https://orcid.org/0000-0001-8574-3724
Figures
Abstract
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.
Author summary
The most medically important snake species in West Africa, Echis ocellatus, was recently taxonomically partitioned into E. ocellatus sensu stricto and E. romani, yet the differences in toxin activities between the venoms and their neutralisation by existing antivenoms remain to be determined. Our phylogenetic analysis revealed several antivenoms were raised against E. romani venom rather than E. ocellatus s. str venom, and the toxin activities differ between the venoms of these two species. Our data shows that whilst all three tested antivenoms recognised and bound to both E. ocellatus s. str. and E. romani venoms, there were neutralising differences between the antivenoms in different in vitro functional assays, and murine preclinical efficacy assays. These findings provide preclinical evidence and highlight potential differences in the efficacy of three antivenoms in neutralising the preclinical lethal effects of medically important E. ocellatus s. str. and E. romani venoms. Clinical evidence is required to confirm these findings, but this work suggests that all three antivenoms may be effective against both species despite their recent taxonomic reclassification.
Citation: Edge RJ, Marriott AE, Keen M, Xie T, Crittenden EP, Dawson CA, et al. (2025) Preclinical evaluation of the neutralising efficacy of three antivenoms against the venoms of the recently taxonomically partitioned E. ocellatus and E. romani. PLoS Negl Trop Dis 19(8): e0013371. https://doi.org/10.1371/journal.pntd.0013371
Editor: Marco Aurélio Sartim, Universidade Federal do Amazonas, BRAZIL
Received: February 26, 2025; Accepted: July 17, 2025; Published: August 4, 2025
Copyright: © 2025 Edge et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All supporting data are available as supplemental files. DNA sequences are available in GenBank (RRID:SCR_002760) under accession numbers (OQ735307-OQ735376).
Funding: The work described in this article is funded by the European Commission under Framework H2020 project ADDovenom (Grant Agreement no. 899670) awarded to NRC https://research-and-innovation.ec.europa.eu/index_en, UK Research and Innovation (UKRI) Future Leaders Fellowships grant MR/S03398X/1 awarded to SA https://www.ukri.org/, Wellcome Trust grant 221712/Z/20/Z awarded to NRC https://wellcome.org/, and National Centre for the Replacement, Refinement and Reduction of Animals in Research grant NC/X001172/1 awarded to SA https://www.ukri.org/. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Stefanie Menzies and Nicholas Casewell 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-Plus-ICP antivenoms, though none of the authors from CSRI were directly involved in this work. Nicholas Casewell was previously employed by the manufacturer of EchiTAbG antivenom (MicroPharm, UK) between 2010 and 2012. Nicholas Casewell and Charlotte Dawson 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.
1. Introduction
Snakebite envenoming, a World Health Organization (WHO) recognised Neglected Tropical Disease, is estimated to affect 2.7 million people each year, with global annual rates of ~138,000 deaths and over 400,000 people suffering life-altering morbidity [1]. Snakes of the genus Echis (common name: saw-scaled or carpet vipers) are one of the most medically important groups of snakes responsible for a large proportion of the global snakebite burden [2], currently consisting of 13 recognised species found throughout much of Africa north of the equator and extending through the Middle East to India and Sri Lanka [3]. Saw-scaled vipers are estimated to be responsible for two-thirds of snakebite envenoming in West Africa [4], with E. ocellatus historically invoked as the species responsible for most of the mortality and morbidity from snakebite in this region [2]. Pathophysiological consequences of envenoming by the Echis genus are characteristic of viperid snakes, consisting predominately of haemotoxic effects defined by frequent bleeding disturbances, venom-induced consumption coagulopathy (VICC) and local tissue necrosis [5,6].
In 2009, a molecular phylogenetic analysis of the genus Echis, including E. ocellatus from multiple regions in West Africa, demonstrated the presence of three distinct phylogroups within the species and, given the large degree of morphological variation within the species, led the authors to hypothesise that “additional organismal lineages” may exist [3]. Based on this molecular phylogeny and the analysis of pattern and scalation characters, E. ocellatus was partitioned into two species E. ocellatus and E. romani, in 2018 [7]. The distribution of E. ocellatus is now thought to extend from eastern Guinea to north-western Nigeria, while E. romani is thought to be resident in northern and north-eastern Nigeria east to at least southern Chad [3,7], with an apparently isolated population in Sudan [8]. For clarity, we will refer to E. ocellatus in its old, pre-partition sense, i.e., including E. romani, as E. ocellatus sensu lato, whereas the post-partition interpretation of E. ocellatus will be referred to as E. ocellatus sensu stricto.
The only specific therapy for treating snakebite envenoming is antivenom, a polyclonal antibody-based serotherapy generated by immunising large animals (equines/ovines) with crude venom to produce anti-toxin antibodies [9,10]. Currently, there are three antivenoms designed specifically for use against the Echis species present in sub-Saharan Africa; EchiTAbG, SAIMR Echis and Echiven, as well as several polyvalent products [11]. The effectiveness of antivenoms in neutralising saw-scaled viper envenoming in Nigeria has been particularly well demonstrated [5,12,13]; EchiTAbG has been robustly examined in controlled trials for clinical efficacy against pre-taxonomic partition E. ocellatus s.l. envenoming [5], and SAIMR Echis has good clinical evidence of efficacy [6,14,15]. Echiven is a recent addition to the sub-Saharan African market and to date, to our knowledge, has not been tested in clinical studies or trials and no publicly available preclinical data exist. As of 2020, the efficacy of nine different monovalent and polyvalent antivenoms raised against, or with suggested efficacy via cross-reactivity against, E. ocellatus s. l. had been examined in 30 different preclinical studies [16], and the findings of this analysis demonstrated a wide range of reported efficacy, both between the different antivenoms and sometimes for the same antivenom against the same species [12,17].
The recent partition of E. ocellatus s. l. means that it is likely that several existing antivenoms indicated for E. ocellatus envenoming have been manufactured either using E. romani venom or E. ocellatus s. str. exclusively, or with a mixture of E. romani and E. ocellatus s.str. venom. Detailed analyses of the biological differences between E. ocellatus s.str. and E. romani venom remain outstanding and therefore it remains to be demonstrated whether existing antivenoms have different efficacies in neutralising the venom of each species, although, given their longstanding use against E. ocellatus s. l. and known paraspecificity of these antivenoms, it is probable that there will be limited clinical impact of the taxonomic split. Consequently, we sought to determine if the snakes and venoms used to manufacture EchiTAbG antivenom and previous iterations of EchiTab-Plus-ICP antivenom, which were collected in northern Nigeria and maintained at the Liverpool School of Tropical Medicine (LSTM) as part of the EchiTAb study group [12], were indeed E. ocellatus s. str., or E. romani, or a mixture of both species. We then investigated the toxin activities of E. ocellatus s. str. and E. romani venoms using in vitro assays, and directly compared the neutralising efficacy of three Echis monospecific antivenoms (EchiTAbG, SAIMR Echis and Echiven) in in vitro and in vivo preclinical assays.
