Search

Venom induced consumption coagulopathy (VICC) is a common complication of snakebite that is associated with hypofibrinogenaemia, bleeding, disability, and death. In remote tropical settings, where most snakebites occur, the 20-minute whole blood clotting test is used to diagnose VICC. Point-of-care (POC) coagulation devices could provide an accessible means of detecting VICC that is better standardised, quantifiable, and more accurate. In this scoping review, the mechanistic reasons that previously studied POC devices have failed in VICC are considered, and evidence-based recommendations are made to prioritise certain devices for clinical validation studies. Four small studies have evaluated a POC international normalised ratio (INR) device in patients with Australian Elapid, Daboia russelii and Echis carinatus envenoming. All of these studies used POC INR devices that rely on a thrombin substrate endpoint, which, unlike laboratory-based INR measurement, is known to underestimate INR in patients with hypofibrinogenaemia. Seventeen commercially available POC devices for measuring INR, activated clotting time (ACT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimer, and fibrin(ogen) degradation products (FDP) have been reviewed. POC INR devices that detect fibrin clot formation, as well as a novel POC device that quantifies fibrinogen were identified, that show promise for use in patients with VICC. These devices could support more accurate allocation of antivenom, reduce the time to antivenom administration, and provide improved clinical trial outcome measurement instruments. There is an urgent need for these promising POC coagulation devices to be validated in prospective clinical snakebite studies., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Snakebite is a major global health concern, for which antivenom remains the only approved treatment to neutralise the harmful effects of the toxins. However, some medically important toxins are poorly immunogenic, resulting in reduced efficacy of the final product. Boosting the immunogenicity of these toxins in the commercial antivenom immunising mixtures could be an effective strategy to improve the final dose efficacy, and displaying snake antigens on Virus-like particles (VLPs) is one method for this. However, despite some applications in the field of snakebite, VLPs have yet to be explored in methods that could be practical at an antivenom manufacturing scale. Here we describe the utilisation of a "plug and play" VLP system to display immunogenic linear peptide epitopes from three finger toxins (3FTxs) and generate anti-toxin antibodies. Rabbits were immunised with VLPs displaying individual consensus linear epitopes and their antibody responses were characterised by immunoassay. Of the three experimental consensus sequences, two produced antibodies capable of recognising the consensus peptides, whilst only one of these could also recognise native whole toxins. Further characterisation of antibodies raised against this peptide demonstrated a sub-class specific response, and that these were able to elicit partially neutralising antibody responses, resulting in increased survival times in a murine snakebite envenoming model., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Simon J Draper reports a relationship with SpyBiotech that includes: board membership and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1 , B4GALT7 , EXT2 , EXTL3 , XYLT2 , NDST1 , and SLC35B2 , which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming.

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.

On the 26 th January 2023, a free to attend, 'improving in vivo snake venom research: a community discussion' meeting was held virtually. This webinar brought together researchers from around the world to discuss current neutralisation of venom lethality mouse assays that are used globally to assess the efficacy of therapies for snakebite envenoming. The assay's strengths and weaknesses were highlighted, and we discussed what improvements could be made to refine and reduce animal testing, whilst supporting preclinical antivenom and drug discovery for snakebite envenoming. This report summarises the issues highlighted, the discussions held, with additional commentary on key perspectives provided by the authors., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Marriott AE et al.)

Introduction: Antivenom is a lifesaving medicine for treating snakebite envenoming, yet there has been a crisis in antivenom supply for many decades. Despite this, substantial quantities of antivenom stocks expire before use. This study has investigated whether expired antivenoms retain preclinical quality and efficacy, with the rationale that they could be used in emergency situations when in-date antivenom is unavailable., Methods: Using WHO guidelines and industry test requirements, we examined the in vitro stability and murine in vivo efficacy of eight batches of the sub-Saharan African antivenom, South African Institute for Medical Research polyvalent, that had expired at various times over a period of 30 years., Results: We demonstrate modest declines in immunochemical stability, with antivenoms older than 25 years having high levels of turbidity. In vitro preclinical analysis demonstrated all expired antivenoms retained immunological recognition of venom antigens and the ability to inhibit key toxin families. All expired antivenoms retained comparable in vivo preclinical efficacy in preventing the lethal effects of envenoming in mice versus three regionally and medically important venoms., Conclusions: This study provides strong rationale for stakeholders, including manufacturers, regulators and health authorities, to explore the use of expired antivenom more broadly, to aid in alleviating critical shortages in antivenom supply in the short term and the extension of antivenom shelf life in the longer term., Competing Interests: Competing interests: GS, GD, AS, MV and GL are all employees of Instituto Clodomiro Picado, University of Costa Rica, a public manufacturer of antivenom products, including for sub-Saharan Africa., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

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., Competing Interests: FG was employed by Eisai Co., Ltd., Tokyo, Japan. The remaining authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hegde, Marriott, Pionnier, Steven, Bulman, Gunderson, Vogel, Koschel, Ehrens, Lustigman, Voronin, Tricoche, Hoerauf, Hübner, Sakanari, Aljayyoussi, Gusovsky, Dagley, Hong, O'Neill, Ward, Taylor and Turner.)

