Article

Preclinical evaluation of three polyspecific antivenoms against the venom of Echis ocellatus: Neutralization of toxic activities and antivenomics

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  • Instituto de Biomedicina de Valencia
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Abstract

Snakebite envenoming has a heavy burden in the public health in sub-Saharan Africa. The viperid species Echis ocellatus (carpet viper or saw-scaled viper) is the medically most important snake in the savannahs of western sub-Saharan Africa. Several antivenoms are being distributed and used in this region for the treatment of envenomings by E. ocellatus, but the preclinical efficacy of some of these antivenoms has not been assessed. The present study evaluated the preclinical efficacy against E. ocellatus venom of three polyspecific antivenoms: (a) Snake Venom Antiserum (Pan Africa), manufactured by Premium Serums and Vaccines (India); (b) Snake Venom Antiserum (Africa), manufactured by VINS Bioproducts (India); and (c) Antivipmyn® Africa, manufactured by Instituto Bioclon (Mexico). Antivenomics analysis revealed the ability of the three antivenoms to immunocapture the majority of components of the venoms of E. ocellatus from Cameroon, Nigeria and Mali, although their maximal immunocapturing capability varied. Bioclon and Premium Serums antivenoms were effective in the neutralization of lethal, hemorrhagic and in vitro coagulant activities of the venom of E. ocellatus from Cameroon, albeit with different potencies. VINS antivenom neutralized hemorrhagic activity of this venom, but failed to neutralize lethality at the highest antivenom dose tested, and had a low neutralizing efficacy against in vitro coagulant effect.

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... Control group of mice were injected venom incubated with saline only. Neutralizing ability of antivenoms were expressed as Median Effective dose i.e. the mg venom/g antivenom ratio in which activity of venom was reduced by 50% (Gutierrez et al., 1990;Calvete et al., 2016). ...
... Control group of mice was injected with same dose of venom incubated with saline only. Neutralizing ability were expressed as Median Effective dose i.e. the mg venom/g antivenom ratio in which activity of venom was reduced by 50% (Gutierrez et al., 1990;Calvete et al., 2016). ...
... Mice were monitored for 24hr and neutralizing ability of antivenoms was expressed as Median Effective Dose (ED 50 ) i.e. the ratio of mg venom/g antivenom in which 50% of injected mice survived. ED50 value was estimated by probits (Sanchez et al., 2015;Calvete et al., 2016). ...
Article
Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested invitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom).
... The case of neutralization of in vitro coagulant activity illustrates this point. When expressed in terms of mg venom neutralized per mL antivenom, the most effective antivenom had an ED 50 of 12.88 ± 0.09 mg/mL, whereas the antivenom with the lowest efficacy had an ED 50 of 0.1 ± 0.01 mg/mL, a 128 fold difference [91]. ...
... However, there are other regions in the world, such as sub-Saharan Africa, in which no parameters for antivenom acceptance have been defined yet. Consequently, antivenoms of highly variable potencies are distributed [91,93]. Hence, there is a need to establish acceptable limits of potency of antivenoms at the preclinical level, a task that demands the concerted work of laboratory scientists, clinical researchers, regulatory agencies, antivenom manufacturers, and the WHO and its regional offices. ...
... % retained proteins = [retained/(retained + non-retained)] × 100 Maximum binding capacity of an antivenom for the different venom antigens can be estimated through a series of experiments where the amount of venom pumped through the immunoaffinity column is varied to estimate the µg of each venom component per mg antivenom protein that saturates their specific antibody binding sites [91]. ...
Article
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Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of “antivenomics”, which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.
... and Echis spp. venoms [29,36,47,48]. According to our findings, however, the venom of B. arietans generated a relatively limited immune response in rabbits when tested against venoms of the other three Bitis species. ...
... The protocol can be used for inferring antigenic similarities of venoms of clinically relevant snake species for other venoms in sub-Saharan Africa and other regions as well. This information can be complemented by other approaches to determine antigenic similarities between venoms, such as antivenomics [8,48]. ...
Article
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Background Snakebite envenomation exerts a heavy toll in sub-Saharan Africa. The design and production of effective polyspecific antivenoms for this region demand a better understanding of the immunological characteristics of the different venoms from the most medically important snakes, to select the most appropriate venom combinations for generating antivenoms of wide neutralizing scope. Bitis spp. and Echis spp. represent the most important viperid snake genera in Africa. Methodology/Principal findings Eight rabbit-derived monospecific antisera were raised against the venoms of four species of Bitis spp. and four species of Echis spp. The effects of immunization in the rabbits were assessed, as well as the development of antibody titers, as judged by immunochemical assays and neutralization of lethal, hemorrhagic, and in vitro coagulant effects. At the end of immunizations, local and pulmonary hemorrhage, together with slight increments in the plasma activity of creatine kinase (CK), were observed owing to the action of hemorrhagic and myotoxic venom components. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within each genus, although some antisera provided a more extensive cross-reactivity than others. The venoms that generated antisera with the broadest coverage were those of Bitis gabonica and B . rhinoceros within Bitis spp. and Echis leucogaster within Echis spp. Conclusions/Significance The methodology followed in this study provides a rational basis for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against viperid venoms in sub-Saharan Africa. Results suggest that the venoms of B . gabonica , B . rhinoceros , and E . leucogaster generate antisera with the broadest cross-reactivity within their genera. These experimental results in rabbits need to be translated to large animals used in antivenom production to assess whether these predictions are reproduced in horses or sheep.
... VINS Snake Venom Antiserum (Pan Africa) was the only antivenom described as lacking efficacy against a venom for which it is indicated for (E. ocellatus [27]). ...
... EchiTAb-Plus-ICP was also shown to be clinically effective in clinical trials against Nigerian E. ocellatus [34]. The ED 50 of VINS Snake Venom Antiserum (Pan Africa) vs. E. ocellatus was examined once and determined to be greater than 4000 μl/mg, a value which the authors decided demonstrated a lack of preclinical efficacy against E. ocellatus venom [27]. Despite this, VINS Snake Venom Antiserum (Pan Africa) is currently indicated for use for treating envenoming by this species (Table 1). ...
Article
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Background The World Health Organization’s strategy to halve snakebite mortality and morbidity by 2030 includes an emphasis on a risk-benefit process assessing the preclinical efficacy of antivenoms manufactured for sub-Saharan Africa. To assist this process, we systematically collected, standardised and analysed all publicly available data on the preclinical efficacy of antivenoms designed for sub-Saharan Africa. Methodology/Principal findings Using a systematic search of publication databases, we focused on publicly available preclinical reports of the efficacy of 16 antivenom products available in sub Saharan Africa. Publications since 1999 reporting the industry standard intravenous pre-incubation method of murine in vivo neutralisation of venom lethality (median effective dose [ED50]) were included. Eighteen publications met the criteria. To permit comparison of the several different reported ED50 values, it was necessary to standardise these to microlitre of antivenom resulting in 50% survival of mice challenged per milligram of venom (μl/mg). We were unable to identify publicly available preclinical data on four antivenoms, whilst data for six polyspecific antivenoms were restricted to a small number of venoms. Only four antivenoms were tested against a wide range of venoms. Examination of these studies for the reporting of key metrics required for interpreting antivenom ED50s were highly variable, as evidenced by eight different units being used for the described ED50 values. Conclusions/Significance There is a disturbing lack of (i) preclinical efficacy testing of antivenom for sub Saharan Africa, (ii) publicly available reports and (iii) independent scrutiny of this medically important data. Where reports do exist, the methods and metrics used are highly variable. This prevents comprehensive meta-analysis of antivenom preclinical efficacy, and severely reduces the utility of antivenom ED50 results in the decision making of physicians treating patients and of national and international health agencies. Here, we propose the use of a standardised result reporting checklist to resolve this issue. Implementation of these straightforward steps will deliver uniform evaluation of products across laboratories, facilitate meta-analyses, and contribute vital information for designing the clinical trials needed to achieve the WHO target of halving snakebite morbidity and mortality by 2030.