2. Methods
2.1 Snake specimens and venoms
Skin sheds were obtained from snakes originating from the Kaltungo (Gombe) region of north-eastern Nigeria that were collected between April 2008 and September 2014 and housed in the LSTM herpetarium as part of the EchiTAb study group collection. An additional shed from an Echis carinatus (Pakistan) specimen housed at LSTM was also used in this study.
Venom from E. ocellatus s. l. (subsequently identified and hereafter referred to as E. romani [Nigeria]) was obtained from pooled (N = 48) venom stocks from the EchiTAb study group collection. Pooled venoms of E. romani from Cameroon (sold as E. ocellatus s. l., hereafter referred to as E. romani [Cameroon]) and E. ocellatus from Ghana were purchased from Latoxan, France (Product ID L1114 for both).
2.2 Total DNA extraction and Sanger sequencing
Total DNA was isolated from 40 individual snakes in the EchiTAb study group collection. Additionally, a shed skin from one Echis carinatus (Pakistan) specimen and lyophilised venoms from E. ocellatus (Ghana) and E. romani (Cameroon) pooled from individual specimens, purchased from Latoxan (France), were examined. Prior to DNA extraction, sheds were stored individually in zip lock bags at room temperature, then total DNA was isolated using a DNeasy Blood & Tissue Kit (Cat 69504, Qiagen, UK) following the manufacturers recommended tissue extraction protocol for snake sheds (using 2.5 mg of shed) and the blood extraction protocol for resuspended venom (starting with 100 µL of 10 mg/mL venom). Polymerase Chain Reaction of NADH4 and CYTB was performed using primer pairs GludgMod2/EchR for CYTB and NADH4/EchR for NADH4 as described in [7]. Amplicons were Sanger sequenced by Source Bioscience (Cambridge, UK) using respective amplicon primer sets, above. Further details can be found in the S1 Text.
2.3 Mitochondrial barcoding and phylogenetic analysis
Resulting sequences were quality checked and aligned using MEGA 11 [18] (RRID:SCR023017). To provide a phylogenetic reference framework, we included in the alignment all sequences of the E. ocellatus group (E. ocellatus, E. romani, E. jogeri), sequences of an E. carinatus from the United Arab Emirates, and, as an outgroup, sequences of a specimen of Cerastes cerastes, which were all sequenced and published as part of a previous phylogenetic study of Echis [3]. We implemented the Model function in MEGA 11, using the Bayesian Information Criterion (BIC) to identify the best substitution model for the unpartitioned data prior to Maximum Likelihood (ML) phylogenetic analysis with 100 bootstrap replicates.
2.4 Antivenoms and control immunoglobulins
Antivenoms used were; (i) EchiTAbG (whole ovine IgG manufactured by MicroPharm UK Ltd) raised against saw-scaled viper venom of Nigerian origin classified at the time as E. ocellatus s.l. (venom provided by LSTM), (ii) snake venom antiserum (Echis) “Echiven” (equine F[ab]’2 manufactured by VINS Bioproducts Ltd, India) raised against the venom of saw-scaled vipers from Cameroon, Ghana and Mali (provided by Latoxan, France, all listed as E. ocellatus) and (iii) “SAIMR Echis carinatus” antivenom (equine F[ab]’2 manufactured by South African Vaccine Producers PTY, South Africa) raised against E. ocellatus s. l. from Nigeria and E. pyramidum from Ethiopia and Eritrea [19]. An overview of antivenoms used in this study is displayed in Table 1, and evaluation of their protein concentration (by BCA assay) and degradation (by size exclusion chromatography) can be found in S1 Text and S1 Fig. In all subsequent experiments, the volume of antivenom was determined based on the maximum volume capacity permissible for each assay and/or the maximum antivenom volume that did not produce interference in antivenom-only control samples.
2.5 End-point ELISA
Venoms from E. ocellatus s. str. (Ghana), E. romani (Nigeria), and E. romani (Cameroon) were coated at 100 ng per well onto Nunc MaxiSorp ELISA plates (ThermoFisher) for one hour at 37 °C, washed with Tris-buffered saline containing 0.1% Tween20 (TBS-T), and then blocked with 5% milk in TBS-T for two hours at room temperature. Plates were washed in TBST before serial dilutions of each antivenom (diluted in blocking solution) were added to the plate and incubated overnight at 4 °C. The following day, plates were washed in TBS-T and anti-horse or anti-sheep IgG secondary antibodies conjugated to horseradish peroxidase (Sigma) were added at 1 in 1000 dilution in PBS for two hours at room temperature. Plates were washed six times with TBS-T and developed with 0.1 mg/mL ABTS substrate (in 0.05 M citrate buffer pH 5.0 with 0.0075% hydrogen peroxide) for 15 minutes at room temperature. The optical density at 405 nm (OD405) was read on a LT-4500 plate reader (Labtech). Control wells consisting of venom-naïve sheep IgG (Biorad) or horse F(ab’)2 (prepared from Biorad horse IgG, using Pierce F[ab’]2 preparation kit in accordance with manufacturers instructions) were diluted 1 in 25 or 1 in 5 respectively to match average protein concentration of antivenom 1 in 500 dilutions, then serial diluted as per antivenom. Secondary antibody only control wells were also included.