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-12Rβ2 + 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pionnier, Furlong-Silva, Colombo, Marriott, Chunda, Ndzeshang, Sjoberg, Archer, Steven, Wanji, Taylor and Turner.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Snakebite envenoming affects more than 250,000 people annually in sub-Saharan Africa. Envenoming by Dispholidus typus (boomslang) results in venom-induced consumption coagulopathy (VICC), whereby highly abundant prothrombin-activating snake venom metalloproteinases (SVMPs) consume clotting factors and deplete fibrinogen. The only available treatment for D. typus envenoming is the monovalent SAIMR Boomslang antivenom. Treatment options are urgently required because this antivenom is often difficult to source and, at US$6000/vial, typically unaffordable for most snakebite patients. We therefore investigated the in vitro and in vivo preclinical efficacy of four SVMP inhibitors to neutralise the effects of D. typus venom; the matrix metalloproteinase inhibitors marimastat and prinomastat, and the metal chelators dimercaprol and DMPS . The venom of D. typus exhibited an SVMP-driven procoagulant phenotype in vitro . Marimastat and prinomastat demonstrated equipotent inhibition of the SVMP-mediated procoagulant activity of the venom in vitro , whereas dimercaprol and DMPS showed considerably lower potency. However, when tested in preclinical murine models of envenoming using mixed sex CD1 mice, DMPS and marimastat demonstrated partial protection against venom lethality, demonstrated by prolonged survival times of experimental animals, whereas dimercaprol and prinomastat failed to confer any protection at the doses tested. The preclinical results presented here demonstrate that DMPS and marimastat show potential as novel small molecule-based therapeutics for D. typus snakebite envenoming. These two drugs have been previously shown to be effective against Echis ocellatus VICC in preclinical models, and thus we conclude that marimastat and DMPS should be further explored as potentially valuable early intervention therapeutics to broadly treat VICC following snakebite envenoming in sub-Saharan Africa., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rachel H Clare reports financial support was provided by Wellcome Trust. Jeroen Kool reports financial support was provided by 10.13039/100010269Wellcome Trust. Nicholas R Casewell reports financial support was provided by 10.13039/100010269Wellcome Trust. Nicholas R Casewell reports financial support was provided by Medical Research Council. Nicholas R Casewell reports financial support was provided by The Royal Society. Chunfang Xie reports financial support was provided by China Scholarship Council., (© 2022 The Authors.)

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.

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.

Filarial nematodes are tissue-dwelling parasitic worms that can cause a range of disfiguring pathologies in humans and potentially lethal infections of companion animals. The bacterial endosymbiont, Wolbachia, is present within most human and veterinary filarial pathogens, including the causative agent of heartworm disease, Dirofilaria immitis. Doxycycline-mediated drug targeting of Wolbachia leads to sterility, clearance of microfilariae and gradual death of adult filariae. This mode of action is attractive in the treatment of filariasis because it avoids severe host inflammatory adverse reactions invoked by rapid-killing anthelmintic agents. However, doxycycline needs to be taken for four weeks to exert curative activity. In this review, we discuss the evidence that Wolbachia drug targeting is efficacious in blocking filarial larval development as well as in the treatment of chronic filarial disease. We present the current portfolio of next-generation anti-Wolbachia candidates discovered through phenotypic screening of chemical libraries and validated in a range of in vitro and in vivo filarial infection models. Several novel chemotypes have been identified with selected narrow-spectrum anti-Wolbachia specificity and superior time-to-kill kinetics compared with doxycycline. We discuss the opportunities of developing these novel anti-Wolbachia agents as either cures, adjunct therapies or new preventatives for the treatment of veterinary filariasis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Filariasis is a global health problem targeted for elimination. Curative drugs (macrofilaricides) are required to accelerate elimination. Candidate macrofilaricides require testing in preclinical models of filariasis. The incidence of infection failures and high intra-group variation means that large group sizes are required for drug testing. Further, a lack of accurate, quantitative adult biomarkers results in protracted timeframes or multiple groups for endpoint analyses. Here we evaluate intra-vital ultrasonography (USG) to identify B. malayi in the peritonea of gerbils and CB.17 SCID mice and assess prognostic value in determining drug efficacy. USG operators, blinded to infection status, could detect intra-peritoneal filarial dance sign (ipFDS) with 100% specificity and sensitivity, when >5 B. malayi worms were present in SCID mice. USG ipFDS was predictive of macrofilaricidal activity in randomized, blinded studies comparing flubendazole, albendazole and vehicle-treated SCID mice. Semi-quantification of ipFDS could predict worm burden >10 with 87-100% accuracy in SCID mice or gerbils. We estimate that pre-assessment of worm burden by USG could reduce intra-group variation, obviate the need for surgical implantations in gerbils, and reduce total SCID mouse use by 40%. Thus, implementation of USG may reduce animal use, refine endpoints and negate invasive techniques for assessing anti-filarial drug efficacy.