.93) and lower QOL (AOR 0.98, 0.98-0.98) showed greatest improvement in mental health indicators over 3-months but was not sustained to the same level at 6-months. CONCLUSIONS: Sustained morbidity management and psychological support is recommended for affected persons to ensure long-term positive mental health and clinical outcomes. CONTEXTE: Cette étude vise à déterminer les principaux indicateurs de santé mentale affectant les personnes atteintes de lymphœdème dû à la filariose lymphatique (FL) en évaluant la prévalence des symptômes dépressifs et la qualité de vie (QV), en identifiant les facteurs de risque sociodémographiques et cliniques associés, et en évaluant l'impact d'une intervention améliorée d'autosoins pour la gestion du lymphœdème. MÉTHODES: Une étude de cohorte prospective d'adultes atteints de lymphoedème filaire dans deux régions du Malawi a été menée pendant six mois en 2021. Les symptômes dépressifs et la qualité de vie ont été évalués à l'aide du questionnaire sur la santé des patients (PHQ-9) et du questionnaire sur la qualité de vie spécifique au lymphœdème, respectivement, au début de l'étude (avant l'intervention), et à 3 puis 6 mois après l'intervention. Une analyse de régression beta a permis d'identifier les facteurs de risque et d'évaluer l'impact de l'intervention. RÉSULTATS: Trois cent onze personnes affectées ont été interrogées, dont 23% (95% CI 18%-29%) ont déclaré des symptômes dépressifs légers/modérés et 31% (95% CI 26%-37%) ont déclaré une qualité de vie modérément faible/faible. Des scores élevés de symptômes dépressifs ont été associés à une fréquence élevée d'épisodes de crises filariennes aiguës. Les personnes présentant des symptômes dépressifs plus élevés (rapport de cotes ajusté (RCA) 0.93, IC à 95 % 0.93-0.93) et une qualité de vie plus faible (RCA 0.98, 0.98-0.98) ont montré la plus grande amélioration des indicateurs de santé mentale au cours des trois mois, mais cette amélioration ne s'est pas maintenue au même niveau au cours des six mois suivants. CONCLUSION: Gestion de la morbidité et soutien psychologique sont des éléments clés pour garantir une santé mentale et des résultats cliniques satisfaisants de personnes atteintes sur le long terme. ANTECEDENTES: Este estudio tuvo como objetivo determinar los indicadores clave de salud mental que afectan a las personas afectadas por linfedema por filariasis linfática (FL) mediante la evaluación de la prevalencia de síntomas depresivos y calidad de vida (CdV), la identificación de factores de riesgo sociodemográficos y clínicos asociados, y la evaluación del impacto de una intervención de autocuidado mejorada para el manejo del linfedema. MÉTODOS: Se realizó un estudio prospectivo de cohortes de adultos con linfedema filarial de dos regiones de Malawi durante seis meses en 2021. Los síntomas depresivos y la calidad de vida se evaluaron mediante el Cuestionario de Salud del Paciente (PHQ-9) y el Cuestionario de Calidad de Vida específico para el LF Cuestionario, respectivamente, al inicio (preintervención) y a los 3 y 6 meses (posintervención). El análisis de regresión beta identificó los factores de riesgo y evaluó el impacto de la intervención. RESULTADOS: Se encuestó a 311 afectados, de los cuales el 23% (IC 95%, 18%-29%) presentaba síntomas depresivos leves/moderados y el 31% (IC 95%, 26%-37%) una CdV moderadamente baja/baja CdV. Las puntuaciones más altas de síntomas depresivos se asociaron con una alta frecuencia de episodios de ataques agudos de filarias. Los individuos con mayores síntomas depresivos (Odds Ratios Ajustados [ORA] 0.93; IC 95%: 0.93-0.93) y menor CdV (ORA 0.98; 0.98-0.98) mostraron la mayor mejoría en los indicadores de salud mental a los 3 meses, pero no se mantuvo al mismo nivel a los 6 meses. CONCLUSIONES: Se recomienda el manejo sostenido de la morbilidad y el apoyo psicológico a las personas afectadas para garantizar resultados clínicos y de salud mental positivos a largo plazo.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi.

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.

NKp46+ natural killer cells develop an activated/memory-like phenotype and contribute to innate immunity against experimental filarial infection.

Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rß2+ ILC1, ST2+ ILC2 and NKp46+ natural killer (NK) innate lymphoid cell population expansions during Brugia malayi experimental peritoneal filarial infections using either immunocompetent or immunodeficient mice. In immunocompetent BALB/c animals, NKp46+ NK cells rapidly expanded representing over 90% of the ILC population in the first week of infection, whereas, surprisingly, ST2+ ILC2 failed to expand. NKp46+ NK cell expansions were confirmed in RAG2 deficient mice lacking adaptive immunity. Ablation of the NKp46+ NK cell compartment in RAG2 common gamma chain (gc) mice led to increased susceptibility to chronic adult B. malayi infection. This data was recapitulated using an Onchocerca ochengi male worm peritoneal implant model. When NKp46+ NK cells were depleted in RAG2 deficient mice using anti-NKp46 or asialo GM1 antibody injections over the first five weeks of B. malayi infection, susceptibility to adult B. malayi infection was significantly increased by 2-3 fold with concomitant impairment in eosinophil or neutrophil recruitments. Finally, we demonstrate that in RAG2 deficient mice, drug clearance of a primary adult B. malayi infection followed by challenge infection leads to resistance against early larval B. malayi establishment. This innate resistance is associated with bolstered NK and eosinophils whereby NKp46+ NK cells express markers of memory-like/enhanced activation (increased expression of interferon gamma and Ly6C). Our data promotes a novel functional role for NKp46+ NK cells in immunoprotection against experimental primary and secondary filarial infection which can proceed in the absence of adaptive immune regulation.