2.6 Phospholipase A2 assay
Neutralisation of venom phospholipase A2 (PLA2) activity was measured using an EnzCheck Phospholipase A2 assay kit (Invitrogen, UK #E10217), as previously described [20]. Optimisation of the amount of venom to be used for each species was first performed to identify the amount of venom that falls within the linear range of enzymatic activity measurements. The relative fluorescence units (RFU) were plotted against the amount of venom per well, and the graphs were manually assessed to identify venom amounts in the linear range of the assay (S2 Fig). The optimal venom amount was determined as 1 µg for all three venoms. Statistical analysis of venom PLA2 activity was by ordinary one-way ANOVA performed in Prism 10 (GraphPad, RRID:SCR_00279) and Tukey’s multiple comparison post-hoc test was performed on pairwise comparisons. Antivenoms were serial diluted in PBS containing the pre-defined amount of venom in a 384-well plate and incubated at 37 °C for 30 minutes, following which 12.5 µL PLA2 substrate was added to each well. Plates were incubated in the dark at room temperature for 10 minutes and then read at excitation 485–15 nm and emission 520–10 nm. RFU measurements were converted to PLA2 activity using the equation of the standard curve, and then expressed as percentage of activity (where the venom only control was 100% activity). For statistical analyses the data were analysed using two-way ANOVA (multiple comparisons) in Prism 9 (GraphPad, RRID:SCR_002798) to compare the antivenoms at each dilution.
2.7 Snake venom metalloproteinase assay
Snake venom metalloproteinase (SVMP) activity and neutralisation of the Echis venoms was measured using the previously described fluorogenic peptide assay [21], although venom amount was reduced to 500 ng to maintain the same signal window but allow greater resolution between antivenoms and dilutions. Briefly, 500 ng venom was added to wells in a 384-well plate, followed by 10 µL of serial dilutions of antivenom or an equal volume of PBS and incubated at 37 °C for 25 minutes. After cooling to room temperature, 90 µL SVMP substrate solution (ES010 [BioTechne] diluted to 7.86 µM in 150 mM NaCl, 50 mM Tris-Cl pH 7.5) was added to each well (7 µM final well concentration). The plate was immediately read at excitation 320–10 nm and emission 420–10 nm on a plate reader. SVMP activity was calculated for each venom, in which ‘venom only’ wells represent 100% activity and the change in SVMP activity in the presence of the test antivenoms was calculated as a percentage of the ‘venom only’ wells. Ordinary one-way ANOVA was performed in Prism 9 (GraphPad, RRID:SCR_00279) and Tukey’s multiple comparison post-hoc test was performed on pairwise comparisons.
2.8 Bovine plasma clotting assay
Plasma clotting activity of the Echis venoms and neutralisation by antivenoms was measured using a previously described bovine plasma clotting assay [22,23]. Briefly, 100 ng venom was added to each well in a 384-well plate, followed by 10 µL of serial dilutions of antivenom or an equal volume of PBS. The assay plate was incubated at 37 °C for 25 minutes then room temperature for 5 minutes, before 20 µL of 20 mM calcium chloride (Sigma, #C1016) followed by 20 µL of citrated bovine plasma (Biowest, VWR #S0260) was added to each well. The optical density was immediately read at a wavelength of 595 nm (OD595) on a plate reader. For analysis, the cross-section at which the ‘normal plasma clotting’ curve intersected the curves of the test conditions was manually identified and the area under the curve at this time point for each condition was calculated (normalised to venom and PBS only controls) before converting to percentage activity. Ordinary one-way ANOVA was performed in Prism 9 (GraphPad, RRID:SCR_002798), and Tukey’s multiple comparison post-hoc test was performed on pairwise comparisons.
2.9. Ethic statement
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, under project licence P24100D38 approved by the UK Home Office in accordance with the UK Animals (Scientific Procedures) Act 1986.
2.9.1 Preclinical experiments.
CD1 mice (male, 6 weeks, 18–20 g, Charles River UK) were grouped in cages of five upon arrival and acclimated for one week before experimentation in specific pathogen-free conditions.
Venom and antivenom were pre-mixed in a volume of 200 µL (diluted in PBS, pH 7.4) and incubated at 37 °C for 30 minutes prior to intravenous injection via the tail vein. Groups of five mice were used as per WHO assay guidelines [9] except in the case of missed or partial doses during injection, as indicated in S6 File. Total numbers used were n = 15 for LD50, 61 for ED50 and 15 for comparative neutralisation of lethality experiments. Mice were challenged with 5 X LD50, using previously reported LD50 values of 17.85 µg/mouse for E. romani (Nigeria) [24], 33.10 µg/mouse for E. romani (Cameroon) [17] and 18.20 µg/mouse E. ocellatus s. str. (Ghana) [25].
The median effective dose (ED50) assay was performed for E. romani (Nigeria) venom to determine the dose of each tested antivenom (µL) that prevented venom-induced lethality in 50% of animals injected with 5 x LD50. To compare the neutralising potential of the antivenoms against venoms of E. romani (Cameroon) and E. ocellatus s. str., we compared survival rates when animals were administered the dose (volume) of antivenom which prevented lethality in 100% of animals when challenged with 5 x LD50 of E. romani (Nigeria) venom (control group). Doses used were 100 µL for EchiTAbG, 25 µL for SAIMR Echis and 30 µL for Echiven.
Upon reaching Humane Endpoints (HEPs) or end of experiment, animals were euthanised using rising concentrations of carbon dioxide or cervical dislocation. Time to HEP, number of deaths and number of survivors over the 6-hour experiment duration were recorded as the experimental outcome. ED50 values were determined by Probit analysis using StatPlus in Microsoft Excel (AnalystSoft) and results are given as (i) volume of antivenom (µL), (ii) potency (mg/mL) using the equation P = (n – 1 * LD50)/ED50 where n = number of LD50 in the challenge dose [26], and (iii) μL of antivenom required to neutralise 1 mg of venom (µL/mg) [27].
3. Results
3.1 Establishing the taxonomic identity of captive E. ocellatus s. str. by mitochondrial barcoding
To categorically define if the saw-scaled viper venoms used for EchiTAbG and historical EchiTAb-Plus-ICP antivenom production (snakes originating from north-eastern Nigeria, housed in the herpetarium at LSTM) and considered as E. ocellatus s. l., are E. ocellatus s. str. or E. romani, we implemented a mitochondrial barcoding approach on 40 individuals collected between April 2008 and September 2014. We aligned 789 base pairs (b.p.) of CYTB and 644 b.p. of NADH4 sequence, the aligned fragments corresponding to those used in the previous phylogenetic analysis of the Echis genus [3]. The Model function in MEGA 11 identified the Tamura-Nei model [28] with gamma-distributed substitution rates (TN93 + G) as the optimal substitution model under the BIC for the data. Phylogenetic analysis of the NADH4 and CYTB sequences amplified from the shed skins or venoms of the 40 individual Nigerian E. ocellatus s. l. specimens in the LSTM collection demonstrate they are all E. romani (Fig 1). Similarly, NADH4 and CYTB sequences amplified from E. ocellatus s. l. venom originating from Cameroon, sourced from Latoxan, demonstrates clearly it has originated from specimens of E. romani, while the sequences amplified from E. ocellatus s. l. venom originating from Ghana, sourced from Latoxan, demonstrates this venom has originated from specimens of E. ocellatus s. str. (Fig 1). As a control, we also sequenced NADH4 and CYTB amplicons from DNA extracted from the skin shed of the E. carinatus specimen originating from Pakistan, with results confirming its E. carinatus designation. All the phylogenetic relationships determined were supported by high bootstrap values (>90). These findings strongly suggest that the EchiTAbG and EchiTAb-Plus-ICP antivenoms made using north-eastern Nigerian saw-scaled viper venoms housed at LSTM [5,12], were directed against E. romani (EchiTAb-Plus-ICP is now made with ‘E. ocellatus’ venom sourced from Latoxan, countries of origin not stated).