... Consequently, while antivenoms are typically marketed as a therapeutic treatment for envenomation by a given species, intraspecific venom variation can reduce antivenom efficacy, dependent upon the difference between the venom composition of the individuals which were used for antivenom manufacture and the individual which delivered the bite (Bénard-Valle et al., 2015;Boyer et al., 2015). It is well documented that the success rates achieved by different antivenoms in treating Echis snakebites can vary significantly depending on the geographical location of the bite due to regional variation of the species' venom (I.S. Calvete et al., 2016;Casewell et al., 2010), which can translate into catastrophic treatment failure and case fatality rates increased by an order of magnitude in a tropical clinical setting (Alirol et al., 2015;Visser et al., 2008;Warrell and Arnett, 1976;Warrell et al., 1980). ...
... Thus the VINS antivenom can be predicted to have little or no likely clinical usefulness against the South Asian or African venoms tested in this study without requiring extremely large doses relative to the other antivenoms studied. These findings agree with a previous study showing that VINS antivenom is highly inefficient in the neutralisation of E. ocellatus from Cameroon (Calvete et al., 2016). ...
Article
Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies.Toxicology Letters http://dx.
... Consequently, while antivenoms are typically marketed as a therapeutic treatment for envenomation by a given species, intraspecific venom variation can reduce antivenom efficacy, dependent upon the difference between the venom composition of the individuals which were used for antivenom manufacture and the individual which delivered the bite (Bénard-Valle et al. 2015;Boyer et al. 2015). It is well documented that the success rates achieved by different antivenoms in treating Echis snakebites can vary significantly depending on the geographical location of the bite due to regional variation of the species' venom Calvete et al., 2016;Casewell et al., 2010), which can translate into catastrophic treatment failure and case fatality rates increased by an order of magnitude in a tropical clinical setting (Alirol et al. 2015;Visser et al., 2008;Warrell and Arnett, 1976;Warrell et al. 1980). ...
... Thus the VINS antivenom can be predicted to have little or no likely clinical usefulness against the South Asian or African venoms tested in this study without requiring extremely large doses relative to the other antivenoms studied. These findings agree with a previous study showing that VINS antivenom is highly inefficient in the neutralisation of E. ocellatus from Cameroon (Calvete et al., 2016). ...
Article
Saw-scaled vipers (genus Echis) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and eected antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid co-factors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections.
... 102 Clinicians often rely on manufacturers' recommendations provided as package inserts or labels, but these can be unreliable. 103 104 We suggest following national protocols or standard regional guidelines for dose. 24 25 Administration is always intravenous, as bolus or diluted in saline solution over 10-60 minutes, at the same dose for adults and children. ...
... Previous preclinical studies using VINS venom antiserum exhibited variations and inconsistency in neutralization capacity of the venom antiserum. This may be due to the differences in the source of venom used in the experiment, or with experimental protocol employed, especially, regarding the multiples of LD 50 used as a 'challenge dose' in the lethality experiments [75,79,80]. This assertion was supported by varying reports on the effectiveness of snake venom neutralization potency of VINS venom antiserum products as follows:Wong et al [75] study showed that VINS African Polyvalent Antivenom (VAPAV) was able to cross-neutralize the lethal effect of N. senegalensis (Senegalese cobra) which is a homologue of N. haje (Egyptian cobra). ...
Article
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Snakebite envenomation is a major health concern in developing countries causing significant mortality and morbidity. With over 1.2 million cases annually caused by medically important snake Original Research Article Djameh et al.; JSRR, 27(11): 25-43, 2021; Article no.JSRR.75395 26 species belonging to the two families Viperidae (Echis spp. and Bitis spp.) and Elapidae (Naja spp. and Dendroaspis spp.). Several antivenoms are being produced and distributed to western sub-Saharan Africa for treatment of envenomation with the absence of preclinical efficacy studies. The present study evaluated the preclinical efficacy of venoms from Echis leucogaster, Echis ocellatus, Bitis arietans, Bitis gabonica, Naja haje, Naja melanoleuca, Naja nigricollis, Dendroaspis jamesoni, Dendroaspis polylepis and Dendroaspis viridis against a polyvalent Snake Venom Antiserum-African IHS (lyophilised), manufactured by VINS Bioproducts Limited (Telangana, India). Our in vitro results showed that, the SVA-AIHS contains antibodies that are capable of recognizing and binding majority of protein components representative of all eight major protein families of venoms of the snake species tested by double immunodiffusion assay and confirmed by western blot. The venom antiserum exhibited high neutralization efficacy against all the viperid and elapid snake species venoms in in vivo studies and confirmed the manufacturer's recommended neutralization capacity. This is clear evidence that the VINS polyvalent SVA-AIHS batch tested has strong neutralizing capacity and will be useful in treating envenoming by most African viperid and some elapid snake species.
... (38), a significant correlation was found (R = 0.492, p = 0.0011, n = 41). Two publications evaluated the neutralization of these effects by seven antivenoms against venoms of Echis ocellatus from various locations in sub-Saharan Africa (39,40). A significant correlation between neutralization of lethality and in vitro coagulant activity was observed (R = 0.7643, p = 0.0009, n = 15). ...
Article
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There is an urgent need to strengthen the implementation of the 3Rs principle (Replacement, Reduction and Refinement) in the use of experimental animals in toxinological research and in the assessment of the neutralizing efficacy of snake antivenoms. This is a challenging task owing to the inherent complexity of snake venoms. The state of the art on this topic is hereby reviewed, with emphasis on the studies in which a correlation has been observed between in vivo toxicity tests and in vitro surrogate assays, particularly in the study of lethal activity of venoms and its neutralization. Correlations have been described with some venoms-antivenoms when using: (a) enzyme immunoassays, (b) hemagglutination, (c) enzyme assays (proteinase, phospholipase A 2 ), (d) in vitro coagulant effect on plasma, (e) cell culture assays for cytotoxicity, (f) functional assays for assessing neurotoxicity in vitro , (g) use of hens’ eggs, and (h) antivenomics. Additionally, the routine introduction of analgesia in these assays and the design of more ‘humane’ protocols for the lethality test are being pursued. It is expected that the next years will witness a growing awareness of the relevance of the 3Rs principles in antivenom testing, and that new in vitro alternatives and more ‘humane’ experimental designs will emerge in this field.
... The degree of immune-recognition for venom components is then quantified by integrating the chromatographic peak areas. Recently, second generation antivenomics has become the preferred approach to characterize of antivenoms [126,127]. In combination with this, several types of immunoassays, including immunoblotting and enzymeimmunoassays HPLC/ELISA, have been used with the venomic approach to characterize antivenom toward specific venom toxins [128][129][130]. ...
Article
Snake venoms are complex chemical mixtures of biologically active proteins and non-protein components. Toxins have a wide range of targets and effects to include ion channels and membrane receptors, and platelet aggregation and platelet plug formation. Toxins target these effectors and effects at high affinity and selectivity. From a pharmacological perspective, snake venom compounds are a valuable resource for drug discovery and development. However, a major challenge to drug discovery using snake venoms is isolating and analyzing the bioactive proteins and peptides in these complex mixtures. Getting molecular information from complex mixtures such as snake venoms requires proteomic analyses, generally combined with transcriptomic analyses of venom glands. The present review summarizes current knowledge and highlights important recent advances in venomics with special emphasis on contemporary separation techniques and bioinformatics that have begun to elaborate the complexity of snake venoms. Several analytical techniques such as two-dimensional gel electrophoresis, RP-HPLC, size exclusion chromatography, ion exchange chromatography, MALDI-TOF-MS, and LC-ESI-QTOF-MS have been employed in this regard. The improvement of separation approaches such as multidimensional-HPLC, 2D-electrophoresis coupled to soft-ionization (MALDI and ESI) mass spectrometry has been critical to obtain an accurate picture of the startling complexity of venoms. In the case of bioinformatics, a variety of software tools such as PEAKS also has been used successfully. Such information gleaned from venomics is important to both predicting and resolving the biological activity of the active components of venoms, which in turn is key for the development of new drugs based on these venom components.
