Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by ...rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.
Snake envenoming causes several potentially lethal pathologies. The specific pathology is dictated by the toxin composition of venom, which varies by species, geography and ontogeny. This variation ...severely restricts the paraspecific efficacy of antivenoms used to treat snakebite victims. With a view to devising pathology-specific snakebite treatments, we assessed the procoagulant activity of 57 snake venoms and investigated the efficacy of various antivenoms. We find that procoagulant venoms act differentially on key steps of the coagulation cascade, and that certain monospecific antivenoms work in a previously unrecognised paraspecific manner to neutralise this activity, despite conventional assumptions of congener-restricted efficacy. Moreover, we demonstrate that the metal chelator EDTA is also capable of neutralising venom-induced lethality in vivo. This study illustrates the exciting potential of developing new, broad-spectrum, toxin-targeting antivenoms capable of treating key snakebite pathologies, and advocates a thorough re-examination of enzyme inhibiting compounds as alternative therapies for treating snakebite victims.
While envenoming by the southern African shield-nosed or coral snakes (genus Aspidelaps) has caused fatalities, bites are uncommon. Consequently, this venom is not used in the mixture of snake venoms ...used to immunise horses for the manufacture of regional SAIMR (South African Institute for Medical Research) polyvalent antivenom. Aspidelaps species are even excluded from the manufacturer's list of venomous snakes that can be treated by this highly effective product. This leaves clinicians, albeit rarely, in a therapeutic vacuum when treating envenoming by these snakes. This is a significantly understudied small group of nocturnal snakes and little is known about their venom compositions and toxicities. Using a murine preclinical model, this study determined that the paralysing toxicity of venoms from Aspidelaps scutatus intermedius, A. lubricus cowlesi and A. l. lubricus approached that of venoms from highly neurotoxic African cobras and mambas. This finding was consistent with the cross-genus dominance of venom three-finger toxins, including numerous isoforms which showed extensive interspecific variation. Our comprehensive analysis of venom proteomes showed that the three Aspidelaps species possess highly similar venom proteomic compositions. We also revealed that the SAIMR polyvalent antivenom cross-reacted extensively in vitro with venom proteins of the three Aspidelaps. Importantly, this cross-genus venom-IgG binding translated to preclinical (in a murine model) neutralisation of A. s. intermedius venom-induced lethality by the SAIMR polyvalent antivenom, at doses comparable with those that neutralise venom from the cape cobra (Naja nivea), which the antivenom is directed against. Our results suggest a wider than anticipated clinical utility of the SAIMR polyvalent antivenom, and here we seek to inform southern African clinicians that this readily available antivenom is likely to prove effective for victims of Aspidelaps envenoming.
Coral and shield-nosed snakes (genus Aspidelaps) comprise two species and several subspecies of potentially medically important venomous snakes distributed in Namibia, Botswana, Zimbabwe, Mozambique and South Africa. Documented human fatalities, although rare, have occurred from both A. lubricus and A. scutatus. However, their venom proteomes and the pathological effects of envenomings by this understudied group of nocturnal snakes remain uncharacterised. Furthermore, no commercial antivenom is made using venom from species of the genus Aspidelaps. To fill this gap, we have conducted a transcriptomics-guided comparative proteomics analysis of the venoms of the intermediate shield-nose snake (A. s. intermedius), southern coral snake (A. l. lubricus), and Cowle's shield snake (A. l. cowlesi); investigated the mechanism of action underpinning lethality by A. s. intermedius in the murine model; and assessed the in vitro immunoreactivity of the SAIMR polyvalent antivenom towards the venom toxins of A. l. lubricus and A. l. cowlesi, and the in vivo capability of this antivenom at neutralising the lethal effect of A. s. intermedius venom. Our data revealed a high degree of conservation of the global composition of the three Aspidelaps venom proteomes, all characterised by the overwhelming predominance of neurotoxic 3FTxs, which induced classical signs of systemic neurotoxicity in mice. The SAIMR polyvalent antivenom extensively binds to Aspidelaps venom toxins and neutralised, with a potency of 0.235 mg venom/mL antivenom, the lethal effect of A. s. intermedius venom. Our data suggest that the SAIMR antivenom could be a useful therapeutic tool for treating human envenomings by Aspidelaps species.
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•The venom proteomes of Aspidelaps scutatus and A. lubricus subspecies were characterized.•Consistent with the predominance of 3FTxs, murine envenoming by A. s. intermedius induced systemic neurotoxicity.•Antivenomics analysis revealed extensively recognision of Aspidelaps venom toxins by the SAIMR polyvalent antivenom.•SAIMR antivenom neutralised mouse lethal activity of A. s. intermedius venom with a potency of 0.235 mg venom/mL antivenom.•Our data suggest that SAIMR antivenom could be a useful therapeutic tool against human envenomings by Aspidelaps species.
Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of ...evolutionary and physiological processes, many taxonomic groups remain under-studied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins.We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.
Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical ...disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa.
We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a 'gold standard' comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom.
None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of 'test' antivenom IgGs to bind venom proteins were not substantially different from that of the 'gold standard' antivenoms. The least effective antivenoms had the lowest IgG content/vial.
Manufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-efficacy testing facilities throughout the continent.