Node labels next to major nodes represent %bootstrap support.
3.2 Antivenom recognition of E. ocellatus s. str. and E. romani venoms by ELISA
High levels of comparable binding by all three antivenoms (EchiTAbG, SAIMR Echis and Echiven) was observed against the three venoms E. ocellatus s. str., E. romani (Nigeria) and E. romani (Cameroon) (S3 Fig). The binding titres remained above naïve control for all antivenoms against all venoms, to at least a 1 in 62,500 dilution of neat antivenom, although this was highest for EchiTAbG. However, there was also a significant difference between the naïve ovine whole IgG and equine F(ab’)2 controls, and therefore the apparent slight increase in recognition by EchiTAbG compared to SAIMR Echis or VINS Echis must be interpreted with caution, and may be due differences in the immunoglobulin format and to the differing efficacy of the two secondary antibodies used to detect their target.
3.3 PLA2 activity and neutralisation by antivenoms
The venoms demonstrated moderate differences in enzymatic PLA2 activity, as shown in Fig 2A. E. ocellatus s. str. activity had the highest activity at 3.25 [U/mL]/µg, compared to 1.75 [U/mL]/µg for E. romani (Cameroon), whereas E. romani (Nigeria) had negligible activity that was not significantly different to buffer only (p > 0.05). This venom was therefore omitted for further analysis regarding neutralisation. The ability of the three antivenoms to neutralise the in vitro PLA2 activity of venoms with significant PLA2 activity was determined using four different volumes of antivenom (Fig 2B-2C). The three antivenoms demonstrated strong neutralisation of PLA2 activity from E. ocellatus s. str. venom and Cameroonian E. romani venom at the highest amount of antivenom tested (Fig 2B and 2C). At the lowest volume of antivenom (0.78 µL), no significant differences were detected between the antivenoms against E. ocellatus s. str. (p > 0.8), whereas for E. romani (Cameroon) SAIMR Echis was significantly more effective than EchiTAbG and Echiven (p < 0.015 for both).
A: PLA2 activity of E. ocellatus (GHA = Ghana) and E. romani (CAM = Cameroon and NGA = Nigeria). Samples were subtracted for background and converted to activity in (U/mL)/μg by extrapolation from a bee venom standard curve. Data show the mean of three replicates and error bars represent standard deviation. Statistical differences in activity compared to ‘buffer only’ were determined by one-way ANOVA, with venom specific p values indicated above the bar (* indicates p < 0.05, *** indicates p < 0.001, ns = not significant, p > 0.05). B -C: Neutralisation of (B) E. ocellatus (Ghana), (C) E. romani (Cameroon) PLA2 activity by the three antivenoms EchiTAbG (ETG), SAIMR Echis (SE) and Echiven (EV) at different doses, expressed as a percentage of a no-antivenom control showing 100% activity. Data show the mean of three replicates and error bars represent standard deviation. Two-way ANOVA was performed to compare differences in PLA2 activity at the 0.78 μL dose of antivenoms. * indicates p < 0.05, ** indicates p < 0.01 *** indicates p < 0.001, ns = not significant (p > 0.05).
3.4 SVMP activity and neutralisation by antivenoms
The Echis venoms exhibited strong SVMP activity in the in vitro assay compared to PBS control (p < 0.0001), as shown in Fig 3A. E. ocellatus s. str. and E. romani (Nigeria) venoms had comparable SVMP activity (p > 0.05, denoted by ^ on Fig 3A), and had significantly greater SVMP activity than the E. romani (Cameroon) venom (p = 0.0002, denoted by #). The ability of the three antivenoms to neutralise the SVMP activity of the three venoms was determined using four different antivenom volumes (Fig 3B-3D). At 2.5 µL of antivenom, all three antivenoms reduced the SVMP activity of the three venoms by at least 30%, albeit with large variability between venoms and between antivenoms. Indeed, for E. ocellatus (Ghana) and E. romani (Cameroon), significant reductions were seen by all antivenoms at all volumes tested (p < 0.0001 for all comparisons to no antivenom). However, against E. romani (Nigeria), only EchTAbG provided significant reductions beyond the highest antivenom dose (p > 0.05 at 1.25 µL for both SAIMR Echis and Echiven). At the lowest volume tested (0.313 µL), there were no significant differences between antivenoms against each of the venoms, but they were all less effective against E. romani (Nigeria) with reductions of less than 12% observed (Fig 3D).
A: SVMP activity of E. ocellatus (GHA = Ghana) and E. romani (CAM = Cameroon and NGA = Nigeria). Data show the mean of four replicates and error bars represent standard deviation. Statistical differences in activity compared to PBS, and between venoms, were determined by one-way ANOVA, with p values against PBS indicated above the bar (**** indicates p < 0.0001), and # indicating significantly lower activity and ^ indicating significantly higher activity. B-D: Neutralisation of (B) E. ocellatus (Ghana), (C) E. romani (Cameroon) and (D) E. romani (Nigeria) SVMP activity by the three antivenoms EchiTAbG (ETG), SAIMR Echis (SE) and Echiven (EV) at different doses, expressed as a percentage of a no antivenom control showing 100% activity. Data show the mean of four replicates and error bars represent standard deviation. One-way ANOVA was performed to compare differences in SVMP activity at the 0.31 μL dose of antivenoms. ns = not significant (p > 0.05).