... Currently, there are difficulties in the distribution of AVs because of the reduction in their production and the lack of cold distribution chains, particularly in Africa, which has forced the search for solutions, such as the production of AVs by other laboratories, particularly in Latin America, 24,30 although with related efficiency and reactivity problems. [31][32][33][34] which implies an overload to the cost of AV. This is even more important considering that the studies analyzed showed that the evaluation of the interventions was made against the use of AV, probably because, to date, there are no other interventions available for this event. ...
Article
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Background: Snakebite is an often-neglected event with a high rate of mortality and is concentrated in poor areas. We aimed to assess the economic impact and health effects of the implementation of interventions for snakebites through a systematic review of the scientific literature. Methods: Thirty search strategies were conducted in seven databases, applying PRISMA's identification, screening, selection, and inclusion phases. The reproducibility of the selection of studies and the extraction of information were guaranteed. The methodological quality was evaluated using the Consolidated Health Economic Evaluation Reporting Standards. Qualitative synthesis and meta-analysis were performed for determining the average cost-effectiveness (ACE) for each death and disability-adjusted life years (DALY) avoided. Results: Six cost-effectiveness studies were included for the supply of antivenom (AV), taken as outcomes on days of hospitalization or in ICU, death and DALYs avoided. All studies only included institutional costs, and majority of them did not specify the analytical model or economic evaluation parameters and did not perform uncertainty analyses. The management protocol standardization with interdisciplinary attention improves ACE of AV. Cost-effectiveness ratio (CER) of treatment with AV was USD 1253 (constant value for the year 2017, adjusted by purchasing power parity) for each death avoided and USD 51 per DALY avoided. Conclusion: High cost-effectiveness of the AV treatment for snakebites was evidenced, which shows that the allocation of resources for this event should be a healthcare priority in addition to implementation of strategies that improve the access to, opportunity, and quality of hospital and pre-hospital care and reduce the cost of AV.
... Despite this, antivenoms have many limitations relating to their specificity, safety and affordability, and thus there is a strong rationale to develop new snakebite therapeutics with higher efficacy and broader species coverage, as well as at a lowered cost [5,6]. Toxicovenomics is a proteomicsbased approach that can be used to analyze snake venoms to provide an overview of which toxins are medically relevant in envenomings, and this approach shows promise for selecting the most effective venom mixtures for immunization [7][8][9]. However, toxicovenomics needs to be combined with complementary analytical approaches, such as animal-based neutralization assays, immunochemical studies [3], and antivenomics [10]; these, together, can provide an in-depth view into the molecular reactivity and potential neutralization of these medically relevant toxins [11,12]. ...
Article
Full-text available
Snakebite envenoming is a major neglected tropical disease that affects millions of people every year. The only effective treatment against snakebite envenoming consists of unspecified cocktails of polyclonal antibodies purified from the plasma of immunized production animals. Currently, little data exists on the molecular interactions between venom-toxin epitopes and antivenom-antibody paratopes. To address this issue, high-density peptide microarray (hdpm) technology has recently been adapted to the field of toxinology. However, analysis of such valuable datasets requires expert understanding and, thus, complicates its broad application within the field. In the present study, we developed a userfriendly, and high-throughput web application named “Snake Toxin and Antivenom Binding Profiles” (STAB Profiles), to allow straight-forward analysis of hdpm datasets. To test our tool and evaluate its performance with a large dataset, we conducted hdpm assays using all African snake toxin protein sequences available in the UniProt database at the time of study design, together with eight commercial antivenoms in clinical use in Africa, thus representing the largest venom-antivenom dataset to date. Furthermore, we introduced a novel method for evaluating raw signals from a peptide microarray experiment and a data normali-zation protocol enabling intra-microarray and even inter-microarray chip comparisons. Finally, these data, alongside all the data from previous similar studies by Engmark et al., were preprocessed according to our newly developed protocol and made publicly available for download through the STAB Profiles web application (http://tropicalpharmacology.com/ tools/stab-profiles/). With these data and our tool, we were able to gain key insights into toxin-antivenom interactions and were able to differentiate the ability of different antivenoms to interact with certain toxins of interest.
... Technologies that show a good correlation coefficient between the venom pro-and anticoagulant effects on mammalian plasmas in vitro and lethality in vivo have been reported [3][4][5][6][7][8][9][10][11][12][13][14][15]. However, these techniques present some limitations: (i) Many studies simply characterize the toxin conversion of isolated substrates, giving a single parameter for one complex enzymatic process. ...
Article
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The sensitivity of vertebrate citrated plasma to pro- and anticoagulant venom or toxins occurs on a microscale level (micrograms). Although it improves responses to agonists, recalcification triggers a relatively fast thrombin formation process in mammalian plasma. As it has a natural factor XII deficiency, the recalcification time (RT) of chicken plasma (CP) is comparatively long [≥ 1800 seconds (s)]. Our objective was to compare the ability of bee venom phospholipase A2 (bvPLA2) to neutralize clot formation induced by an activator of coagulation (the aPTT clot) in recalcified human and chicken plasmas, through rotational thromboelastometry. The strategy used in this study was to find doses of bvPLA2 that were sufficient enough to prolong the clotting time (CT) of these activated plasmas to values within their normal RT range. The CT of CP was prolonged in a dose-dependent manner by bvPLA2, with 17 ± 2.8 ng (n = 6) being sufficient to displace the CT values of the activated samples to ≥ 1800 s. Only amounts up to 380 ± 41 ng (n = 6) of bvPLA2 induced the same effect in activated human plasma samples. In conclusion, the high sensitivity of CP to agonists and rotational thromboelastometry could be useful. For example, during screening procedures for assaying the effects of toxins in several stages of the coagulation pathway, such as clot initiation, formation, stability, strength, or dissolution.
... However, due to ethical considerations, neutralisation assays were performed only for one of the commercially marketed Indian antivenoms. The effectiveness of the other antivenoms in neutralising bites from the neglected species requires further validation through preclinical and clinical research [96,97]. Since none of these antivenom manufacturers utilize the venoms of the neglected species in the immunisation mixture, we do not expect them to perform any better than the antivenom tested in this study. ...
Article
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Background Snakebite in India causes the highest annual rates of death (46,000) and disability (140,000) than any other country. Antivenom is the mainstay treatment of snakebite, whose manufacturing protocols, in essence, have remained unchanged for over a century. In India, a polyvalent antivenom is produced for the treatment of envenomations from the so called ‘big four’ snakes: the spectacled cobra (Naja naja), common krait (Bungarus caeruleus), Russell’s viper (Daboia russelii), and saw-scaled viper (Echis carinatus). In addition to the ‘big four’, India is abode to many other species of venomous snakes that have the potential to inflict severe clinical or, even, lethal envenomations in their human bite victims. Unfortunately, specific antivenoms are not produced against these species and, instead, the ‘big four’ antivenom is routinely used for the treatment. Methods We characterized the venom compositions, biochemical and pharmacological activities and toxicity profiles (mouse model) of the major neglected yet medically important Indian snakes (E. c. sochureki, B. sindanus, B. fasciatus, and two populations of N. kaouthia) and their closest ‘big four’ congeners. By performing WHO recommended in vitro and in vivo preclinical assays, we evaluated the efficiencies of the commercially marketed Indian antivenoms in recognizing venoms and neutralizing envenomations by these neglected species. Findings As a consequence of dissimilar ecologies and diet, the medically important snakes investigated exhibited dramatic inter- and intraspecific differences in their venom profiles. Currently marketed antivenoms were found to exhibit poor dose efficacy and venom recognition potential against the ‘neglected many’. Premium Serums antivenom failed to neutralise bites from many of the neglected species and one of the ‘big four’ snakes (North Indian population of B. caeruleus). Conclusions This study unravels disturbing deficiencies in dose efficacy and neutralisation capabilities of the currently marketed Indian antivenoms, and emphasises the pressing need to develop region-specific snakebite therapy for the ‘neglected many’.