The preparatory phase for ground larviciding implementation for chocerciasis control in the Meme River Basin in South West Cameroon: the COUNTDOWN Consortium alternative strategy implementation trial.

BACKGROUND: Onchocerciasis control using ivermectin alone has been achieved in some endemic savannah zones of Africa. In the forest regions, the co-endemicity with Loa loa has led to severe adverse events (SAEs) resulting in poor adherence of community members to ivermectin mass drug administration (MDA). This may jeopardize achieving the interruption of transmission of onchocerciasis. Therefore, to accelerate the elimination of onchocerciasis in L. loa co-endemic zones, alternative treatment strategies (ATS) including ground larviciding may be necessary. This study aimed at identifying Simulium breeding sites, cytospecies, transmission profile, susceptibility of Simulium larvae to insecticide (temephos) and identification of some non-target aquatic fauna prior to the implementation of the COUNTDOWN consortium ground larviciding alternative strategy in the Meme River Basin in South West Cameroon. METHODS: A topographic map and entomological survey were used to determine breeding sites. Larvae and adults were identified using standard identification keys. Susceptibility tests were carried out on collected larvae by exposing them to decreasing concentrations of temephos and assessing survival rates while the cytospecies were identified using cytotaxonomy. Various entomological indicators were assessed from dissected flies. Fishing was used as proxy to traps to assess some aquatic fauna at different sites. RESULTS: Twenty-two breeding sites were prospected in the Meme River Basin with eight productive for larvae. A concentration of 0.5-0.1 mg/l temephos induced 100% larval mortality. As the concentration of temephos decreased from 0.05 to 0.0025 mg/l, mortality of larvae also decreased from 98.7 to 12%. Nine cytospecies were observed in the Meme River Basin; 13,633 flies were collected and 4033 dissected. A total of 1455 flies were parous (36.1%), 224 flies were infected (5.5%), and 64 were infective (1.6%). Aquatic fauna observed included Cyprinus spp., Clarias spp., crabs, tadpoles, beetles and larvae of damsel fly. CONCLUSIONS: Onchocerciasis is being actively transmitted within the Meme River Basin. Simulium larvae are susceptible to temephos, and nine cytospecies are present. Non-target fauna observed included fishes, frogs, crabs and insects. Besides treatment with ivermectin, vector control through ground larviciding may be a complementary strategy to accelerate onchocerciasis elimination in the study area.

Anti-Wolbachia drugs for filariasis.

The mutualistic association between Wolbachia endosymbionts and their filarial nematode hosts has been exploited as a validated drug target delivering macrofilaricidal outcomes. Limitations of existing antibiotics to scale-up have driven the search for new drugs, which are effective in shorter regimens of 7 days or less. Here, we review the last 14 years of anti-Wolbachia drug discovery by the anti-Wolbachia (A·WOL) consortium, which has screened more than two million compounds, delivering thousands of hit compounds. Refined screening models integrated with robust pharmacokinetic/pharmacodynamic (PK/PD) driven optimisation and selection strategies have delivered the first two drug candidates specifically designed to target Wolbachia. AWZ1066S and ABBV-4083 are currently progressing through clinical trials with the aim of delivering safe and effective macrofilaricides to support the elimination of onchocerciasis and lymphatic filariasis.

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor-mediated lymphangiogenesis.

Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2-positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin.

Advances in preclinical platforms of Loa loa for filarial neglected tropical disease drug and diagnostics research.

The tropical disease, loiasis, caused by the filarial parasite, Loa, has gained prominence in global public health as a cause of excess mortality and a barrier to the elimination of the related prioritized neglected tropical diseases (NTDs), lymphatic filariasis and onchocerciasis, within Central Africa. There are no effective drug cures or vaccines available to treat loiasis safely. Here we review recent advances in loiasis preclinical platform technologies, including novel in vitro culturing systems, animal models and innovations in experimental infections of the L. loa vector, Chrysops, that have facilitated access to all L. loa filarial life-cycle stages. We detail applications of these new model systems in anti-filarial drug screening, diagnostic development, immunology, and pathophysiology research. Finally, we provide an overview of how loiasis preclinical platforms may be further utilized in translational medicine applications to support the development of much needed new interventions against filarial NTDs.

Structural Requirements for Dihydrobenzoxazepinone Anthelmintics: Actions against Medically Important and Model Parasites: Trichuris muris, Brugia malayi, Heligmosomoides polygyrus, and Schistosoma mansoni.

Nine hundred million people are infected with the soil-transmitted helminths Ascaris lumbricoides (roundworm), hookworm, and Trichuris trichiura (whipworm). However, low single-dose cure rates of the benzimidazole drugs, the mainstay of preventative chemotherapy for whipworm, together with parasite drug resistance, mean that current approaches may not be able to eliminate morbidity from trichuriasis. We are seeking to develop new anthelmintic drugs specifically with activity against whipworm as a priority and previously identified a hit series of dihydrobenzoxazepinone (DHB) compounds that block motility of ex vivo Trichuris muris. Here, we report a systematic investigation of the structure-activity relationship of the anthelmintic activity of DHB compounds. We synthesized 47 analogues, which allowed us to define features of the molecules essential for anthelmintic action as well as broadening the chemotype by identification of dihydrobenzoquinolinones (DBQs) with anthelmintic activity. We investigated the activity of these compounds against other parasitic nematodes, identifying DHB compounds with activity against Brugia malayi and Heligmosomoides polygyrus. We also demonstrated activity of DHB compounds against the trematode Schistosoma mansoni, a parasite that causes schistosomiasis. These results demonstrate the potential of DHB and DBQ compounds for further development as broad-spectrum anthelmintics.