3.5 Plasma clotting activity and neutralisation by antivenoms
Each venom possessed a significant ability to cause clotting of bovine plasma compared to PBS control (p < 0.0001), with comparable activity between the venoms (p > 0.05, Fig 4A). When tested at 2.5 µL per well, each antivenom was able to reduce clotting activity against all three venoms, however, the percentage of clotting activity remaining was variable, particularly for antivenoms against the same venom. Whilst SAIMR Echis and Echiven were able to significantly reduce coagulopathic activity to approximately 50% for all three venoms (p < 0.0001 for all antivenom volume comparisons to no antivenom), EchiTAbG could only achieve this against E. ocellatus s. str. and E. romani (Cameroon) with declining significant reductions against E. romani (Nigeria) (p = 0.0034 at 0.63 µL and p = 0.017 at 0.313 µL). At the lowest volume of antivenom tested (0.313 µL), there were significant differences between the effectiveness of the antivenoms to modulate clotting activity. SAIMR Echis was significantly better at reducing the clotting activity of the Nigerian locality of E. romani than Echiven or EchiTAbG (reduced to < 50% vs 70–90% respectively, p < 0.0001) (Fig 4D), whilst against the E. ocellatus (Fig 4B) and Cameroon locality (Fig 4C), SAIMR Echis and Echiven were both significantly better than EchiTAbG (~50% compared to ~70%, p < 0.0001).
A: Plasma clotting activity of E. ocellatus (GHA = Ghana) and E. romani (CAM = Cameroon and NGA = Nigeria). Data show the mean of four replicates and error bars represent standard deviation. Statistical differences in activity compared to PBS, and between venoms, were determined by one-way ANOVA, with p values against PBS indicated above the bar (**** indicates p < 0.0001). B-D: Neutralisation of (B) E. ocellatus (Ghana), (C) E. romani (Cameroon) and (D) E. romani (Nigeria) plasma clotting activity by the three antivenoms EchiTAbG (ETG), SAIMR Echis (SE) and Echiven (EV) at different doses, expressed as a percentage of a no antivenom control showing 100% activity. Data show the mean of four replicates and error bars represent standard deviation. One-way ANOVA was performed to compare differences in plasma clotting activity at the 0.31 μL dose of antivenoms. **** indicates p < 0.0001, *** indicates p < 0.001, ns = not significant (p > 0.05).
3.6 Ability of antivenoms to neutralise murine venom induced lethality
The antivenom efficacies and potencies were determined for each antivenom against the venom of E. romani (Nigeria) and are presented in Table 2. These experiments demonstrated notable differences in the capacity of antivenoms to neutralise E. romani (Nigeria) venom induced lethality. By volume of antivenom and potency, SAIMR Echis was the most efficacious antivenom. In comparison, Echiven was two-fold less potent and EchiTAbG was seven-fold less potent.
We next implemented a comparative neutralisation of lethality assay, similar to one previously utilised for comparing the efficacy of various polyvalent antivenoms for East Africa [29]. We examined the ability of each antivenom to neutralise 5 x LD50s of E. romani (Cameroon) and E. ocellatus s. str. venom compared to a fixed venom dose - the volumes of antivenoms that conferred 100% survival against Nigerian E. romani venom (EchiTAbG 100 µL, SAIMR Echis 25 µL and Echiven 30 µL).
All three antivenoms provided only partial protection against 5 x LD50 of E. romani (Cameroon) venom at these doses (SAIMR Echis 80% survival, Echiven 40% survival, and EchiTAbG 20% survival) (Fig 5A). When using the same doses against 5 x LD50 of E. ocellatus (Ghana) venom, both SAIMR Echis and Echiven provided complete protection, with 100% of mice surviving until the end of experiment (Fig 5B). In contrast, EchiTAbG failed to prevent lethality but further increased time to humane endpoints (Fig 5C, mean survival times 95 minutes, compared to 6 minutes with no antivenom).
A: E. ocellatus (Ghana) B: E. romani (Cameroon) C: Mean survival time of animals – bars indicate mean survival time and markers indicate individual survival times for each animal. Each experiment used five mice per dose group, challenged with a dose of 5 x venom LD50 and monitored for 6 hours. EchiTAbG shown in grey, SAIMR Echis shown in magenta, Echiven shown in teal.
Discussion
Given the medical significance of envenoming by snakes of the genus Echis [30], the identification of efficacious antivenoms suitable to treat such snakebites is integral to the WHO’s objectives to halve snakebite mortality and morbidity by 2030 [31]. This preclinical study aimed to test available Echis-specific monospecific antivenoms and directly compare their ability to neutralise E. ocellatus sensu lato venoms, both in vitro and in preclinical murine models of envenoming. This is particularly important considering the recent changes in taxonomy to the genus which has seen E. ocellatus, historically viewed as the most medically important species of the genus in Africa [6], split into E. romani and E. ocellatus s. str. [7]. The recent taxonomic change raised important questions regarding differences in venom composition between the newly identified E. romani and E. ocellatus, and naturally raised questions around potential efficacy of antivenoms indicated for E. ocellatus pre-species partition, which we sought to address in this study.
The saw-scaled vipers housed in the LSTM herpetarium that were barcoded in this study all originate from the Kaltungo (Gombe) region of north-eastern Nigeria. The barcoding results presented here clearly demonstrate that all these animals, historically considered E. ocellatus s. l. prior to partition, are E. romani, further evidencing the apparent distinct geographical ranges of the newly partitioned species [3,7]. Furthermore, based on the genetic barcoding results presented, it is likely that several existing antivenoms indicated for E. ocellatus envenoming are highly likely to have been manufactured using a mixture of E. romani and E. ocellatus venom or solely E. romani venom. The latter will certainly be the case with EchiTAbG and previous iterations of the trivalent antivenom EchiTAb-Plus-ICP, which have been manufactured using the venom of some of the snakes barcoded in this study [12].
The main proteinaceous components of Echis venoms are SVMPs, PLA2, C-type lectin-like proteins, serine proteases, disintegrins, and L-amino acid oxidases [17,32,33]. In particular, the Echis genus of snakes have a remarkably high abundance of SVMPs, and specifically for E. ocellatus/romani venoms which comprise up to 70% of the venom [34]. These zinc-dependent proteinases play a fundamental role in driving venom-induced consumption coagulopathy and systemic haemorrhage [35]. The in vitro SVMP activity of each of the three venoms were significantly neutralised by the highest dose of each antivenom. With regards to PLA2, a previous transcriptome analysis of various Echis species [36] demonstrated proportional differences in relative Group II PLA2 abundance across different species, with markedly less detected in E. romani (previously E. ocellatus) [36]. Similarly, venomic analyses of E. romani (previously E. ocellatus) from different locales demonstrated intraspecies differences in the abundance of PLA2 [37], and this was demonstrated by evident differences observed in our in vitro PLA2 assay. Bearing in mind the known variation in venom toxins and subsequent activity, this reiterates the importance of carefully evaluating the source of venoms used for antivenom production and the need for thorough and transparent preclinical testing of proposed species efficacy. Our ELISA results demonstrated little to no difference with regards to the ability of each antivenom to recognise each venom, and suggests that, for Echis species at least, this may not be an appropriate method for estimating neutralising capabilities given the differences observed in the functional in vitro assays.
