The study of snake venom toxicity and evaluation of the neutralisation ability of antivenoms and novel therapeutic agents for envenoming has largely relied on animal tests, especially the analysis of the lethal activity of venoms and its neutralisation. In addition, other animal-based assays are used to assess pathology-specific effects of venoms. Despite their demonstrated value, these assays have several limitations. They involve acute stress and pain in animals, and their validity, vis-à-vis the characteristics of human snakebite envenoming, is limited owing to the nature of the assays. Therefore, urgent innovations are required in this field. Ensuring the implementation of the 3Rs (Replacement, Reduction, and Refinement) is essential when animal studies are required. These include: (a) refinement of the tests (e.g., use of analgesia, reduction of assay duration, and refining of humane endpoints); (b) reduction in the number of animals used by more thorough in vitro assessment of antivenoms prior to in vivo assays, and improved statistical analyses and group sizing; and, most importantly, and (c) replacement of in vivo rodent tests with validated in vitro approaches, such as immunoassays, enzymatic and cell-based assays, the use of invertebrate models, and the implementation of New Approach Methodologies (NAMs), i.e., novel in vitro or in silico techniques that model complex pathophysiological processes of envenoming. A roadmap involving researchers, manufacturers, quality control groups, regulators, and funding agencies is proposed, with the aim of driving significant changes in this field.

Snakebite envenoming is a neglected tropical disease that causes high mortality and morbidity. The current treatment, intravenous antivenom, comes with numerous disadvantages, making new therapeutics important. Optimized small molecules offer the possibility for oral use at the onset of envenoming, and the highly pathogenic, zinc-dependent snake venom metalloproteinase toxin family represents an attractive target for drug discovery. Through systematic chemical modification guided by molecular modeling, we describe the development of hydroxamic acid 23 (DC-174), a molecule that displays potent broad-spectrum metalloproteinase inhibition (IC50s < 10 nM) and neutralizes the procoagulant activities of multiple snake venoms. In oral dosing studies, 23 showed preclinical efficacy in a mouse model of severe envenoming, with efficacy boosted by a pharmacokinetically informed multiple dosing regimen. This rationally designed, orally bioavailable metalloproteinase inhibitor represents an excellent lead compound for the development of a small-molecule drug treatment for snakebite.

Preclinical murine models remain the primary method for assessing the efficacy of antivenoms, yet the quality and transparency of reporting in these studies is often varied and inconsistent. This undermines reproducibility, limits the potential for refinement, and hinders the translation of findings into clinical benefit. To address this, we present a reporting guide tailored specifically to in vivo venom neutralisation experiments. This reporting guide aligns with the ARRIVE 2.0 Essential 10 and incorporate recent World Health Organization recommendations, offering a practical framework to support rigorous and transparent reporting. The guidance is structured into two tiers: an "essential" set that outlines the minimum information required for robust methodological scrutiny, and a "recommended" set designed to promote refinement and reduction in animal use. Together, these checklists aim to support researchers, reviewers, and editors in improving the quality and reproducibility of pre-clinical venom research, without requiring additional experimental work. While developed with lethality neutralisation assays in mind, the guidelines are adaptable to other common in vivo models and experimental designs. By encouraging more complete reporting, they facilitate cross-study comparisons, support meta-analyses, and help build confidence in both existing and novel envenoming therapies. Ultimately, this approach promotes better science, more ethical animal use, and improved prospects for clinical translation.