... Prompt recognition of systemic envenomation and timely administration of anti-venom serum are effective life saving measures aimed at neutralization of snake venom, reverses acute venom-induced inflammation, haemorrhagic syndrome [28][29][30], reversing severe coagulopathy [19], reducing renal damage [10] and preventing necrosis. Currently, anti-venom serum is the only safe and efficacious specific treatment for snake envenomation [1,31]. ...
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Background Snake envenomation is an underestimated pathology in sub-Saharan Africa associated with severe emergencies, and even death in case of late presentation. We herein present a case of severe envenomation managed at the surgical emergency department of the Yaoundé Central Hospital. Case presentation We report a case of a 47-year-old female farmer with no relevant past history who sustained a snakebite by an Echis occellatus viper during an agricultural activity. Her initial management consisted in visiting a traditional healer who administered her some herbal remedies orally and applied a white balm on the affected limb. Due to progressive deterioration of her condition, she was rushed to our surgical department where she arrived 20 h after the snakebite incident. On admission she presented in a state of shock (suggestive of an anaphylactic shock), coagulopathy, renal impairment, and gangrene of the entire right upper limb. Emergency management consisted of fluid resuscitation, repeated boluses of adrenaline, a total of three vials of polyvalent anti-venom sera, promethazine, analgesics, corticosteroids, and administration of fresh frozen plasma. Within four hours of emergency department hospitalisation she developped signs of sepsis and persistent hypotension refractory to fluid resuscitation, suggestive of an associated septic shock. Management pursued with antiobiotherapy and administration of noradrenaline through an electric pump syringe to achieve a mean arterial blood pressure above 65 mmHg. The patient deceased at the 10th hour of hospitalisation in a state of circulatory collapse unresponsive to vasopressors, coagulopathy, renal failure, sepsis and gangrene of the right forearm. Conclusion The authors highlight this unusual presentation but equally pinpoint how late presentation to the emergency department, harmful tradition practices, poverty and cultural beliefs can adversely affect the prognosis of snakebite in our setting.
... Despite its relatively recent introduction, numerous studies aimed at assessing the immunological profiles of antivenoms from different manufacturers and a range of different snake species from Europe, Asia, Oceania, Latin America, and Africa have demonstrated the usefulness and validity of antivenomics in complementing existing assays [67,68,[73][74][75][76][77][78][79][80][81][82]. However, antivenomics can still merely suggest likely explanations of observed cross-reactivity, such as the presence of the same subfamily of important toxins in two given venoms, where one venom is used in antivenom production [83]. ...
Article
Full-text available
Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology. Antivenomics involves a top-down assessment of the toxin-binding capacities of antivenoms, whereas high-density peptide microarray technology may be harnessed to provide in-depth knowledge on which toxin epitopes are recognized by antivenoms. This review provides an overview of both the classical and new methods used to investigate antivenom cross-reactivity, the advantages and disadvantages of each method, and examples of studies using the methods. A special focus is given to antivenomics and high-density peptide microarray technology as these high-throughput methods have recently been introduced in this field and may enable more detailed assessments of antivenom cross-reactivity.
... Survivors are often afflicted by psychological disorders (e.g., post-traumatic stress) and left handicapped with amputations, blindness, or other sequelae [3][4][5][6][7]. The situation is further complicated by antivenom shortages and the undesirable traits of some antivenoms, including immunogenicity and low efficacy [8][9][10][11][12][13][14]. Antivenoms from hyperimmunized animals were first envisioned by A. Calmette and C. Phisalix in 1894 [15]. ...
Article
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With the inclusion of snakebite envenoming on the World Health Organization's list of Neglected Tropical Diseases, an incentive has been established to promote research and development effort in novel snakebite antivenom therapies. Various technological approaches are being pursued by different research groups, including the use of small molecule inhibitors against enzymatic toxins as well as peptide-and oligonucleotide-based aptamers and antibody-based biotherapeutics against both enzymatic and non-enzymatic toxins. In this article, the most recent advances in these fields are presented, and the advantages, disadvantages, and feasibility of using different toxin-neutralizing molecules are reviewed. Particular focus within small molecules is directed towards the inhibitors varespladib, batimastat, and marimastat, while in the field of antibody-based therapies, novel recombinant polyclonal plantivenom technology is discussed.
... New technologies that show good correlation coefficient between antivenom's ability in inactivating venom pro-or anticoagulant effects of venoms in vitro and lethality in vivo have been reported (Gen e et al., 1989;Laing et al., 1992;Clemens et al., 1995;Sprivulis et al., 1996;Sells, 2003;Isbister et al., 2007Isbister et al., , 2010Segura et al., 2010;Pla et al., 2014Pla et al., , 2017Pornmuttakun and Ratanabanangkoon, 2014;S anchez et al., 2015;Calvete et al., 2016). ...
... For instance, only four antivenoms specific to Deinagkistrodon acutus, Agkistrodon halys, Bungarus multicinctus, and Naja atra are manufactured in China, whereas there are about 50 species of venomous snakes. To complicate matters, some marketed antivenoms appear clinically ineffective [3,4]. Snakebite envenomation was added to the priority list of neglected tropical diseases (NTDs) by the World Health Organization (WHO) in 2017, calling for increased scientific investigation and financial input [5]. ...
Article
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Phospholipase A2s (PLA2) is a major component of snake venom with diverse pathologic toxicities and, therefore, a potential target for antivenom therapy. Varespladib was initially designed as an inhibitor of mammal PLA2s, and was recently repurposed to a broad-spectrum inhibitor of PLA2 in snake venom. To evaluate the protective abilities of varespladib to hemorrhage, myonecrosis, and systemic toxicities that are inflicted by different crude snake venoms, subcutaneous ecchymosis, muscle damage, and biochemical variation in serum enzymes derived from the envenomed mice were determined, respectively. Varespladib treatment showed a significant inhibitory effect to snake venom PLA2, which was estimated by IC50 in vitro and ED50 in vivo. In animal models, the severely hemorrhagic toxicity of D. acutus and A. halys venom was almost fully inhibited after administration of varespladib. Moreover, signs of edema in gastrocnemius muscle were remarkably attenuated by administration of varespladib, with a reduced loss of myonecrosis and desmin. Serum levels of creatine kinase, lactate dehydrogenase isoenzyme 1, aspartate transaminase, and alanine transaminase were down-regulated after treatment with varespladib, which indicated the protection to viscera injury. In conclusion, varespladib may be a potential first-line drug candidate in snakebite envenomation first aid or clinical therapy.
... At present date, anti-venom immunotherapy is the only specific treatment for snake envenomation [1,13]. Evidence from local clinical trials has confirmed the safety and efficacy of these sera in the neutralization of snake venom, reversal of acute venom-induced inflammation and haemorrhagic syndrome [14][15][16][17]. In general, AVS should be administered as a matter of urgency in the presence of signs of envenomation [18,19]. ...
Article
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Objectives Snakebite is an underestimated medical and surgical emergency in developing countries responsible for a high disease burden. Optimal management of snake envenomation in these resource-limited settings is precluded by several public health challenges. In this review, we discuss the disease burden of snakebites in Cameroon and the public health challenges of its management in view of making recommendations essential for policy-making. MEDLINE, African Journals Online and Google Scholar were searched from January 1990 to February 2017 for studies addressing snakebite in Cameroon. Our search extended to include grey literature from book chapters, conference proceedings, theses and documents from organizations. Results Our results suggest that snakebites pose a significant health and economic burden in Cameroon. A composite of factors contributes to the challenge of managing snakebites in Cameroon and include: inadequate disease surveillance; poor health-seeking behaviours of patients; under-production and scarcity of anti-venom serum and the relatively high cost of anti-venom serum. There is an urgent need to revamp the current health policies through health education, promotion and building of sustainable health systems. Disease surveillance and management can be improved by providing refresher courses for healthcare providers and subsidization of the prices of anti-venom serum in pharmacies in the country.