Whilst in vitro assays are an important tool to identify potential efficacious snakebite treatments, preclinical efficacy testing remains heavily reliant on murine neutralisation of lethality assays due to the complexity and multiplicity of venom activities in vivo. The in vivo efficacy results presented here demonstrate that each Echis monospecific antivenom was capable of neutralising the lethal effects of Nigerian E. romani, however notable differences are seen in their comparative preclinical potency against venoms from E. ocellatus and other geographic locales of E. romani. The calculated ED50 values were in broad agreement with previously calculated ED50 values for EchiTAbG and SAIMR Echis against Nigerian E. romani (formerly E. ocellatus) venom [38], with SAIMR Echis possessing the most potent venom-neutralising ability. When accounting for total protein concentration of the three Echis specific antivenoms (Table 1), the differences in neutralising efficacy between products were more modest, although the trends in neutralising ability remained. In addition to total protein content, the antivenoms also had different formulations which can be difficult to control for. However, previous studies comparing whole IgG and F(ab)’2 against envenomation by Bothrops asper found no significant differences between the two formats [27,39].
When the antivenoms were assessed further for cross-reactivity, intra-country and intra-species differences became apparent. The dose of each antivenom that neutralised 100% of lethality against E. romani (Nigeria) was unable to fully protect mice challenged with E. romani venom from Cameroon, whilst SAIMR Echis and Echiven fully prevented lethality from E. ocellatus s. str., but EchiTAbG could not at the dose tested. The results mirrored the in vitro and ED50 findings, with substantial differences in dose-matched potency of antivenoms against other species. The most notable finding was the lower ability of antivenoms to protect mice from E. romani (Cameroon) envenoming when using a protective E. romani (Nigeria) antivenom dose, with 60% and 80% of mice succumbing to venom effects when dosed with Echiven and EchiTAbG, respectively. This suggests that E. romani venoms from different localities have different potencies and thus differences in their ability to be neutralised by antivenoms, meriting further research to understand the impact of intraspecific E. romani venom variation on antivenom efficacy [40].
These findings illustrates how, given the frequency of sometimes extreme venom variation within species, even in the face of extensive gene flow [41], taxonomic revisions should be seen as broad roadmaps for additional research into antivenom efficacy, but not interpreted as robust predictors of venom composition or antivenom effectiveness [42]. In view of the public health importance of the E. ocellatus complex, further research into variation in venom composition within the group would be advisable. It is also important to note here that only a single dose has been examined to enable comparative analysis of the ability of an antivenom to neutralise lethal pathology of different venoms at that dose, and results should therefore be viewed in this context and treated with caution and not extrapolated to the human scenario. EchiTAbG has been proven to be clinically effective in
Nigeria [5,11,12], and has a WHO positive risk-benefit assessment [43] for treatment against E. ocellatus from Ghana, E. romani from Cameroon and E. pyramidum venoms from Egypt and Kenya (personal correspondence). Whilst previous polyvalent products produced by VINS for use in Africa have not proven efficacious in independent testing [29,37] our murine model of systemic envenoming demonstrated both SAIMR Echis and the sample of Echiven provided by VINS had relative superior dose efficacy for all venoms investigated compared to EchiTAbG.
In this study we chose to implement a comparator model of envenoming – where animals are given the same dose of antivenom against different venoms and survival is compared - to reduce the number of animals required to compare whether the antivenoms demonstrated similar preclinical neutralising efficacy against closely related venoms. Such comparator models of envenoming therapy efficacy have been used successfully in the past to discriminate between different antivenom efficacies whilst substantially reducing the number of animals and capital cost required as compared to standard ED50 testing [29]. Due to their inherent limitations, such models cannot provide direct comparison of the neutralising potency of different antivenoms against a particular venom, as can ED50 experiments, but they do allow inference of potential efficacy and limitations in neutralising potential at a single comparable dose. The comparison of expired and non-expired antivenom could be considered as a limitation. However, the efficacy of antivenoms has been demonstrated to be maintained decades after expiry [44–46], and therefore we are confident the use of expired antivenoms has had minimal to no impact on this study. Additionally, we used SEC analysis of the antivenoms (see S1 File) which demonstrated most of the protein content in each antivenom sample was of the expected size, i.e., non-degraded.
In summary, all antivenoms conveyed a degree of intra-genus preclinical neutralisation amongst the sub-Saharan African Echis venoms, although this was highly variable across the different Echis species and the three Echis monospecific antivenoms tested. Ultimately, our data provides the first empirical evidence of differences in venom potencies and antivenom efficacies against the recently partitioned medically important west African saw-scaled viper species E. romani and E. ocellatus.
Supporting information
S1 Text. Contains further methodological detail on: venom storage, DNA extraction (from venoms and skins), PCR, Sanger sequencing, antivenom reconstitution and control immunoglobulins, size exclusion chromatography, ELISA, PLA2 assay, SVMP assay, plasma clotting assay, preclinical assays.
https://doi.org/10.1371/journal.pntd.0013371.s001
(DOCX)
S1 Fig. Size exclusion chromatography (SEC) analysis of the three antivenoms and control proteins.
Analysis was carried out using a Superdex 200 SEC column equilibrated in PBS and elution was monitored at 214 nm. Fifty μL (50 μg) of sample was loaded. The native molecular weights (kDa) of the proteins are indicated above the respective peak.: SEC analysis of (A) control immunoglobulins whole sheep IgG (blue) and equine F(ab’)2 (gold), (B) whole sheep IgG (blue) and EchiTAbG (gold) and (C) equine F(ab’)2 (blue), SAIMR Echis (gold) and Echiven (‘VINS’, green).
https://doi.org/10.1371/journal.pntd.0013371.s008
(TIF)
S2 Fig. PLA2 assay optimisation.