Snakebite is a significant public health concern in Africa, with the viperid species Echis ocellatus being responsible for the majority of snakebite deaths in West Africa. Recently E. ocellatus underwent taxonomic revision and was split into two species, E. ocellatus sensu stricto and E. romani, leading to questions regarding differences in venom bioactivities and the efficacy of antivenoms indicated for treatment of 'E. ocellatus' envenoming against the two redefined species. Using a range of in vitro assays we compared the toxin activities of the two species and the venom-neutralising efficacy of three antivenoms (EchiTAbG, SAIMR Echis and Echiven) raised against 'E. ocellatus'. We then used murine preclinical assays to compare the in vivo efficacy of these antivenoms against E. romani and E. ocellatus s. str venoms. Mitochondrial barcoding of snake skins and venom revealed that E. romani, and not E. ocellatus, is used in the manufacture of several antivenoms raised against 'E. ocellatus'. There were also a number of differences in specific toxin activity between the venoms of the two species in the three in vitro assays utilised in this study.; E. ocellatus (Ghana) had the strongest phospholipase A2 (PLA2) activity, followed by weak PLA2 activity for E. romani (Cameroon) and insignificant activity by E. romani (Nigeria). E. ocellatus (Ghana) and E. romani (Nigeria) demonstrated comparable snake venom metalloproteinase activity, whilst E. romani (Cameroon) had reduced, albeit still significant, activity in comparison. However no differences were observed in a plasma clotting assay measuring coagulopathy between the venoms and localities. Venoms from E. ocellatus (Ghana) and E. romani (Cameroon and Nigeria) were all recognised comparably by the three antivenoms, and there were only modest differences between antivenoms in neutralising the various in vitro toxin effects. In murine preclinical assays, each antivenom could neutralise the lethal effects of E. romani (Nigeria), but differences were seen in their comparative potency when the same antivenom doses were tested against E. romani (Cameroon) and E. ocellatus (Ghana). In these comparative potency assays, all three antivenoms were unable to confer 100% survival when tested against E. romani (Cameroon), but SAIMR Echis provided the best protection with 80% survival. When tested against E. ocellatus (Ghana), the comparative doses of SAIMR Echis and Echiven provided 100% protection whereas EchiTAbG failed to prevent lethality beyond three hours. This represents the first detailed analysis of differences between E. ocellatus and E. romani venom bioactivities and the efficacy of existing antivenoms against these two species. Our findings demonstrate that EchiTAbG, SAIMR Echis and Echiven antivenoms are preclinically efficacious against the lethal effects of E. ocellatus and E. romani venom across a number of localities.

Background Across North America an estimated 3,800–6,500 snakebite envenomings occur annually, resulting in 7–15 deaths and an unknown number of disfigurements and disabilities. Most bites are caused by Crotalid snake species. The variable diversity and toxin complexity of crotalid venoms presents a considerable challenge to developing broadly effective small molecule therapeutics to better treat snakebite in this region. Methods We evaluated the ability of three small molecule, toxin inhibiting, repurposed drugs to inhibit the venom activities of six medically important crotalid snake species ( Agkistrodon contortrix, Crotalus atrox, C. adamanteus, C. horridus, C. scutulatus and Sistrurus miliarius ). These drugs target two pathologically relevant venom toxin families, the snake venom metalloproteinases (SVMPs; marimastat and DMPS) and phospholipases A2 (PLA2s; varespladib), and venom inhibition was measured using in vitro enzymatic and phenotypic plasma coagulation assays. Thereafter we evaluated the efficacy of individual drugs and dual drug combinations in in vivo preclinical models of snakebite envenoming, using both preincubation and rescue model formats. Results In vitro bioassays demonstrated that the selected small molecules showed potent inhibition of the enzymatic activity of different toxin families to the nanomolar (varespladib vs PLA2 and marimastat vs SVMP) or micromolar (DMPS vs SVMP) level. Three of the venoms had anticoagulant activity, which varespladib restored to normal coagulation profiles, suggesting this activity is mostly driven by PLA2 toxins. Preclinical experiments revealed that pre-incubation of representative venoms with single drugs was insufficient to completely protect against lethality, except for varespladib against C. scutulatus . Superior efficacy was observed when drugs were used in a combination approach, with the combination of marimastat and varespladib providing greatest protection against lethality in both pre-incubation and rescue models. Conclusions Venom variation among snake species makes the development of generic snakebite therapeutics challenging. In this study we showed that while SVMP and PLA2 inhibiting drugs show inhibitory potency against diverse North American snake venoms, drug combinations consisting of an SVMP inhibitor together with a PLA2 inhibitor are required to confer broad in vivo protection against lethality caused by envenoming. This study highlights the potential long-term value of drug combinations as next-generation therapeutics for snakebite envenoming. ### Competing Interest Statement NRC is named as an inventor on a patent application describing the use of the DMPS and varespladib drug combination as a therapeutic for snakebite indication. * WHO : World Health Organisation FDA : Food and Drug Administration SVMP : Snake Venom Metalloproteinase PLA2 : Phospholipase A2 CLPs : C-type lectin-like proteins LMIC : Low and Middle-Income Country DMPS : 2,3-dimercaptopropane-1-sulfonic acid Wellcome Trust, , 221712/Z/20/Z Medical Research Council, , MC/PC/15040

