Plug and play virus-like particles for the generation of anti-toxin antibodies.

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.

Reported snakebite mortality and state compensation payments in Madhya Pradesh, India, from 2020 to 2022.

BACKGROUND: India experiences the highest snakebite burden globally, with 58 000 predicted deaths annually. The central Indian state of Madhya Pradesh is thought to have a substantial snakebite burden and provides compensation to families who can demonstrate by postmortem and hospital treatment reports that their relatives have died due to snakebite. This study represents the first report on the frequency of distribution of compensation for snakebite deaths in Madhya Pradesh. METHODS: Statewide snakebite death compensation data from 2020-2021 and 2021-2022, provided by the Madhya Pradesh health authorities, were analysed alongside interviews with 15 families that described the events that ultimately led to their compensation claims. RESULTS: Compensation was paid to a total of 5728 families, with a total value equating to 22 912 Lakhs (approximately US${\$}$27.94 million). Families described commonly recognised snakebite risk factors and behaviours in the events that resulted in their relatives' deaths. CONCLUSIONS: The snakebite burden in Madhya Pradesh is significant, both in terms of mortality and economic expenditure of the state. Sustained investment in preventative interventions, as well as monitoring of the rate of compensation payouts due to snakebite death as a measure of intervention effectiveness, should be considered to substantially reduce snakebite incidence and mortality.

Handheld point-of-care devices for snakebite coagulopathy: a scoping review.

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.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences.

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

Improving in vivo assays in snake venom and antivenom research: A community discussion.

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.

Noma finally recognised as a neglected tropical disease.

In December 2023, after decades of tireless advocacy from stakeholders and partners, the World Health Organization (WHO) gave noma the long overdue recognition as a neglected tropical disease. The significance of this official recognition cannot be overstated, and it is hoped this will serve as a turning point in our battle against this devastating disease.

African polyvalent antivenom can maintain pharmacological stability and ability to neutralise murine venom lethality for decades post-expiry: evidence for increasing antivenom shelf life to aid in alleviating chronic shortages.

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.

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Characterisation of Putative Outer Membrane Proteins from Leptospira borgpetersenii Serovar Hardjo-Bovis Identifies Novel Adhesins and Diversity in Adhesion across Genomospecies Orthologs.

Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans, this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii, which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host-pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host-pathogen interactions of a genus responsible for substantial global disease burden.

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products.

As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.

Virus-like particles displaying conserved toxin epitopes stimulate polyspecific, murine antibody responses capable of snake venom recognition.

Antivenom is currently the first-choice treatment for snakebite envenoming. However, only a low proportion of antivenom immunoglobulins are specific to venom toxins, resulting in poor dose efficacy and potency. We sought to investigate whether linear venom epitopes displayed on virus like particles can stimulate an antibody response capable of recognising venom toxins from diverse medically important species. Bioinformatically-designed epitopes, corresponding to predicted conserved regions of group I phospholipase A2 and three finger toxins, were engineered for display on the surface of hepatitis B core antigen virus like particles and used to immunise female CD1 mice over a 14 weeks. Antibody responses to all venom epitope virus like particles were detectable by ELISA by the end of the immunisation period, although total antibody and epitope specific antibody titres were variable against the different epitope immunogens. Immunoblots using pooled sera demonstrated recognition of various venom components in a diverse panel of six elapid venoms, representing three continents and four genera. Insufficient antibody yields precluded a thorough assessment of the neutralising ability of the generated antibodies, however we were able to test polyclonal anti-PLA2 IgG from three animals against the PLA2 activity of Naja nigricollis venom, all of which showed no neutralising ability. This study demonstrates proof-of-principle that virus like particles engineered to display conserved toxin linear epitopes can elicit specific antibody responses in mice which are able to recognise a geographically broad range of elapid venoms.

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms.

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

Correction: Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms.

[This corrects the article DOI: 10.1371/journal.pntd.0009659.].

Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms.

BACKGROUND: Snakebite is a neglected tropical disease that causes high global rates of mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapeutic for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Two different immunogen mixtures, consisting of seven and twelve haemotoxic venoms sourced from geographically diverse and/or medically important snakes, were used to raise ovine polyclonal antibodies, prior to characterisation of their immunological binding characteristics and in vitro neutralisation profiles against each of the venoms. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom. These findings suggest that a core set of venom immunogens may be sufficient to stimulate antibodies capable of broadly neutralising a geographically diverse array of haemotoxic snake venoms, and that adding additional venom immunogens may impact negatively on the dose efficacy of the resulting antivenom. CONCLUSIONS/SIGNIFICANCE: Although selection of appropriate immunogens that encapsulate venom toxin diversity without diluting antivenom potency remains challenging and further optimisation is required, the findings from this pilot study suggest that the generation of pathology-specific antivenoms with global utility is likely to feasible, thereby highlighting their promise as future modular treatments for the world's tropical snakebite victims.

Hexapod Assassins' Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus.

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

The new COST Action European Venom Network (EUVEN)-synergy and future perspectives of modern venomics.

Venom research is a highly multidisciplinary field that involves multiple subfields of biology, informatics, pharmacology, medicine, and other areas. These different research facets are often technologically challenging and pursued by different teams lacking connection with each other. This lack of coordination hampers the full development of venom investigation and applications. The COST Action CA19144-European Venom Network was recently launched to promote synergistic interactions among different stakeholders and foster venom research at the European level.

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.