Graphs show the fluorescence intensity measured in the EnzCheck PLA2 assay with different amounts of each venom. Amounts of venom that fall in the linear range were used for subsequent assays of venom PLA2 neutralisation by antivenoms. E. ocellatus (Ghana) shown in magenta, E. romani (Cameroon) shown in black and E. romani (Nigeria) shown in teal. Samples were subtracted for background fluorescence. Data show the mean of three replicates and error bars represent standard deviation.
https://doi.org/10.1371/journal.pntd.0013371.s009
(TIF)
S3 Fig. The titre of three antivenoms against E. ocellatus s. str. and E. romani venoms determined by end-point titration ELISA.
EchiTAbG shown in grey, SAIMR Echis shown in magenta, and Echiven shown in teal. Venom-naïve equine F(ab)’2 shown in dark purple and venom-naïve ovine IgG shown in light purple. Panel A: E. ocellatus (Ghana). Panel B: E. romani (Cameroon). Panel C: E. romani (Nigeria). Data points represent the mean of two replicates and error bars show the standard deviation.
https://doi.org/10.1371/journal.pntd.0013371.s010
(TIF)
Acknowledgments
We are grateful to Paul Rowley (LSTM) for the care of, and venom extraction from, some of the snakes used in this study. We also acknowledge the EchiTAb Study Group in partnership with the Federal Ministry of Health, Republic of Nigeria who were responsible for the collection of the individual E. ocellatus sensu lato snakes that underwent mitochondrial barcoding in this study. We gratefully acknowledge VINS Bioproducts (India) for the provision of Echiven antivenom for use in this study. The authors acknowledge use of the Biomedical Services Unit provided by Liverpool Shared Research Facilities, Faculty of Health and Life Sciences, University of Liverpool.
References
- 1. Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nat Rev Dis Primers. 2017;3:17063. pmid:28905944
- 2. Warrell DA, Arnett C. The importance of bites by the saw-scaled or carpet viper (Echis carinatus): epidemiological studies in Nigeria and a review of the world literature. Acta Trop. 1976;33(4):307–41. pmid:14490
- 3. Pook CE, Joger U, Stümpel N, Wüster W. When continents collide: phylogeny, historical biogeography and systematics of the medically important viper genus Echis (Squamata: Serpentes: Viperidae). Mol Phylogenet Evol. 2009;53(3):792–807. pmid:19666129
- 4. Hamza M, Idris MA, Maiyaki MB, Lamorde M, Chippaux J-P, Warrell DA, et al. Cost-Effectiveness of Antivenoms for Snakebite Envenoming in 16 Countries in West Africa. PLoS Negl Trop Dis. 2016;10(3):e0004568. pmid:27027633
- 5. Abubakar IS, Abubakar SB, Habib AG, Nasidi A, Durfa N, Yusuf PO, et al. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria. PLoS Negl Trop Dis. 2010;4(7):e767. pmid:20668549
- 6. Warrell DA, Davidson NM c D, Greenwood BM, Ormerod LD, Pope HM, Watkins BJ. Poisoning by bites of the saw-scaled or carpet viper (Echis carinatus) in Nigeria. Q J Med. 1977;46:33–62.
- 7.
Trape JF. Partition d’Echis ocellatus Stemmler, 1970 (Squamata, Viperidae), avec la description d’une espèce nouvelle. 2018;13–34.
- 8.
Sprawls S, Mohammad A, Mazuch T. Handbook of Amphibians and Reptiles of Northeast Africa. London: Bloomsbury Wildlife. 2023.
- 9.
WHO. Guidelines for the production, control and regulation of snake antivenom immunoglobulins, Annex 5, TRS No 1004. 2013. https://www.who.int/publications/m/item/snake-antivenom-immunoglobulins-annex-5-trs-no-1004
- 10. León G, Vargas M, Segura Á, Herrera M, Villalta M, Sánchez A, et al. Current technology for the industrial manufacture of snake antivenoms. Toxicon. 2018;151:63–73. pmid:29959968
- 11. Potet J, Smith J, McIver L. Reviewing evidence of the clinical effectiveness of commercially available antivenoms in sub-Saharan Africa identifies the need for a multi-centre, multi-antivenom clinical trial. PLoS Negl Trop Dis. 2019;13(6):e0007551. pmid:31233536
- 12. Abubakar SB, Abubakar IS, Habib AG, Nasidi A, Durfa N, Yusuf PO, et al. Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria. Toxicon. 2010;55(4):719–23. pmid:19874841
- 13. Hamman NA, Ibrahim AD, Hamza M, Jahun MG, Micah M, Lawal HA, et al. A Randomized Controlled Trial to Optimize Antivenom Therapy for Carpet Viper (Echis romani)-Envenomed Children in Nigeria. Am J Trop Med Hyg. 2024;111(4):904–10. pmid:39106853
- 14. Warrell DA, Davidson NM, Omerod LD, Pope HM, Watkins BJ, Greenwood BM, et al. Bites by the saw-scaled or carpet viper (Echis carinatus): trial of two specific antivenoms. Br Med J. 1974;4(5942):437–40. pmid:4154124
- 15. Swinson C. Control of antivenom treatment in Echis carinatus (Carpet Viper) poisoning. Trans R Soc Trop Med Hyg. 1976;70(1):85–7. pmid:1265825
- 16. Ainsworth S, Menzies SK, Casewell NR, Harrison RA. An analysis of preclinical efficacy testing of antivenoms for sub-Saharan Africa: Inadequate independent scrutiny and poor-quality reporting are barriers to improving snakebite treatment and management. PLoS Negl Trop Dis. 2020;14(8):e0008579. pmid:32817682
- 17. Sánchez LV, Pla D, Herrera M, Chippaux JP, Calvete JJ, Gutiérrez JM. Evaluation of the preclinical efficacy of four antivenoms, distributed in sub-Saharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria. Toxicon. 2015;106:97–107. pmid:26415904
- 18. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38(7):3022–7. pmid:33892491
- 19. Rogalski A, Soerensen C, Op den Brouw B, Lister C, Dashevsky D, Arbuckle K, et al. Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies. Toxicol Lett. 2017;280:159–70. pmid:28847519
- 20. Ainsworth S, Petras D, Engmark M, Süssmuth RD, Whiteley G, Albulescu L-O, et al. The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms. J Proteomics. 2018;172:173–89. pmid:28843532
- 21. Albulescu L-O, Hale MS, Ainsworth S, Alsolaiss J, Crittenden E, Calvete JJ, et al. Preclinical validation of a repurposed metal chelator as an early-intervention therapeutic for hemotoxic snakebite. Sci Transl Med. 2020;12(542):eaay8314. pmid:32376771
- 22. Albulescu L-O, Xie C, Ainsworth S, Alsolaiss J, Crittenden E, Dawson CA, et al. A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite. Nat Commun. 2020;11(1):6094. pmid:33323937
- 23. Still KBM, Nandlal RSS, Slagboom J, Somsen GW, Casewell NR, Kool J. Multipurpose HTS Coagulation Analysis: Assay Development and Assessment of Coagulopathic Snake Venoms. Toxins (Basel). 2017;9(12):382. pmid:29186818
- 24. Ainsworth S, Slagboom J, Alomran N, Pla D, Alhamdi Y, King SI, et al. The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms. Commun Biol. 2018;1:34. pmid:30271920
- 25. Arias AS, Rucavado A, Gutiérrez JM. Peptidomimetic hydroxamate metalloproteinase inhibitors abrogate local and systemic toxicity induced by Echis ocellatus (saw-scaled) snake venom. Toxicon. 2017;132:40–9. pmid:28400263
- 26.