... Despite more than 45 commercial or government antivenom producers existing around the world [6], the shortage of independently validated, demonstrably safe, effective, and affordable snake antivenoms in many affected regions [7,8] is an important contributor to this tropical disease burden [9]. To complicate matters, there is growing evidence suggesting that some marketed antivenoms are clinically ineffective [10,11]. Although necessarily multicomponent in nature, strategies to address this neglect should include the improvement of antivenom availability and the preclinical testing of antivenom efficacy [5,12]. ...
Article
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Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms.
... Antivenomic studies have been performed on antivenoms against a range of different snake species from Europe, Asia, Oceania, Latin America, and Africa [108,[119][120][121][122][123][124][125][126][127][128][129]. They are yet to be performed on non-snake species, possibly owing to the scarcity of venoms from smaller venomous animals. ...
Article
In this review, the different approaches that have been employed with the aim of developing novel antivenoms against animal envenomings are presented and discussed. Reported efforts have focused on the use of innovative immunization strategies, small molecule inhibitors against enzymatic toxins, endogenous animal proteins with toxin-neutralizing capabilities, and recombinant monoclonal antibodies. Harnessing either of these approaches, antivenom development may benefit from an in-depth understanding of venom compositions and the medical importance of individual venom toxins. Focus is thus also directed towards the different omics technologies (particularly venomics, antivenomics, and toxicovenomics) that are being used to uncover novel animal toxins, shed light on venom complexity, and provide directions for how to determine the medical relevance of individual toxins within whole venoms. Finally, techniques for assessing antivenom specificity and cross-reactivity are reviewed, with special focus on antivenomics and high-density peptide microarray technology.
... The second-generation antivenomic strategy outlined above has been used most often in recent characterizations of antivenoms [66][67][68]. Additional types of immunoassays have also been combined with venomic analyses in order to evaluate the specificity of antibodies present in an antivenom toward particular venom proteins. ...
Article
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This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.
... In addition to a sustained international cooperative effort in the preclinical testing of antivenom efficacy being performed in Latin America for many years (e.g., [28]), it was also reported to the workshop that several research groups have been/are performing preclinical efficacy assessment of several antivenoms distributed in sub-Saharan Africa. Examples include studies on the neutralization of venoms of the saw-scaled viper, Echis ocellatus from various regions [29][30][31] and of the black mamba, Dendroaspis polylepis [32]. It was agreed that the acknowledged usefulness of these independent research activities would be substantially enhanced by greater coordination and division of the work load to ensure continent-wide antivenom quality control (QC) testing. ...
Article
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The deliberations and conclusions of a Hinxton Retreat convened in September 2015, entitled “Mechanisms to reverse the public health neglect of snakebite victims” are reported. The participants recommended that the following priority actions be included in strategies to reduce the global impact of snake envenoming: (a) collection of accurate global snakebite incidence, mortality and morbidity data to underpin advocacy efforts and help design public health campaigns; (b) promotion of (i) public education prevention campaigns; (ii) transport systems to improve access to hospitals and (iii) establishment of regional antivenom-efficacy testing facilities to ensure antivenoms’ effectiveness and safety; (c) exploration of funding models for investment in the production of antivenoms to address deficiencies in some regions; (d) establishment of (i) programs for training in effective first aid, hospital management and post-treatment care of victims; (ii) a clinical network to generate treatment guidelines and (iii) a clinical trials system to improve the clinical management of snakebite; (e) development of (i) novel treatments of the systemic and local tissue-destructive effects of envenoming and (ii) affordable, simple, point-of-care snakebite diagnostic kits to improve the accuracy and rapidity of treatment; (f) devising and implementation of interventions to help the people and communities affected by physical and psychological sequelae of snakebite.
... PLA2s have, additionally, involvement in hemolysis and hemorrhage related to the complement system in the setting of sepsis and vasodilatory anaphylotoxins [53] in some Micrurus (e.g., Figure 2b) and cobra venoms [54][55][56][57][58][59][60]. In addition to widening the diversity of snake venoms tested, future experiments would likely benefit from in-depth venomic analyses for the venoms to be tested, including second-generation venomics [61][62][63]. Ultimately, however, clinical testing where antivenoms are available for clinical study and comparison are necessary and are the only way to answer the critical questions of safety and efficacy in the setting of snakebite [2]. ...
Article
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Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2) activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2) inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases) could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.
... PLA2s have, additionally, involvement in hemolysis and hemorrhage related to the complement system in the setting of sepsis and vasodilatory anaphylotoxins [53] in some Micrurus (e.g., Figure 2b) and cobra venoms [54][55][56][57][58][59][60]. In addition to widening the diversity of snake venoms tested, future experiments would likely benefit from in-depth venomic analyses for the venoms to be tested, including second-generation venomics [61][62][63]. Ultimately, however, clinical testing where antivenoms are available for clinical study and comparison are necessary and are the only way to answer the critical questions of safety and efficacy in the setting of snakebite [2]. ...
Article
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Access to safe, effective, quality-assured antivenom products that are tailored to endemic venomous snake species is a crucial component of recent coordinated efforts to reduce the global burden of snakebite envenoming. Multiple access barriers may affect the journey of antivenoms from manufacturers to the bedsides of patients. Our review describes the antivenom ecosystem at different levels and identifies solutions to overcome these challenges. At the global level, there is insufficient manufacturing output to meet clinical needs, notably for antivenoms intended for use in regions with a scarcity of producers. At national level, variable funding and deficient regulation of certain antivenom markets can lead to the procurement of substandard antivenom. This is particularly true when producers fail to seek registration of their products in the countries where they should be used, or where weak assessment frameworks allow registration without local clinical evaluation. Out-of-pocket expenses by snakebite victims are often the main source of financing antivenoms, which results in the underuse or under-dosing of antivenoms, and a preference for low-cost products regardless of efficacy. In resource-constrained rural areas, where the majority of victims are bitten, supply of antivenom in peripheral health facilities is often unreliable. Misconceptions about treatment of snakebite envenoming are common, further reducing demand for antivenom and exacerbating delays in reaching facilities equipped for antivenom use. Multifaceted interventions are needed to improve antivenom access in resource-limited settings. Particular attention should be paid to the comprehensive list of actions proposed within the WHO Strategy for Prevention and Control of Snakebite Envenoming.
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Lethality and cytotoxicity assays of snake venoms and their neutralization by antivenom require many mice for the experiments. Recent developments have prompted researchers to seek alternative strategies that minimize the use of mice in line with Russel and Burch's 3Rs philosophy (Replacement, Reduction, and Refinement). Artemia salina is an animal model widely used for toxicity screening. However, its use in snake venom toxinology is limited by a lack of data. The present study compared the toxicity of venoms from Bitis arietans, Naja ashei, and Naja subfulva using mice and Artemia salina. In the Artemia salina test at 24 h and the dermonecrotic test in mice, the toxicity of the venoms was in the order Naja ashei ∼ Naja subfulva > Bitis arietans. In the lethality test in mice, the toxicity of the venoms was in the order Naja subfulva > Naja ashei > Bitis arietans. These findings suggest that the toxicity of the venoms in Artemia salina and the dermonecrotic bioassay in mice have a similar trend but differ from the lethality test in mice. Therefore, it may be relevant to further explore the Artemia salina bioassay as a potential surrogate test of dermonecrosis in mice. Studies with more venoms may be needed to establish the correlation between the Artemia salina bioassay and the dermonecrotic assay in mice.