Routine laboratory animal tests necessary to assess the toxicity of snake venoms and the preclinical neutralizing ability of antivenoms and other inhibitory substances induce significant pain and ...distress. This has prompted initiatives to introduce the routine use of analgesia. In this study, the analgesic effect of morphine and tramadol was assessed in tests assessing the lethal, hemorrhagic, myotoxic and edema-forming activities of the venom of the viperid snake Bothrops asper. The Mouse Grimace Scale (MGS) and mouse-exploration activity were used to assess pain and its inhibition by the analgesics. Results demonstrate that tests assessing lethality and myotoxicity induce higher levels of pain than assays quantifying hemorrhagic and edema-forming activities. Our observations also indicate that pretreatment of mice with both analgesics, at the doses used, were similarly effective in reducing the MGS magnitude and increase mouse-exploration activity after the administration of B. asper venom. Moreover, the analgesic effect of both drugs was more evident in the myotoxic and lethality assays. Combined with previous observations showing that these analgesics do not alter the extent of toxic effects induced by B. asper venom, our results strongly indicate that the use of analgesia (using either morphine or tramadol) should be considered in the routine assessment of venom toxicity and antivenom efficacy.
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•The analgesics morphine and tramadol were evaluated in mice envenomed with Bothrops asper venom.•Venom induced pain when injected by the intradermal, intramuscular, subcutaneous and intraperitoneal routes.•Both analgesics were effective at reducing pain in these models.•Precautionary analgesia should be considered when testing the toxicity of snake venoms.
Snakebite envenoming (SBE) leads to significant morbidity and mortality, resulting in over 90,000 deaths and approximately 400,000 amputations annually. In sub-Saharan Africa (SSA) alone, SBE ...accounts for over 30,000 deaths per annum. Since 2017, SBE has been classified as a priority Neglected Tropical Disease (NTD) by the World Health Organisation (WHO). The major species responsible for mortality from SBE within SSA are from the Bitis, Dendroaspis, Echis and Naja genera. Pharmacologically active toxins such as metalloproteinases, serine proteinases, 3-finger toxins, kunitz-type toxins, and phospholipase A
s are the primary snake venom components. These toxins induce cytotoxicity, coagulopathy, hemorrhage, and neurotoxicity in envenomed victims. Antivenom is currently the only available venom-specific treatment for SBE and contains purified equine or ovine polyclonal antibodies, collected from donor animals repeatedly immunized with low doses of adjuvanted venom. The resulting plasma or serum contains a high titre of specific antibodies, which can then be collected and stored until required. The purified antibodies are either whole IgG, monovalent fragment antibody (Fab) or divalent fragment antibody F(ab')
. Despite pharmacokinetic and pharmacodynamic differences, all three are effective in the treatment of SBE. No antivenom is without adverse reactions but, the level of their impact and severity varies from benign early adverse reactions to the rarely occurring fatal anaphylactic shock. However, the major side effects are largely reversible with immediate administration of adrenaline and corticosteroids. There are 16 different antivenoms marketed within SSA, but the efficacy and safety profiles are only published for less than 50% of these products.
Antivenom is the only effective treatment for snakebite and comprise immunoglobulins obtained from venom-immunised horses or sheep. Globally, more than 45 manufacturers make over 120 snake ...antivenoms; it is a regulatory requirement that the venom-neutralising efficacy of all antivenoms are assessed preclinically. The World Health Organisation (WHO) recommended preclinical tests of efficacy are the median lethal venom dose (LD50) and median effective antivenom dose (ED50) assays performed in mice. They result in substantial pain and suffering to the mice with death/survival as their metric. With NC3R-funding, we sought to apply the ‘Refine, Reduce and Replace’ principles of animal experimentation to these murine assays. Pain is a near-universal symptom of snake envenoming, and one of our objectives was to identify an effective analgesic that could be utilised without invalidating the assay results. The Mouse Grimace Scale and Activity scores were used to measure pain. We examined the effects of two opioid analgesics, buprenorphine and morphine, in a range of venom LD50 and ED50 assays. Both were effective at reducing pain scores, but death rates were higher in those which had received buprenorphine, hence morphine is preferable.We demonstrate that each venom exhibits a distinct set of lesions, the severity of which appears time and dose dependent, and that the observed murine pathological lesions show significant similarities to those reported in envenomed human victims. Applying the 3R principles, we have used pathological observations, in combination with ante mortem observations, to establish more humane end-points, consequently reducing the duration of LD50 and ED50 assays from 24 to 6 hours. In addition, we have implemented a ‘dose-staging’ element into experimental design in which one dose is given and the next dose(s) selected based on the results of the previous dose, reducing total mice required. To reduce the numbers of assays, and therefore mice, we have shown an excellent correlation between in vitro binding assays, cytotoxicity neutralisation assays and in vivo ED50 using antivenoms derived from the same pool of donor animals. Comparison of the results of in vitro binding assays between 35 different venom/antivenom combinations showed a poor correlation overall, but the correlation improved when each of five venoms were considered separately. The possibility of replacing the in vivo LD50 and ED50 tests, using a cell-based neutralisation assay was investigated using two cell lines from diverse tissues of origin, namely VERO epithelial-type cells and neural SH SY5Y. All venoms studied produced a cytopathic effect in both cell lines, with the VERO cells being more sensitive to viper venoms and SH SY5Y cells to the effects of elapid venoms, when both cell lines were grown in co-culture. However, variability of results made optimisation of a neutralisation assay inadequate for use as an alternative to in vivo tests.
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