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

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

Comparison of Phylogeny,Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A₂ reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.     

Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG₁ – κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/μL and 2.1 ng/μL in spiked buffer samples and 28.7 ng/μL and 110 ng/μL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.     

The action of Echis carinatus and Naja naja venoms on human neutrophils; an emphasis on NETosis

BackgroundNeutrophils are the first line defense cells of the innate immunity. As a final defense, they discharge their de-condensed chromatin/DNA fibers, the NETs (Neutrophil Extracellular Traps), by a process called NETosis. Two types of NETosis have been currently described: the suicidal/delayed/classical-type, which is ROS dependent that results in the ejection of nuclear DNA, and the vital/rapid/early-type, which may or may not require ROS but, eject nuclear/mitochondrial DNA or both. Thus, Echis carinatus and Naja naja venoms are comparatively studied for their NET inducing property.MethodsFormation of NETs, cell viability, ROS, and Ca²⁺ levels are estimated. An in vivo toxicity study and possible cellular signaling have been addressed using immunoblots and pharmacological inhibitors.ResultsE. carinatus and N. naja venoms respectively induce suicidal and vital NETosis. E. carinatus venom induces NETosis by activating NOX and PAD-4 enzymes in a ROS dependent manner via PKC/ERK/JNK signaling axis, while N. naja venom does it by activating PAD-4 enzyme, but independent of ROS requirement and as well as PKC/ERK/JNK activation.ConclusionFor the first time our study demonstrates the distinct action of E. carinatus and N. naja venoms on the process of NETosis. NETosis being a newly explored area in snake venom pharmacodynamics, it is important to study its impact on the various pathophysiological properties induced by snake venoms.SignificanceUnderstanding the varied actions of snake venoms on neutrophils/blood cells and the role of DNase are likely to provide insights for better management of snakebite pathophysiology.     

Treatment outcomes among snakebite patients in north-west Ethiopia—A retrospective analysis

BackgroundMillions of people are bitten by venomous snakes annually, causing high mortality and disability, but the true burden of this neglected health issue remains unknown. Since 2015, Médecins Sans Frontières has been treating snakebite patients in a field hospital in north-west Ethiopia. Due to the poor market situation for effective and safe antivenoms for Sub-Saharan Africa, preferred antivenom was not always available, forcing changes in choice of antivenom used. This study describes treatment outcomes and the effectiveness and safety of different antivenoms used.Methodology / Principal findingsThis retrospective observational study included 781 snakebite patients presenting at the field hospital between 2015 and 2019. Adjusted odds ratios, 95%-CI and p-values were used to compare the treatment outcome of patients treated with Fav-Afrique (n = 149), VacSera (n = 164), and EchiTAb-PLUS-ICP (n = 156) antivenom, and to identify the risk of adverse reactions for each antivenom. Whereas only incidental snakebite cases presented before 2015, after treatment was made available, cases rapidly increased to 1,431 in 2019. Envenomation was mainly attributed to North East African saw-scaled viper (Echis pyramidum) and puff adder (Bitis arietans). Patients treated with VacSera antivenom showed lower chance of uncomplicated treatment outcome (74.4%) compared to Fav-Afrique (93.2%) and EchiTAb-PLUS-ICP (90.4%). VacSera and EchiTAb-PLUS-ICP were associated with 16- and 6-fold adjusted odds of treatment reaction compared to Fav-Afrique, respectively, and VacSera was weakly associated with higher odds of death.Conclusions / SignificanceSnakebite frequency is grossly underreported unless treatment options are available. Although EchiTAb-PLUS-ICP showed favorable outcomes in this retrospective analysis, prospective randomized trials are needed to evaluate the effectiveness and safety of the most promising antivenoms for Sub-Saharan Africa. Structural investment in sustained production and supply of antivenom is urgently needed.     

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4–115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20–25 main toxins belonging to the following protein families: disintegrin, PLA₂, serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), l-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA₂ crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA₂s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for ～ 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (～ 60%) recognition of PLA₂ molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA₂ molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.     

Antivenoms for the treatment of snakebite envenomings: The road ahead

The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders.     

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization,isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A₂ activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A₂ homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A₂ variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.     

Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa

The development of snake antivenoms more than a century ago should have heralded effective treatment of the scourge of snakebite envenoming in impoverished, mostly rural populations around the world. That snakebite still exists today, as a widely untreated illness that maims, kills and terrifies men, women and children in vulnerable communities, is a cruel anachronism. Antivenom can be an effective, safe and affordable treatment for snakebites, but apathy, inaction and the politicisation of public health have marginalised both the problem (making snakebite arguably the most neglected of all neglected tropical diseases) and its solution. For lack of any coordinated approach, provision of antivenoms has been pushed off the public health agenda, leading to an incongruous decline in demand for these crucial antidotes, excused and fed by new priorities, an absence of epidemiological data, and a poor regulatory framework. These factors facilitated the infiltration of poor quality products that degrade user confidence and undermine legitimate producers. The result is that tens of thousands are denied an essential life-saving medicine, allowing a toll of human suffering that is a summation of many individual catastrophes. No strategy has been developed to address this problem and to overcome the intransigence and inaction responsible for the global tragedy of snakebite. Attempts to engage with the broader public health community through the World Health Organisation (WHO), GAVI, and other agencies have failed. Consequently, the toxinology community has taken on a leadership role in a new approach, the Global Snakebite Initiative, which seeks to mobilise the resources, skills and experience of scientists and clinicians for whom venoms, toxins, antivenoms, snakes and snakebites are already fields of interest. Proteomics is one such discipline, which has embraced the potential of using venoms in bio-discovery and systems biology. The fields of venomics and antivenomics have recently evolved from this discipline, offering fresh hope for the victims of snakebites by providing an exciting insight into the complexities, nature, fundamental properties and significance of venom constituents. Such a rational approach brings with it the potential to design new immunising mixtures from which to raise potent antivenoms with wider therapeutic ranges. This addresses a major practical limitation in antivenom use recognised since the beginning of the 20th century: the restriction of therapeutic effectiveness to the specific venom immunogen used in production. Antivenomic techniques enable the interactions between venoms and antivenoms to be examined in detail, and if combined with functional assays of specific activity and followed up by clinical trials of effectiveness and safety, can be powerful tools with which to evaluate the suitability of current and new antivenoms for meeting urgent regional needs. We propose two mechanisms through which the Global Snakebite Initiative might seek to end the antivenom drought in Africa and Asia: first by establishing a multidisciplinary, multicentre, international collaboration to evaluate currently available antivenoms against the venoms of medically important snakes from specific nations in Africa and Asia using a combination of proteomic, antivenomic and WHO-endorsed preclinical assessment protocols, to provide a validated evidence base for either recommending or rejecting individual products; and secondly by bringing the power of proteomics to bear on the design of new immunising mixtures to raise Pan-African and Pan-Asian polyvalent antivenoms of improved potency and quality. These products will be subject to rigorous clinical assessment. We propose radically to change the basis upon which antivenoms are produced and supplied for the developing world. Donor funding and strategic public health alliances will be sought to make it possible not only to sustain the financial viability of antivenom production partnerships, but also to ensure that patients are relieved of the costs of antivenom so that poverty is no longer a barrier to the treatment of this important, but grossly neglected public health emergency.     

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom

ABSTRACT

Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra.

Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies.

Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A₂ activity. Phospholipase A₂ and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs.

Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.     

A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.     

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity

BackgroundThe plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study.MethodsE. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC–MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico.ResultsThe bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A₂, hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom.ConclusionFor the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity.General significanceExploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.     

Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom

Background

Snakebite is a significant cause of death and disability in subsistent farming populations of sub-Saharan Africa. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses/sheep but, because of complex fiscal reasons, there is a paucity of antivenom in sub-Saharan Africa. To address the plight of thousands of snakebite victims in savannah Nigeria, the EchiTAb Study Group organised the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region. The Echis saw-scaled vipers have a wide African distribution and medical importance. In an effort to maximise the clinical utility of scarce antivenom resources in Africa, we aimed to ascertain, at the pre-clinical level, to what extent the E. ocellatus-specific EchiTAbG antivenom, which was designed specifically for Nigeria, neutralised the lethal activity of venom from two other African species, E. pyramidum leakeyi and E. coloratus.

Methodology/Principal Findings

Despite apparently quite distinctive venom protein profiles, we observed extensive cross-species similarity in the immuno-reactivity profiles of Echis species-specific antisera. Using WHO standard pre-clinical in vivo tests, we determined that the monospecific EchiTAbG antivenom was as effective at neutralising the venom-induced lethal effects of E. pyramidum leakeyi and E. coloratus as it was against E. ocellatus venom. Under the restricted conditions of this assay, the antivenom was ineffective against the lethal effects of venom from the non-African Echis species, E. carinatus sochureki.