WHO. Target product profiles for animal plasma-derived antivenoms: antivenoms for treatment of snakebite envenoming in sub-Saharan Africa. 2023. https://www.who.int/publications-detail-redirect/9789240074569
- 27. León G, Rojas G, Lomonte B, Gutiérrez JM. Immunoglobulin G and F(ab’)2 polyvalent antivenoms do not differ in their ability to neutralize hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom. Toxicon. 1997;35(11):1627–37. pmid:9428109
- 28. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10(3):512–26. pmid:8336541
- 29. Harrison RA, Oluoch GO, Ainsworth S, Alsolaiss J, Bolton F, Arias A-S, et al. Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa. PLoS Negl Trop Dis. 2017;11(10):e0005969. pmid:29045429
- 30. Habib AG, Kuznik A, Hamza M, Abdullahi MI, Chedi BA, Chippaux J-P, et al. Snakebite is Under Appreciated: Appraisal of Burden from West Africa. PLoS Negl Trop Dis. 2015;9(9):e0004088. pmid:26398046
- 31. Minghui R, Malecela MN, Cooke E, Abela-Ridder B. WHO’s Snakebite Envenoming Strategy for prevention and control. Lancet Glob Health. 2019;7(7):e837–8. pmid:31129124
- 32. Tasoulis T, Isbister GK. A Review and Database of Snake Venom Proteomes. Toxins (Basel). 2017;9(9):290. pmid:28927001
- 33. Wagstaff SC, Sanz L, Juárez P, Harrison RA, Calvete JJ. Combined snake venomics and venom gland transcriptomic analysis of the ocellated carpet viper, Echis ocellatus. J Proteomics. 2009;71(6):609–23. pmid:19026773
- 34. Damm M, Hempel B-F, Süssmuth RD. Old World Vipers-A Review about Snake Venom Proteomics of Viperinae and Their Variations. Toxins (Basel). 2021;13(6):427. pmid:34204565
- 35. Howes J-M, Kamiguti AS, Theakston RDG, Wilkinson MC, Laing GD. Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets. Biochim Biophys Acta. 2005;1724(1–2):194–202. pmid:15863354
- 36. Casewell NR, Harrison RA, Wüster W, Wagstaff SC. Comparative venom gland transcriptome surveys of the saw-scaled vipers (Viperidae: Echis) reveal substantial intra-family gene diversity and novel venom transcripts. BMC Genomics. 2009;10:564. pmid:19948012
- 37. Calvete JJ, Arias AS, Rodríguez Y, Quesada-Bernat S, Sánchez LV, Chippaux JP, et al. Preclinical evaluation of three polyspecific antivenoms against the venom of Echis ocellatus: Neutralization of toxic activities and antivenomics. Toxicon. 2016;119:280–8. pmid:27377229
- 38. Casewell NR, Cook DAN, Wagstaff SC, Nasidi A, Durfa N, Wüster W, et al. Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom. PLoS Negl Trop Dis. 2010;4(10):e851. pmid:21049058
- 39. León G, Monge M, Rojas E, Lomonte B, Gutiérrez JM. Comparison between IgG and F(ab’)(2) polyvalent antivenoms: neutralization of systemic effects induced by Bothrops asper venom in mice, extravasation to muscle tissue, and potential for induction of adverse reactions. Toxicon. 2001;39(6):793–801. pmid:11137538
- 40. Sánchez A, Coto J, Segura Á, Vargas M, Solano G, Herrera M, et al. Effect of geographical variation of Echis ocellatus, Naja nigricollis and Bitis arietans venoms on their neutralization by homologous and heterologous antivenoms. Toxicon. 2015;108:80–3. pmid:26450770
- 41. Zancolli G, Calvete JJ, Cardwell MD, Greene HW, Hayes WK, Hegarty MJ, et al. When one phenotype is not enough: divergent evolutionary trajectories govern venom variation in a widespread rattlesnake species. Proc Biol Sci. 2019;286(1898):20182735. pmid:30862287
- 42. Malhotra A, Wüster W, Owens JB, Hodges CW, Jesudasan A, Ch G, et al. Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base. Toxicon X. 2021;12:100081. pmid:34522881
- 43.
WHO. List of Product Assessment Outcomes | WHO - Prequalification of Medical Products (IVDs, Medicines, Vaccines and Immunization Devices, Vector Control). 2019. [cited 2024 March 12]. Available from: https://extranet.who.int/prequal/vaccines/list-product-assessment-outcomes
- 44. Solano G, Cunningham S, Edge RJ, Duran G, Sanchez A, Villalta M, et al. 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. BMJ Glob Health. 2024;9(3):e014813. pmid:38485142
- 45. Soopairin S, Patikorn C, Taychakhoonavudh S. Preclinical testing of expired antivenoms and its uses in real-world experience: a systematic review. Emerg Med J. 2024;41(9):551–9. pmid:38844330
- 46. Blessmann J, Hanlodsomphou S, Santisouk B, Krumkamp R, Kreuels B, Ismail AK, et al. Experience of using expired lyophilized snake antivenom during a medical emergency situation in Lao People’s Democratic Republic--A possible untapped resource to tackle antivenom shortage in Southeast Asia. Trop Med Int Health. 2023;28(1):64–70. pmid:36416013