Antivenom Cross-reactivity Research Articles

Promoting co-existence between humans and venomous snakes through increasing the herpetological knowledge base.

Snakebite incidence at least partly depends on the biology of the snakes involved. However, studies of snake biology have been largely neglected in favour of anthropic factors, with the exception of taxonomy, which has been recognised for some decades to affect the design of antivenoms. Despite this, within-species venom variation and the unpredictability of the correlation with antivenom cross-reactivity has continued to be problematic. Meanwhile, other aspects of snake biology, including behaviour, spatial ecology and activity patterns, distribution, and population demography, which can contribute to snakebite mitigation and prevention, remain underfunded and understudied. Here, we review the literature relevant to these aspects of snakebite and illustrate how demographic, spatial, and behavioural studies can improve our understanding of why snakebites occur and provide evidence for prevention strategies. We identify the large gaps that remain to be filled and urge that, in the future, data and relevant metadata be shared openly via public data repositories so that studies can be properly replicated and data used in future meta-analyses.

An interactive database for the investigation of high-density peptide microarray guided interaction patterns and antivenom cross-reactivity.

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 user-friendly, 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 normalization 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.The data, as well as the web application, we present in this article should be of significant value to the venom-antivenom research community. Knowledge gained from our current and future analyses of this dataset carry the potential to guide the improvement and optimization of current antivenoms for maximum patient benefit, as well as aid the development of next-generation antivenoms.

Basal but divergent: Clinical implications of differential coagulotoxicity in a clade of Asian vipers

Envenomations by Asian pitvipers can induce multiple clinical complications resulting from coagulopathic and neuropathic effects. While intense research has been undertaken for some species, functional coagulopathic effects have been neglected. As these species' venoms affect the blood coagulation cascade we investigated their effects upon the human clotting cascade using venoms of species from the Azemiops, Calloselasma, Deinagkistrodon and Hypnale genera. Calloselasma rhodostoma, Deinagkistrodon acutus, and Hypnale hypnale produced net anticoagulant effects through pseudo-procoagulant clotting of fibrinogen, resulting in weak, unstable, transient fibrin clots. Tropidolaemus wagleri was only weakly pseudo-procoagulant, clotting fibrinogen with only a negligible net anticoagulant effect. Azemiops feae and Tropidolaemus subannulatus did not affect clotting. This is the first study to examine in a phylogenetic context the coagulotoxic effects of related genera of basal Asiatic pit-vipers. The results reveal substantial variation between sister genera, providing crucial information about clinical effects and implications for antivenom cross-reactivity.

Habu coagulotoxicity: Clinical implications of the functional diversification of Protobothrops snake venoms upon blood clotting factors

Venom can affect any part of the body reachable via the bloodstream. Toxins which specifically act upon the coagulation cascade do so either by anticoagulant or procoagulant mechanisms. Here we investigated the coagulotoxic effects of six species within the medically important pit viper genus Protobothrops (Habu) from the Chinese mainland and Japanese islands, a genus known to produce hemorrhagic shock in envenomed patients. Differential coagulotoxicity was revealed: P. jerdonii and P. mangshanensis produced an overall net anticoagulant effect through the pseudo-procoagulant clotting of fibrinogen; P. flavoviridis and P. tokarensis exhibit a strong anticoagulant activity through the destructive cleavage of fibrinogen; and while P. elegans and P. mucrosquamatus both cleaved the A-alpha and B-beta chains of fibrinogen they did not exhibit strong anticoagulant activity. These variations in coagulant properties were congruent with phylogeny, with the closest relatives exhibiting similar venom effects in their action upon fibrinogen. Ancestral state reconstruction indicated that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state, while anticoagulation produced by destructive cleavage of fibrinogen is the derived state within this genus. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one genus surrounding one target site, governed by variability in co-factor dependency. The documentation that the same net overall function, anticoagulation, is mediated by differential underlying mechanics suggests limited antivenom cross-reactivity, although this must be tested in future work. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient.

Antibody Cross-Reactivity in Antivenom Research.

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.

Assessment of quality, safety, and pre-clinical toxicity of an equine polyvalent anti-snake venom (Pan Africa): Determination of immunological cross-reactivity of antivenom against venom samples of Elapidae and Viperidae snakes of Africa

Snakebite causes a large amount of morbidities and mortalities in Africa. The safety, efficacy, and homogeneity of anti-snake venoms are crucial for snakebite treatments to be effective with minimal adverse effects. We assessed the homogeneity of preparations of three different batches of Combipack snake venom antiserums (Pan Africa) [CSVAPA] by quantitatively analysing F(ab’)2, IgG, and other contaminating proteins of plasma. LC-MS/MS analysis showed that approximately 92.4% of the proteins from the CSVAPA samples was IgG/F(ab’)2 and the percent composition of contaminating proteins in CSVAPA varied from 0.07 to 4.6%. Batch 1 of the CSVAPA also contained a minor amount of undigested IgG and F(ab’)2 aggregates. CSVAPA contained more than 60% venom-specific antibodies, showed moderate complement activation, no IgE contamination, safe level of endotoxin, and also showed pre-clinical safety. The immuno cross-reactivity of CSVAPA against 14 Viperidae and Elapidae snake venoms of Africa was tested by ELISA and immunoblotting, and the neutralization of major enzymatic venom activities, demonstrating that high molecular weight (>50 kDa) venom proteins are better recognized/neutralized compared to relatively low molecular weight (<20 kDa) venom proteins. CSVAPA at a dose of 3–12 times higher than the clinical dose did not cause deaths or adverse reaction of treated rabbits. The results suggest the satisfactory quality, safety, and efficacy of CSVAPA.