Preprint
With the inclusion of snakebite envenoming on the World Health Organisation’s list of Neglected Tropical Diseases, an incentive has been established to promote research and development effort in novel snakebite antivenom therapies. Different technological approaches are being pursued by different research groups, including the use of small molecule inhibitors against enzymatic toxins, as well as peptide and oligonucleotide-based aptamers and antibody-based biotherapeutics against both enzymatic and non-enzymatic toxins. In this article, the most recent advances in these fields are presented, and the advantages, disadvantages, and feasibility of using different toxin-neutralizing molecules are reviewed. Particular focus within small molecules is directed towards the inhibitors, varespladib, batimastat, and marimastat, while in the field of antibody-based therapies, novel recombinant polyclonal plantivenom technology is discussed.
Article
Snakebite envenoming represents a major issue in rural areas of tropical and subtropical regions across sub-Saharan Africa, South to Southeast Asia, Latin America and Oceania. Antivenoms constitute the only scientifically validated therapy for snakebite envenomings, provided they are safe, effective, affordable, accessible and administered appropriately. However, the lack of financial incentives in a technology that has remained relatively unchanged for more than a century, has contributed to some manufacturers leaving the market and others downscaling production or increasing the prices, leading to a decline in the availability and accessibility for these life-saving antidotes to millions of rural poor most at risk from snakebites in low income countries. The shortage of antivenoms can be significantly alleviated by optimizing the use of current antivenoms (through the assessment of their specific and paraspecific efficacy against the different medically relevant homologous and heterologous snake venoms) and by generating novel polyspecific antivenoms exhibiting broad clinical spectrum and wide geographic distribution range. Research on venoms has been continuously enhanced by advances in technology. Particularly, the last decade has witnessed the development of omics strategies for unravelling the toxin composition of venoms ("venomics") and to assess the immunorecognition profile of antivenoms ("antivenomics"). Here, we review recent developments and reflect on near future innovations that promise to revolutionize the mutually enlightening relationship between evolutionary and translational venomics.
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Background: Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. Methods: To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. Results: A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. Conclusions: Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. General significance: This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.
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Vulnerable populations need urgent access to effective and affordable treatments For many years snake bite experts have sought to raise the profile of this forgotten problem with public health authorities and donors. Lamentably, however, it has taken news of the departure of antivenom manufacturer Sanofi-Pasteur from the sub-Saharan African market to focus the spotlight on the calamity of snake bite among the world’s poorest people. Nevertheless, beyond this successful media storm,1 2 the reality is that for the majority of people bitten by snakes in Africa the loss of Sanofi’s FAV-Afrique polyvalent antivenom will mean little, if anything at all. This is because the product simply never reached them in the first place. In a region where median gross domestic product per capita is $550 (£360; €490), Sanofi’s product was simply too expensive (a four vial treatment cost about $540) and produced in insufficient quantities to meet the needs of more than a small part of the African continent.3 4 The harsh fact is that the continent is largely devoid of safe, effective, and affordable treatments for something that is eminently treatable. For decades there have …
Article
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Of the 24 neglected tropical diseases (NTDs) and conditions listed by WHO, snakebite is among the top killers [1]. Tens of thousands of people die each year as a result of snakebite envenoming, with close to 50,000 deaths in India alone [2] and up to 32,000 in sub-Saharan Africa [3]. Yet there are few sources of effective, safe, and affordable antivenoms. The regions that bear the highest snakebite burden are especially underserved [4]. The Fav-Afrique antivenom, produced by Sanofi Pasteur (France), is considered safe and effective and is one of the few antivenoms to be approved by a Stringent Regulatory Authority (French National Regulatory Authority), although limited formal evidence has been published [5,6]. It is polyvalent, targeting most of the medically important snake species in sub-Saharan Africa. In particular, it is highly effective in treating envenoming by Echis ocellatus, the West African saw-scaled viper [5–7] that causes great morbidity and mortality throughout the West and Central African savannah. The venom of E. ocellatus may induce systemic haemorrhage, coagulopathy, and shock, as well as extensive local tissue damage. In the absence of treatment, the case fatality rate is 10%–20% [8]. Medecins Sans Frontieres (MSF) uses Fav-Afrique in its projects in sub-Saharan Africa, notably in Paoua in Central African Republic (CAR), where E. ocellatus envenoming is frequent [9]. Worryingly, MSF has been informed that the production of Fav-Afrique by Sanofi Aventis will be permanently discontinued. The last batch was released in January 2014, with an expiry date of June 2016. All the vials produced have already been sold by Sanofi Pasteur. Although several alternative antivenom products target a similar list of species as Fav-Afrique, there is currently no evidence of their safety and effectiveness. We aimed to review the evidence for the efficacy and safety of existing and in-development snake antivenoms, and to list the alternatives to Fav-Afrique in sub-Saharan Africa.
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Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxins.
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Venom research has been continuously enhanced by technological advances. High-throughput technologies are changing the classical paradigm of hypothesis-driven research to technology-driven approaches. However, the thesis advocated in this paper is that full proteome coverage at locus-specific resolution requires integrating the best of both worlds into a protocol that includes decomplexation of the venom proteome prior to liquid chromatography-tandem mass spectrometry matching against a species-specific transcriptome. This approach offers the possibility of proof-checking the species-specific contig database using proteomics data. Immunoaffinity chromatography constitutes the basis of an antivenomics workflow designed to quantify the extent of cross-reactivity of antivenoms against homologous and heterologous venom toxins. In the author's view, snake venomics and antivenomics form part of a biology-driven conceptual framework to unveil the genesis and natural history of venoms, and their within- and between-species toxicological and immunological divergences and similarities. Understanding evolutionary trends across venoms represents the Rosetta Stone for generating broad-ranging polyspecific antivenoms.
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Snakebite envenoming is a major public health problem among rural communities of the Nigerian savanna. The saw-scaled or carpet viper (Echis ocellatus) and, to a lesser extent, the African cobras (Naja spp.) and puff adders (Bitis arietans) have proved to be the most important cause of mortality and morbidity. The main clinical features of E. ocellatus envenoming are systemic hemorrhage, incoagulable blood, shock, local swelling, bleeding and, occasionally, necrosis. Bites may be complicated by amputation, blindness, disability, disfigurement, mutilation, tissue destruction and psychological consequences. Antivenom remains the hallmark and mainstay of envenoming management while studies in Nigeria confirm its protection of over 80% against mortality from carpet-viper bites. However, the availability, distribution and utilization of antivenom remain challenging although two new antivenoms (monospecific EchiTab G and trispecific EchiTab ICP-Plus) derived from Nigerian snake venoms have proven very effective and safe in clinical trials. A hub-and-spoke strategy is suggested for broadening antivenom access to endemic rural areas together with instituting quality assurance, standardization and manpower training. With the advent of antivenomics, national health authorities must be aided in selecting and purchasing antivenoms appropriate to their national needs while manufacturers should be helped in practical ways to improve the safety, efficacy and potential coverage against snake venoms and pricing of their products.
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In West Africa, envenoming by saw-scaled or carpet vipers (Echis ocellatus) causes great morbidity and mortality, but there is a crisis in supply of effective and affordable antivenom (ISRCTN01257358). In a randomised, double-blind, controlled, non-inferiority trial, "EchiTAb Plus-ICP" (ET-Plus) equine antivenom made by Instituto Clodomiro Picado was compared to "EchiTAb G" (ET-G) ovine antivenom made by MicroPharm, which is the standard of care in Nigeria and was developed from the original EchiTAb-Fab introduced in 1998. Both are caprylic acid purified whole IgG antivenoms. ET-G is monospecific for Echis ocellatus antivenom (initial dose 1 vial) and ET-Plus is polyspecific for E. ocellatus, Naja nigricollis and Bitis arietans (initial dose 3 vials). Both had been screened by pre-clinical and preliminary clinical dose-finding and safety studies. Patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus, were recruited in Kaltungo, north-eastern Nigeria. Those eligible and consenting were randomly allocated with equal probability to receive ET-Plus or ET-G. The primary outcome was permanent restoration of blood coagulability 6 hours after the start of treatment, assessed by a simple whole blood clotting test repeated 6, 12, 18, 24 and 48 hr after treatment. Secondary (safety) outcomes were the incidences of anaphylactic, pyrogenic and late serum sickness-type antivenom reactions. Initial doses permanently restored blood coagulability at 6 hours in 161/194 (83.0%) of ET-Plus and 156/206 (75.7%) of ET-G treated patients (Relative Risk [RR] 1.10 one-sided 95% CI lower limit 1.01; P = 0.05). ET-Plus caused early reactions on more occasions than did ET-G [50/194 (25.8%) and 39/206 (18.9%) respectively RR (1.36 one-sided 95% CI 1.86 upper limit; P = 0.06). These reactions were classified as severe in 21 (10.8%) and 11 (5.3%) of patients, respectively. At these doses, ET-Plus was slightly more effective but ET-G was slightly safer. Both are recommended for treating E. ocellatus envenoming in Nigeria. Current Controlled Trials ISRCTN01257358.