Each year, snakebite envenoming claims thousands of lives and causes severe injury to victims across sub-Saharan Africa, many of whom depend on antivenoms derived from animal plasma as their sole treatment option1. Traditional antivenoms are expensive, can cause adverse immunological reactions, offer limited efficacy against local tissue damage and are often ineffective against all medically relevant snake species2. There is thus an urgent unmet medical need for innovation in snakebite envenoming therapy. However, developing broad-spectrum treatments is highly challenging owing to the vast diversity of venomous snakes and the complex and variable composition of their venoms3. Here we addressed this challenge by immunizing an alpaca and a llama with the venoms of 18 different snakes, including mambas, cobras and a rinkhals, constructing phage display libraries, and identifying high-affinity broadly neutralizing nanobodies. We combined eight of these nanobodies into a defined oligoclonal mixture, resulting in an experimental polyvalent recombinant antivenom that was capable of neutralizing seven toxin families or subfamilies. This antivenom effectively prevented venom-induced lethality in vivo across 17 African elapid snake species and markedly reduced venom-induced dermonecrosis for all tested cytotoxic venoms. The recombinant antivenom performed better than a currently used plasma-derived antivenom and therefore shows considerable promise for comprehensive, continent-wide protection against snakebites by all medically relevant African elapids.

Snakebite envenoming is a neglected tropical disease that causes high mortality and morbidity. The current treatment, intravenous antivenom, comes with numerous disadvantages making new therapeutics important. Optimised small molecules offer the possibility for oral use at the onset of envenoming, and the highly pathogenic, zinc-dependent, snake venom metalloproteinase toxin family represents an attractive target for drug discovery. Through systematic chemical modification guided by molecular modelling, we describe the development of hydroxamic acid DC-174, a molecule that displays potent broad spectrum metalloproteinase inhibition and neutralises the procoagulant activities of multiple snake venoms. In oral-dosing studies, DC-174 showed preclinical efficacy in a mouse model of severe envenoming, with efficacy boosted by a pharmacokinetically-informed multiple dosing regimen. This rationally designed metalloproteinase inhibitor offers a potential paradigm shift from delayed treatment with antivenom in tertiary hospitals to a contemporary approach using oral drugs amenable for rapid use in snakebite-affected communities. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, 221712/Z/20/Z

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.

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.

Venom-induced consumption coagulopathy (VICC) is a common complication of snakebite that is associated with hypofibrinogenemia, 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 standardized, 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 prioritize certain devices for clinical validation studies. Four small studies have evaluated a POC international normalized ratio (INR) device in patients with Australian Elapid, Daboia russelii, and Echis carinatus envenoming. The devices assessed in these studies either relied on a thrombin substrate endpoint, which is known to underestimate INR in patients with hypofibrinogenemia, have been recalled due to poor accuracy, or have since been discontinued. Sixteen commercially available POC devices for measuring INR, activated clotting time, activated partial thromboplastin time, fibrinogen, D-dimer, and fibrin(ogen) degradation products have been reviewed. POC INR devices that detect fibrin clot formation, as well as a novel POC device that quantifies fibrinogen were identified, which 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.