Differential procoagulant effects of saw-scaled viper (Serpentes: Viperidae: Echis) snake venoms on human plasma and the narrow taxonomic ranges of antivenom efficacies

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 restricted 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 cofactors 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.

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping.

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.

Antivenomics as a tool to improve the neutralizing capacity of the crotalic antivenom: a study with crotamine

BackgroundSnakebite treatment requires administration of an appropriate antivenom that should contain antibodies capable of neutralizing the venom. To achieve this goal, antivenom production must start from a suitable immunization protocol and proper venom mixtures. In Brazil, antivenom against South American rattlesnake (Crotalus durissus terrificus) bites is produced by public institutions based on the guidelines defined by the regulatory agency of the Brazilian Ministry of Health, ANVISA. However, each institution uses its own mixture of rattlesnake venom antigens. Previous works have shown that crotamine, a toxin found in Crolatus durissus venom, shows marked individual and populational variation. In addition, serum produced from crotamine-negative venoms fails to recognize this molecule.MethodsIn this work, we used an antivenomics approach to assess the cross-reactivity of crotalic antivenom manufactured by IVB towards crotamine-negative venom and a mixture of crotamine-negative/crotamine-positive venoms.ResultsWe show that the venom mixture containing 20% crotamine and 57% crotoxin produced a strong immunogenic response in horses. Antivenom raised against this venom mixture reacted with most venom components including crotamine and crotoxin, in contrast to the antivenom raised against crotamine-negative venom.ConclusionsThese results indicate that venomic databases and antivenomics analysis provide a useful approach for choosing the better venom mixture for antibody production and for the subsequent screening of antivenom cross-reactivity with relevant snake venom components.

Genus Calliophis of Asiatic coral snakes: A deficiency of venom cross-reactivity and neutralization against seven regional elapid antivenoms

Venoms of Calliophis bivirgata and Calliophis intestinalis exhibited moderate binding activities toward Neuro Bivalent Antivenom (Taiwan) but not the other six elapid monovalent or bivalent antivenoms available in the region. All antivenoms failed to neutralize C. bivirgata venom lethality in mice. The findings indicate the need to validate antivenom cross-reactivity with in vivo cross-neutralization, and imply that distinct antigens of Calliophis venoms should be incorporated in the production of a pan-regional poly-specific antivenom.

Immunological profile of antivenoms: Preclinical analysis of the efficacy of a polyspecific antivenom through antivenomics and neutralization assays

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. 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.

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.

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.

Impact of Regional Variation in Bothrops asper Snake Venom on the Design of Antivenoms: Integrating Antivenomics and Neutralization Approaches

Intraspecific snake venom variations have implications in the preparation of venom pools for the generation of antivenoms. The impact of such variation in the cross-reactivity of antivenoms against Bothrops asper venom was assessed by comparing two commercial and four experimental antivenoms. All antivenoms showed similar immunorecognition pattern toward the venoms from adult and neonate specimens. They completely immunodepleted most P-III snake venom metalloproteinases (SVMPs), l-amino acid oxidases, serine proteinases, DC fragments, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like proteins, and partially immunodepleted medium-sized disintegrins, phospholipases A(2) (PLA(2)s), some serine proteinases, and P-I SVMPs. Although all antivenoms abrogated the lethal, hemorrhagic, coagulant, proteinase, and PLA(2) venoms activities, monospecific experimental antivenoms were more effective than the polyspecific experimental antivenom. In addition, the commercial antivenoms, produced in horses subjected to repeated immunization cycles, showed higher neutralization than experimental polyspecific antivenom, produced by a single round of immunization. Overall, a conspicuous pattern of cross-neutralization was evident for all effects by all antivenoms, and monospecific antivenoms raised against venom from the Caribbean population were effective against venom from the Pacific population, indicating that geographic variations in venom proteomes of B. asper from Costa Rica do not result in overt variations in immunological cross-reactivity between antivenoms.

Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming

Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact, particularly in Asia, Africa and Latin America. A global initiative, aimed at increasing antivenom production and accessibility, is being promoted by the World Health Organization and others. This work discusses several aspects of antivenom manufacture and control in which the proteomic analysis of snake venoms, for which the term 'snake venomics' has been coined, might play a relevant supporting role. Snake venomics has already shown its usefulness for generating knowledge at different levels (ontogenetic, individual, and geographic) on inter- and intraspecies venom variability. This information has applications for the quality control of venom preparations used in antivenom manufacture. Moreover, the design of the best venom mixtures for immunization, aimed at increasing the effectiveness of antivenoms, may also be guided by venom proteome analysis, including molecular studies of the cross-reactivity of antivenoms and heterologous venoms through a recently developed methodological approach termed 'antivenomics'. Results generated by proteomic protocols should be complemented by preclinical testing of antivenom efficacy using functional neutralization assays. Snake venomics might be also helpful in designing alternative in vitro tests for the assessment of antivenom efficacy that would eventually substitute current in vivo tests.