Preclinical validation of a repurposed metal chelator as an early intervention therapeutic for hemotoxic snakebite

Snakebite envenoming causes 138,000 deaths annually, and ~400,000 victims are left with permanent disabilities. Envenoming by saw-scaled vipers (Viperidae: Echis) leads to systemic hemorrhage and coagulopathy, and represents a major cause of snakebite mortality and morbidity in Africa and Asia. The only specific treatment for snakebite, antivenom, has poor specificity, low affordability, and must be administered in clinical settings due to its intravenous delivery and high rates of adverse reactions. This requirement results in major treatment delays in resource-poor regions and substantially impacts on patient outcomes after envenoming. Here we investigated the value of metal ion chelators as pre-hospital therapeutics for snakebite. Among the tested chelators, dimercaprol (British anti-Lewisite) and its derivative 2,3-dimercapto-1-propanesulfonic acid (DMPS), were found to potently antagonize the activity of Zn2+-dependent snake venom metalloproteinases in vitro. Moreover, DMPS prolonged or conferred complete survival in murine preclinical models of envenoming against a variety of saw-scaled viper venoms. DMPS also considerably extended survival in a ‘challenge and treat’ model, where drug administration was delayed after venom injection, and the oral administration of this chelator provided partial protection against envenoming. Finally, the potential clinical scenario of early oral DMPS therapy combined with a delayed, intravenous dose of conventional antivenom provided prolonged protection against the lethal effects of envenoming in vivo. Our findings demonstrate that the safe and affordable repurposed metal chelator DMPS can effectively neutralize saw-scaled viper venoms in vitro and in vivo and highlights the promise of this drug as an early, pre-hospital, therapeutic intervention for hemotoxic snakebite envenoming

Diagnostic characteristics of the 20-minute whole blood clotting test in detecting venom-induced consumptive coagulopathy following carpet viper envenoming

Introduction Envenoming by Echis spp. (carpet or saw-scaled vipers) causes haemorrhage and coagulopathy and represents a significant proportion of snakebites in the savannah regions of West Africa. Early diagnosis of envenoming is crucial in the management of these patients and there is limited evidence on the utility of the 20-minute whole blood clotting test (20WBCT) in diagnosing venom-induced consumptive coagulopathy (VICC) following envenoming by Echis ocellatus. Methods A prospective observational cohort study was conducted at the Kaltungo General Hospital in North-eastern Nigeria from September 2019 to September 2021. Standardised 20WBCTs were conducted by trained hospital staff and citrated plasma samples were collected at numerous timepoints. Prothrombin time (PT) and international normalised ratio (INR) were determined using a semi-automated analyser and INR values were calculated using international sensitivity indices (ISI). The sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and likelihood ratios of the 20WBCT compared to an INR ≥ 1.4 were calculated, alongside 95% confidence intervals. Results We enrolled 121 patients into our study, with a median age of 26 (18.0–35.0) years and a male predominance (75.2%). The 20WBCT was positive (abnormal) in 101 out of 121 patients at timepoint 0h, of which 95 had an INR ≥ 1.4, giving a sensitivity of 87.2% (95%CI 79.4–92.8). Among patients with a negative 20WBCT (normal), six had an INR < 1.4 giving a specificity of 50% (95%CI 21.1–78.9%). The positive and negative likelihood ratios were 1.7 (95%CI 1.6–1.9) and 0.3 (95%CI 0.1–0.4) respectively. Conclusion The 20WBCT is a simple, cheap, and easily accessible bedside test with a high sensitivity for the detection of patients with venom induced consumptive coagulopathy (VICC) following envenoming by E. ocellatus, although false positives do occur. Repeated 20WBCTs can identify patients with new, persistent, and rebound coagulopathy

Profiling the Murine Acute Phase and Inflammatory Responses to African Snake Venom: An Approach to Inform Acute Snakebite Pathology

Snake envenoming causes rapid systemic and local effects that often result in fatal or long-term disability outcomes. It seems likely that acute phase and inflammatory responses contribute to these haemorrhagic, coagulopathic, neurotoxic, nephrotoxic and local tissue destructive pathologies. However, the contributory role of acute phase/inflammatory responses to envenoming is under-researched and poorly understood—particularly for envenoming by sub-Saharan African venomous snakes. To provide data to help guide future studies of human patients, and to explore the rationale for adjunct anti-inflammatory medication, here we used an in vivo murine model to systematically assess acute phase and inflammatory responses of mice to ten African snake venoms. In addition to investigating snake species-specific effects of venom on the cardiovascular system and other key organs and tissues, we examined the response to intravascular envenoming by acute phase reactants, including serum amyloid A, P-selectin and haptoglobin, and several cytokines. Venoms of the spitting (Naja nigricollis) and forest (N. melanoleuca) cobras resulted in higher acute phase and inflammatory responses than venoms from the other cobras, mambas and vipers tested. Naja nigricollis venom also stimulated a 100-fold increase in systemic interleukin 6. Thin blood films from venom-treated mice revealed species-specific changes in red blood cell morphology, indicative of membrane abnormalities and functional damage, lymphopenia and neutrophil leukocytosis. Our ex vivo assays with healthy human blood treated with these venoms identified that N. nigricollis venom induced marked levels of haemolysis and platelet aggregation. We conclude that African snake venoms stimulate very diverse responses in this mouse model of acute systemic envenoming, and that venoms of the African cobras N. nigricollis and N. melanoleuca, in particular, cause marked inflammatory and non-specific acute phase responses. We also report that several African snake venoms cause haemolytic changes. These findings emphasise the importance of understanding acute responses to envenoming, and that further research in this area may facilitate new diagnostic and treatment approaches, which in turn may lead to better clinical outcomes for snakebite patients

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T. albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims

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