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Envenoming resulting from snakebites is an important public health problem in many tropical and subtropical countries. Few attempts have been made to quantify the burden, and recent estimates all suffer from the lack of an objective and reproducible methodology. In an attempt to provide an accurate, up-to-date estimate of the scale of the global problem, we developed a new method to estimate the disease burden due to snakebites. The global estimates were based on regional estimates that were, in turn, derived from data available for countries within a defined region. Three main strategies were used to obtain primary data: electronic searching for publications on snakebite, extraction of relevant country-specific mortality data from databases maintained by United Nations organizations, and identification of grey literature by discussion with key informants. Countries were grouped into 21 distinct geographic regions that are as epidemiologically homogenous as possible, in line with the Global Burden of Disease 2005 study (Global Burden Project of the World Bank). Incidence rates for envenoming were extracted from publications and used to estimate the number of envenomings for individual countries; if no data were available for a particular country, the lowest incidence rate within a neighbouring country was used. Where death registration data were reliable, reported deaths from snakebite were used; in other countries, deaths were estimated on the basis of observed mortality rates and the at-risk population. We estimate that, globally, at least 421,000 envenomings and 20,000 deaths occur each year due to snakebite. These figures may be as high as 1,841,000 envenomings and 94,000 deaths. Based on the fact that envenoming occurs in about one in every four snakebites, between 1.2 million and 5.5 million snakebites could occur annually. Snakebites cause considerable morbidity and mortality worldwide. The highest burden exists in South Asia, Southeast Asia, and sub-Saharan Africa.
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As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn((R)) Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab')(2)-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.
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This study compares two antivenoms used to treat Echis ocellatus snake bite patients at Mathias Hospital, Yeji, central Ghana. FAV-Afrique antivenom (Aventis Pasteur) was given to 278 patients during 2001--2003, whilst Asna Antivenom C (Bharat Serum and Vaccines Ltd) was used in 2004 to treat 66 patients. The two groups had comparable patient attributes, time from snake bite to treatment and staff adherence to the tested treatment protocol. The antivenom C group required more repeat doses and twice the amount of antivenom to treat coagulopathy. Of greater concern, the antivenom C mortality rate was 12.1%, a marked rise from the 1.8% rate in the earlier FAV-Afrique antivenom group. In this study, antivenom C was ineffective as treatment for West African E. ocellatus snake venom. This illustrates the absolute need for regional pilot tests to assess the effectiveness of a new antivenom against local snake venoms before its sole and general distribution in a region is initiated.
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We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.
Article
Objective: Understanding the molecular basis of complex adaptive traits, such as snake venom, demands qualitative and quantitative comparisons of the temporal and spatial patterns of venom variation. Here, we assessed the proof-of-concept that locus-resolved reference venom proteome maps can be achieved through efficient pre-MS venom proteome decomplexation, peptide-centric MS/MS analysis and species-specific database searching. Methods: Venom proteome components were fractionated and quantified by RP-HPLC, SDS-PAGE and 2DE prior to LC-MS/MS matching against a species-specific transcriptomic dataset. Results: Combination of RP-HPLC/SDS-PAGE and 2DE followed by LC-MS/MS showed the existence of ∼178-180 venom protein species generated from ∼48 unique transcripts. Conclusions: Our results underscore that if sufficient pre-MS and MS efforts are applied, comprehensive venom maps can be achieved. And - equally important - dissociating the venom decomplexing steps from the protein identification process represents the key to achieving a quantitative and locus-resolved insight of the venom proteome.
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Snakebite envenoming causes a heavy toll in sub-Saharan Africa in terms of mortality and sequelae. In the West African savannah, the viperid Echis ocellatus is responsible for the vast majority of bites. In the last decades, several new antivenoms have been introduced for the treatment of these envenomings, although the assessment of their preclinical efficacy against the venom of E. ocellatus has been studied only for some of them. This work analyzed comparatively the ability of four antivenoms (FAV Afrique, EchiTAb G, EchiTAB-Plus-ICP(®), and Inoserp™ Panafricain) to neutralize lethal, hemorrhagic, and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria. In addition, an immunoaffinity chromatography antivenomic protocol was used to assess the ability of the four antivenoms to bind to the proteins of these venoms. Results showed that all the antivenoms were effective in the neutralization of the three effects investigated, and were able to immunocapture, completely or partially, the most abundant components in the E. ocellatus venoms from the geographical origins sampled. Our observations also highlighted quantitative differences between antivenoms in their neutralizing and antivenomics profiles, especially regarding neutralization of in vitro coagulant activity, suggesting that different doses of these antivenoms are probably needed for an effective treatment of human envenomings by this species.
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Health specialists warn that stocks of antivenom will run out in 2016.
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Unlabelled: Parenteral administration of animal-derived antivenoms constitutes the mainstay in the treatment of snakebite envenomings. Despite the fact that this therapy has been available for over a century, the detailed understanding of the neutralizing and immunoreactivity profiles of the majority of antivenoms is pending. Currently, a combination of preclinical neutralization tests and 'antivenomics', i.e. a proteomic-based assessment of antivenom immunoreactivity, provides a powerful analytical platform to investigate the preclinical efficacy of antivenoms. In this review, the studies performed on the polyvalent antivenom manufactured by Instituto Clodomiro Picado, Costa Rica, are summarized. This antivenom is prepared by immunizing horses with a mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys, and is used in Central America for the treatment of envenomings by viperid species. Overall, the antivenom shows a widespread pattern of immunological reactivity against homologous and heterologous venoms, which correlates with its ability to neutralize lethal, hemorrhagic, myotoxic, coagulant, defibrinogenating, phospholipase A2 and proteinase activities of viperid venoms. At the same time, antivenomics detected several venom components against which the antivenom shows only partial or negligible immunorecognition, such as low molecular mass vasoactive peptides, disintegrins, and some phospholipases A2, P-I metalloproteinases and serine proteinases. Such information can be used to design strategies for enhancing the antibody response of horses against poorly immunogenic, toxicologically-relevant venom components in order to further improve the efficacy of this antivenom. Biological significance: The timely parenteral administration of an appropriate antivenom remains, more than a century after the development of the first serum antivenimeux by Calmette and Phisalix and Bertrand, the only currently effective treatment for snakebite envenomings. A key technical issue in the generation of novel antivenoms is the design of optimized immunization venom mixtures that ensure that the resulting antidotes will be effective against the highest number of venoms from snakes of medical concern across the geographical range where they will be used. Antivenomics is a proteomics-based protocol developed to complement in vitro and in vivo standard preclinical tests in the qualitative and quantitative characterization of the immunological profile and the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. Antivenomics is translational venomics. The combination of antivenomics and neutralization assays represents a powerful analytical platform to investigate the efficacy of antivenoms at the molecular and preclinical levels. This article is part of a Special Issue entitled: Proteomics of non-model organisms.