Conclusions/Significance

Using WHO-recommended pre-clinical tests we have demonstrated that the new anti-E. ocellatus monospecific antivenom EchiTAbG, developed in response to the considerable snakebite-induced mortality and morbidity in Nigeria, neutralised the lethal effects of venoms from Echis species representing each taxonomic group of this genus in Africa. This suggests that this monospecific antivenom has potential to treat envenoming by most, perhaps all, African Echis species.     

Effective Equine Immunization Protocol for Production of Potent Poly-specific Antisera against Calloselasma rhodostoma,Cryptelytrops albolabris and Daboia siamensis

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma “Malayan pit viper” (CR), and Daboia siamensis “Russell’s viper” (DS). Four horses were immunized with a mixture of the 3 viper venoms using the ‘low dose, low volume multi-site’ immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.     

Hemostatic interference of Indian king cobra (<Emphasis Type="Italic">Ophiophagus hannah</Emphasis>) venom. Comparison with three other snake venoms of the subcontinent

Unlike Naja naja, Bungarus caeruleus, Echis carinatus, and Daboia/Vipera russellii venoms, Ophiophagus hannah venom is medically ignored in the Indian subcontinent. Being the biggest poisonous snake, O. hannah has been presumed to inject several lethal doses of venom in a single bite. Lack of therapeutic antivenom to O. hannah bite in India makes any attempt to save the victim a difficult exercise. This study was initiated to compare O. hannah venom with the above said venoms for possible interference in hemostasis. Ophiophagus hannah venom was found to actively interfere in hemostatic stages such as fibrin clot formation, platelet activation/aggregation, and fibrin clot dissolution. It decreased partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin clotting time (TCT). These activities are similar to that shown by E. carinatus and D. russellii venoms, and thus O. hannah venom was found to exert procoagulant activity through the common pathway of blood coagulation, while N. naja venom increased aPTT and TCT but not PT, and hence it was found to exert anticoagulant activity through the intrinsic pathway. Venoms of O. hannah, E. carinatus, and D. russellii lack plasminogen activation property as they do not hydrolyze azocasein, while they all show plasmin-like activity by degrading the fibrin clot. Although N. naja venom did not degrade azocasein, unlike other venoms, it showed feeble plasmin-like activity on fibrin clot. Venom of E. carinatus induced clotting of human platelet rich plasma (PRP), while the other three venoms interfered in agonist-induced platelet aggregation in PRP. Venom of O. hannah least inhibited the ADP induced platelet aggregation as compared to D. russellii and N. naja venoms. All these three venoms showed complete inhibition of epinephrine-induced aggregation at varied doses. However, O. hannah venom was unique in inhibiting thrombin induced aggregation.     

Availability,affordability and stock-outs of commodities for the treatment of snakebite in Kenya

BackgroundAnnually, about 2.7 million snakebite envenomings occur globally. Alongside antivenom, patients usually require additional care to treat envenoming symptoms and antivenom side effects. Efforts are underway to improve snakebite care, but evidence from the ground to inform this is scarce. This study, therefore, investigated the availability, affordability, and stock-outs of antivenom and commodities for supportive snakebite care in health facilities across Kenya.Methodology/principal findingsThis study used an adaptation of the standardised World Health Organization (WHO)/Health Action International methodology. Data on commodity availability, prices and stock-outs were collected in July-August 2020 from public (n = 85), private (n = 36), and private not-for-profit (n = 12) facilities in Kenya. Stock-outs were measured retrospectively for a twelve-month period, enabling a comparison of a pre-COVID-19 period to stock-outs during COVID-19. Affordability was calculated using the wage of a lowest-paid government worker (LPGW) and the impoverishment approach. Accessibility was assessed combining the WHO availability target (≥80%) and LPGW affordability (<1 day’s wage) measures. Overall availability of snakebite commodities was low (43.0%). Antivenom was available at 44.7% of public- and 19.4% of private facilities. Stock-outs of any snakebite commodity were common in the public- (18.6%) and private (11.7%) sectors, and had worsened during COVID-19 (10.6% versus 17.0% public sector, 8.4% versus 11.7% private sector). Affordability was not an issue in the public sector, while in the private sector the median cost of one vial of antivenom was 14.4 days’ wage for an LPGW. Five commodities in the public sector and two in the private sector were deemed accessible.ConclusionsAccess to snakebite care is problematic in Kenya and seemed to have worsened during COVID-19. To improve access, efforts should focus on ensuring availability at both lower- and higher-level facilities, and improving the supply chain to reduce stock-outs. Including antivenom into Universal Health Coverage benefits packages would further facilitate accessibility.     