Background: The genus Echis is of high medical importance across Africa. Recently the taxonomy of its most medically important species, Echis ocellatus, underwent a revision, resulting in a splitting of the species into E. romani and E. ocellatus, and leading to uncertainty of the efficacy of antivenoms indicated for treatment of 'E. ocellatus' envenomings against the two redefined species.Methods: We compared the in vitro and murine preclinical venom-neutralising efficacy of three antivenoms (EchiTAbG, SAIMR Echis and Echiven) raised against E. ocellatus sensu lato against the venoms of E. romani and E. ocellatus, and investigated cross-reactivity to E. coloratus, E. leucogaster, and E. pyramidum leakeyi.Findings: In preclinical assays of envenoming, all three antivenoms neutralised Nigerian E. romani venom, though all three were less protective against Cameroonian E. romani. SAIMR Echis and Echiven neutralised E. ocellatus venom whereas EchiTAbG was less protective. SAIMR Echis and Echiven showed strong cross-reactivity to E. p. leakeyi and E. leucogaster, whilst EchiTAbG showed weaker cross-reactivity. All three antivenoms exhibited poor neutralisation of E. coloratus venom.Interpretation: This represents the first detailed analysis of differences between E. ocellatus and E. romani venom bioactivities and the impact of antivenom on these two species. Our findings demonstrate that SAIMR Echis and Echiven antivenoms are preclinically efficacious against the lethal effects of several species of Echis. These products, in addition to EchiTAbG, seem likely to meet the WHO recommendation of three antivenoms required for treatment of Echis envenomings across sub-Saharan Africa, though clinical evidence is required to confirm these findings.Funding: Horizon 2020 FET Open #899670, UKRI FLF grant MR/S03398X/1, Wellcome Trust grant 221712/Z/20/Z and NC3Rs grant NC/X001172/1.Declaration of Interest: SKM and NRC communicated with the antivenom manufacturer VINS Bioproducts to obtain a sample of Echiven antivenom for testing. The antivenom manufacturer had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The Centre for Snakebite Research and Interventions (CSRI) at LSTM was historically involved in the development of EchiTAbG and EchiTAb-Plus0ICP antivenoms, though none of the authors from CSRI were directly involved in this work. NRC was previously employed by the manufacturer of EchiTAbG antivenom (MicroPharm, UK) between 2010 and 2012. NRC and CAD are currently collaborators of the EchiTAbG manufacturer MicroPharm, UK. Micropharm had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.Ethical Approval: Animal experiments were conducted under protocols approved by the Animal Welfare and Ethical Review Boards of the Liverpool School of Tropical Medicine and the University of Liverpool, and under project licence P24100D38 approved by the UK Home Office in accordance with the UK Animal (Scientific Procedures) Act 1986.

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.

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.

On the 26th 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.

ABSTRACT Use of experimental cats and dogs in veterinary heartworm preclinical drug research is increasing. As a potential alternative primary in vivo heartworm preventative drug screen, we assessed lymphopenic mice with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of D. immitis . Non-obese diabetic (NOD) Severe Combined ImmunoDeficient (SCID)γc -/- (NSG / NXG) mice consistently yielded viable D. immitis larvae at 2-4 weeks post-infection across multiple experiments, different batches of infectious larvae inoculates, different isolates of D. immitis and at independent laboratories. Mice did not display any overt clinical signs associated with infection up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, the natural site of this stage of heartworm in dogs. Larvae retrieved from NSG / NXG mice were mid-L4 stage of development. Compared with 14-day in vitro propagated larvae, in vivo derived L4 were significantly larger and contained expanded Wolbachia endobacteria titres, determined by QPCR and Fluorescent in situ Hybridisation (FISH). We established an ex vivo 6-day L4 paralytic screening system against nematodicidal agents (moxidectin, levamisole) which highlighted discrepancies in relative drug sensitivities in comparison with in vitro reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70-90% in D. immitis L4 following 2-7 day oral in vivo exposures of NSG / NXG infected mice with doxycycline or the rapid-acting investigational anti- Wolbachia drug, AWZ1066S. We validated the NSG / NXG mouse model as a filaricide drug screen by in vivo treatments with single injections of moxidectin, which mediated 60-88% reduction in L4 larvae at 14-28 days. Future adoption of the mouse model as a first-line efficacy screen will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround and reduced costs whilst simultaneously decreasing need for experimental cat or dog use.

IntroductionDirofilariasis, including heartworm disease, is a major emergent veterinary parasitic infection and a human zoonosis. Currently, experimental infections of cats and dogs are used in veterinary heartworm preclinical drug research.MethodsAs a refined alternative in vivo heartworm preventative drug screen, we assessed lymphopenic mouse strains with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of Dirofilaria immitis.ResultsNon-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc−/− (NSG and NXG) and recombination-activating gene (RAG)2−/−γc−/− mouse strains yielded viable D. immitis larvae at 2–4 weeks post-infection, including the use of different batches of D. immitis infectious larvae, different D. immitis isolates, and at different laboratories. Mice did not display any clinical signs associated with infection for up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, which is the natural site of this stage of heartworm in dogs. Compared with in vitro-propagated larvae at day 14, in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expanded Wolbachia endobacteria titres. We established an ex vivo L4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison with in vitro-reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70%−90% in D. immitis L4 following 2- to 7-day oral in vivo exposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXG D. immitis mouse models as a filaricide screen by in vivo treatments with single injections of moxidectin, which mediated a 60%−88% reduction in L4 larvae at 14–28 days.DiscussionFuture adoption of these mouse models will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround, and reduced costs and may simultaneously decrease the need for experimental cat or dog use.

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.