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A second generation antivenomics protocol, based on affinity chromatography, was compared with a previously (first generation) immunodepletion protocol using as a proof of principle the pan-African EchiTAb-Plus-ICP(®) IgG antivenom and the venoms of Echis ocellatus, Bitis arietans, and African spitting cobras. The antivenom showed qualitatively similar immunoreactivity patterns using either antivenomic approach. Quantitative departures were noticed between both methods, which may be ascribed to differences in calculating the relative amounts of the non-recognized venom proteins. The smoother baseline in chromatograms of the affinity column allowed better resolution and more accurate quantification of the antivenomic outcome than the original immunodepletion protocol. Our results indicate that both methods can be used interchangeably to investigate the in vitro immunoreactivity of antivenoms. However, advantages of the second generation antivenomics are the possibility of analyzing F(ab')(2) antivenoms and the reusability of the affinity columns. These features contribute to the generalization, economy and reproducibility of the method.
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Snakebites represent an important neglected public health problem in many developing countries. There is a lack of epidemiological data, which would be very useful for the organisation of snakebite management and provision of antivenom. An extensive literature search for the years 1970-2010 was performed. Data were analysed using meta-analysis to take into account the heterogeneity between the studies and their respective weight. Incidence, mortality and population at risk were estimated after stratification according to the environment (urban or rural) and survey methodologies (national, hospital or community studies). The incidence of snakebite was inversely correlated with population density. The number of envenomings was estimated at 314,078 [CI95% = 251,513-377,462], of which 95% occurred in rural areas. The remainder occurred in cities. The annual mortality was estimated at 7,331 [5,148-9,568], of which 97% occurred in a rural environment. The annual number of amputations ranged from 5,908 to 14,614. The population most at risk was young men engaged in agricultural or pastoral labours. Household surveys indicated that actual incidence and mortality were likely 3-5 times higher. The difference maybe explained by treatment seeking behaviour. However, incidences and mortalities reported here reflect the number of patients who attend modern health facilities, giving underestimated figures of the burden of snakebites in sub-Saharan Africa but realistic current requirements for antivenoms.
Article
A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.
Article
Snakebite in Africa causes thousands of deaths annually and considerable permanent physical disability. The saw-scaled viper, Echis ocellatus, represents the single most medically important snake species in West Africa. To provide a detailed compositional analysis of the venom of E. ocellatus for designing novel toxin-specific immunotherapy and to delineate sequence structure-function relationships of individual toxins, we characterised the venom proteome and the venom gland transcriptome. Whole E. ocellatus venom was fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction using a combination of SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS of tryptic peptides. This analysis identified around 35 distinct proteins of molecular masses in the range of 5.5-110 kDa belonging to 8 different toxin families (disintegrin, DC-fragment, phospholipase A(2), cysteine-rich secretory protein, serine proteinase, C-type lectin, l-amino acid oxidase, and Zn(2+)-dependent metalloprotease). Comparison of the toxin composition of E. ocellatus venom determined using a proteomic approach, with the predicted proteome derived from assembly of 1000 EST sequences from a E. ocellatus venom gland cDNA library, shows some differences. Most notably, peptides derived from 26% of the venom proteins could not be ascribed an exact match in the transcriptome. Similarly, 64 (67%) out of the 95 putative toxin clusters reported in the transcriptome did not match to peptides detected in the venom proteome. These data suggest that the final composition of venom is influenced by transcriptional and post-translational mechanisms that may be more complex than previously appreciated. This, in turn, emphasises the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the precise composition of snake venom, than would be gleaned from using one analysis alone. From a clinical perspective, the large amount of SVMPs (66.5% of the total venom proteins) is consistent with the haemorrhagic pathology associated with E. ocellatus envenoming. More significantly, whilst the proteomic analysis confirms the majority of these metalloproteinases (58%) belong to the SVMP PIII class, MS/MS derived peptide sequencing also demonstrates a major constituent (32%) of E. ocellatus venom is a PIV-SVMP with a quaternary structure comprising a 48 kDa (Q2UXQ4 or Q2UXQ5) PIII-SVMP subunit, and two 14-16 kDa C-type lectin-like domains [EOC_00087 and EOC_00124] which display similarity to echicetin alpha [P81017] and beta [P81996] subunits.
Article
The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten species of Costa Rican crotaline snakes were studied, together with the neutralization of these effects by a polyvalent antivenom. The venoms of Bothrops asper, B. schlegelii, B. nummifer, B. godmani, Lachesis muta and Crotalus durissus induced a coagulant effect in vitro, and all of them, with the exception of B. nummifer, also induced defibrination in vivo. The four non-coagulant venoms (B. lateralis, B. ophryomegas, B. nasuta and B. picadoi) induced a degradation of the alpha (A) chain of fibrinogen, thereby inhibiting coagulation. However, they did not induce defibrination upon i.v. injection. All of the venoms showed fibrinolytic activity in vitro. Polyvalent antivenom was effective in the neutralization of coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of these venoms, with the exception of coagulant effect induced by C. durissus venom. Since only three venoms are used in the immunization of horses, these results demonstrate the high degree of immunological cross reactivity between components affecting coagulation in Costa Rican crotaline snake venoms.
Article
The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ED50 ranging from 50 to 300 microliters antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently (ED50 = 760 microliters/mg.) Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi (ED = 430 microliters/mg) and its highest towards the venom of B. asper (Pacific region) (ED50 = 47 microliters/mg). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.
Article
Echis carinatus is the most important cause of morbidity and mortality from snake bite in Nigeria and in many other parts of the world. Forty-six patients with systemic poisoning by this snake were given echis antivenom made either by the South African Institute for Medical Research (S.A.I.M.R.) or by Behringwerke (North and West African polyvalent antivenom). A simple test of blood coagulability was used to assess whether an adequate neutralizing dose of antivenom had been given. An average of 15.2 ml S.A.I.M.R. antivenom restored normal coagulability permanently in all 23 patients in one group, but in the other group receiving an average dose of 37.9 ml Behringwerke antivenom normal clotting resulted in only 18 out of 23 patients. Local tissue swelling was similar in both groups, but local necrosis occurred in three patients treated with Behringwerke antivenom and in none given S.A.I.M.R. antivenom.
Article
A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two different methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of effective doses 50% (ED(50)) differed markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD(50)s is used, than with the method of IB, where a challenge dose of 5 LD(50)s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was effectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were effective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive cross reactivity between these antivenoms and Central and South American crotaline snake venoms.
Article
A study of the applicability of a colorimetric biuret procedure for protein determination to a variety of biological preparation has revealed that the practice of using this procedure is undesirable because markedly apparent high values are obtained in this way. In some cases, this interference was mainly due to lipids. Use of suitable controls to account for the interfering opalescence in biuret color measurements considerably improved the method. Conditions affecting the reliability of the procedure are also described. For crude extracts the colorimetric phenol method was found to be more reliable. However, a prior separation of protein by TCA is often advantageous not alone for Kjeldahl analysis but also for colorimetric estimations. The actual chromogenicity of the protein mixtures of different biological extracts studied was found to be comparable to that of the commonly employed standard protein, bovine serum albumin.
Article
Snake bite envenoming, mainly caused by the saw-scaled or carpet viper (Echis ocellatus), is a neglected disease of West Africa. Specific antivenoms can save life and limb but, for various reasons, supply of these essential drugs to Africa has dwindled to less than 2% of estimated requirements. Other problems include maldistribution, inadequate conservation and inappropriate clinical use of antivenoms. In the face of this crisis, several promising new antivenoms have been developed. However, some dangerously inappropriate products of Indian origin are being marketed by unscrupulous manufacturers or distributors in Africa and Papua New Guinea, with disastrous results. A major source of confusion is labelling antivenom with ambiguous snake names that fail to distinguish the Asian species whose venoms are used in their production from the local snakes whose venoms are antigenically dissimilar.
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Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa
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Evaluation of the preclinical efficacy of four antivenoms
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Sánchez, L.V., Pla, D., Herrera, M., Chippaux, J.P., Calvete, J.J., Gutiérrez, J.M., 2015. 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 106, 97-107.