Proteomic tools against the neglected pathology of snake bite envenoming

This article covers the application of proteomic tools (‘venomics’, ‘antivenomics’ and ‘venom phenotyping’) to study the composition and natural history of snake venoms, and the cross-reactivity of antivenoms with homologous and heterologous venoms, to help address the neglected pathology of snake bite envenoming. The identification of evolutionary and immunological trends may help to replace the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies with a more rational approach based on proteome phenotype and immunological profile similarities. Antivenomics and venom phenotyping may also contribute to expand the clinical range of currently existing antidotes.     

A painful journey to antivenom: The therapeutic itinerary of snakebite patients in the Brazilian Amazon (The QUALISnake Study)

Access to antivenoms is not guarranteed for vulnerable populations that inhabit remote areas in the Amazon. The study of therapeutic itineraries (TI) for treatment of snakebites would support strategies to provide timely access to users. A TI is the set of processes by which individuals adhere to certain forms of treatment, and includes the path traveled in the search for healthcare, and practices to solve their health problems. This study aims to describe TIs of snakebite patients in the Brazilian Amazon. This study was carried out at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado, in Manaus, state of Amazonas, Brazil. The itinerary from the moment of the bite to the patient’s admission to the reference unit was analyzed. Sample size was defined by saturation. After an exploratory survey to collect epidemiological variables, in-depth interviews were conducted following a semi-structured guide. Patients originated from rural areas of 11 different municipalities, including ones located >500 kilometers from Manaus. A great fragmentation was observed in the itineraries, marked by several changes of means of transport along the route. Four themes emerged from the analysis: exposure to snakebite during day-to-day activities, use of traditional therapeutic practices, and personal perception of the severity, as well as the route taken and its contingencies. Access to healthcare requires considerable effort on the part of snakebite patients. Major barriers were identified, such as the low number of hospitals that offer antivenom treatment, poor access to healthcare due to long distances and geographic barriers, low acceptability of healthcare offered in countryside, lack of use of personal protective equipment, common use of ineffective or deleterious self-care practices, late recognition of serious clinical signs and resistance to seeking medical assistance. Health education, promotion of immediate transport to health centers and decentralization of antivenom from reference hospitals to community healthcare centers in the Brazilian Amazon are more effective strategies that would to maximize access to antivenom treatment.     

Cross neutralization of Afro-Asian cobra and Asian krait venoms by a Thai polyvalent snake antivenom (Neuro Polyvalent Snake Antivenom)

Background

Snake envenomation is a serious public health threat in the rural areas of Asian and African countries. To date, the only proven treatment for snake envenomation is antivenom therapy. Cross-neutralization of heterologous venoms by antivenom raised against venoms of closely related species has been reported. The present study examined the cross neutralizing potential of a newly developed polyvalent antivenom, termed Neuro Polyvalent Snake Antivenom (NPAV). NPAV was produced by immunization against 4 Thai elapid venoms.

Principal Findings

In vitro neutralization study using mice showed that NPAV was able to neutralize effectively the lethality of venoms of most common Asiatic cobras (Naja spp.), Ophiophagus hannah and kraits (Bungarus spp.) from Southeast Asia, but only moderately to weakly effective against venoms of Naja from India subcontinent and Africa. Studies with several venoms showed that the in vivo neutralization potency of the NPAV was comparable to the in vitro neutralization potency. NPAV could also fully protect against N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in anesthetized rats, demonstrating that the NPAV could neutralize most of the major lethal toxins in the Naja venom.

Conclusions/Significance

The newly developed polyvalent antivenom NPAV may find potential application in the treatment of elapid bites in Southeast Asia, especially Malaysia, a neighboring nation of Thailand. Nevertheless, the applicability of NPAV in the treatment of cobra and krait envenomations in Southeast Asian victims needs to be confirmed by clinical trials. The cross-neutralization results may contribute to the design of broad-spectrum polyvalent antivenom.