Pharmacokinetic evaluation of a single chain antibody fragment against scorpion toxins in sheep.

A key aspect during the development of antivenoms is the evaluation of the efficiency and security of the therapeutic molecules. In this work, we report the pharmacokinetic analysis of a neutralizing single chain antibody fragment named LR (scFv LR) where three sheep were used as a large animal model. The animals were injected through i.v. route with 2 mg of scFv LR. Blood samples were drawn every minute within the first 15 min, the sampling continues at 20, 25, 30, 45, 60, 90, 120 min, subsequently at 1-h intervals, 3, 4, 5, 6 h, two more samples at 9 and 12 h and, two more samples at 24 and 48 h and finally at one-day intervals during 4 days. scFv LR levels were measured from blood serum and urine samples by an ELISA. The pharmacokinetics of the experimental data was analyzed using the three-exponential kinetics. The value of the fast initial component (τ1=0.409±0.258min) indicated that the scFv is distributed rapidly into the tissues. The mean residence time, MRT, was 45 ± 0.51 min and the clearance (CL), 114.3 ± 14.3 mL/min. From urine samples it was possible to detect significant amounts of scFv LR, which is evidence of renal elimination.

Quantifying venom production: A study on Micrurus snakes in Mexico.

Our study quantifies venom production in nine Mexican coral snake species (Micrurus), encompassing 76 specimens and 253 extractions. Noteworthy variations were observed, with M. diastema and M. laticollaris displaying diverse yields, ranging from 0.3 mg to 59 mg. For animals for which we have length data, there is a relationship between size and venom quantity. Twenty-eight percent of the observed variability in venom production can be explained by snake size, suggesting that other factors influence the amount of obtained venom. These findings are pivotal for predicting venom effects and guiding antivenom interventions. Our data offer insights into Micrurus venom yields, laying the groundwork for future research and aiding in medical response strategies. This study advances understanding coral snake venom production, facilitating informed medical responses to coral snake bites.

Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Ontogenetic change in the venom composition of one Mexican black-tailed rattlesnake (Crotalus molossus nigrescens) from Durango, Mexico.

To corroborate the ontogenetic shift in the venom composition of the Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) previously reported through the census approach, we evaluated the shift in the protein profile, lethality, and proteolytic and phospholipase activities of four venom samples obtained in 2015, 2018, 2019, and 2021 from one C. m. nigrescens individual (CMN06) collected in Durango, Mexico. We demonstrated that the venom of C. m. nigrescens changed from a myotoxin-rich venom to a phospholipase A2 and snake venom metalloproteinase-rich venom. Additionally, the proteolytic and phospholipase activities increased with age, but the lethality decreased approximately three times.

Longitudinal anti-SARS-CoV-2 antibody immune response in acute and convalescent patients.

Despite global efforts to assess the early response and persistence of SARS-CoV-2 antibodies in patients infected with or recovered from COVID-19, our understanding of the factors affecting its dynamics remains limited. This work aimed to evaluate the early and convalescent immunity of outpatients infected with SARS-CoV-2 and to determine the factors that affect the dynamics and persistence of the IgM and IgG antibody response. Seropositivity of volunteers from Mexico City and the State of Mexico, Mexico, was evaluated by ELISA using the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein for 90 days, at different time points (1, 15, 45, 60, and 90 days) after molecular diagnosis (RT-qPCR). Gender, age range, body mass index (BMI), comorbidities, and clinical spectrum of disease were analyzed to determine associations with the dynamics of anti-SARS-CoV-2 antibodies. On 90 days post-infection, individuals with moderate and asymptomatic disease presented the lowest levels of IgM, while for IgG, at the same time, the highest levels occurred with mild and moderate disease. The IgM and IgG levels were related to the clinical spectrum of disease, BMI, and the presence/absence of comorbidities through regression trees. The results suggest that the dynamics of anti-SARS-CoV-2 IgM and IgG antibodies in outpatients could be influenced by the clinical spectrum of the disease. In addition, the persistence of antibodies against SARS-CoV-2 could be related to the clinical spectrum of the disease, BMI, and the presence/absence of comorbidities.

Proteomic and toxicological characterization of the venoms of the most enigmatic group of rattlesnakes: The long-tailed rattlesnakes.

The most enigmatic group of rattlesnakes is the long-tailed rattlesnake group, consisting of three species: Crotalus ericsmithi, Crotalus lannomi and Crotalus stejnegeri. These species have been the least studied rattlesnakes in all aspects, and no study on the characterization of their venoms has been carried out to date. Our main objective was to investigate the proteomic composition, as well as some of the biochemical and toxic activities of these venoms, and their neutralization by commercial antivenom. The venom proteome of C. ericsmithi mainly contains metalloproteinases (SVMP; 49.3%), phospholipases A2 (PLA2; 26.2%), disintegrins (Dis; 12.6%), and snake venom serine proteases (SVSP; 6.8%), while C. lannomi venom mainly consists of SVMP (47.1%), PLA2 (19.3%), Dis (18.9%), SVSP (6%) and l-amino acid oxidase (LAAO; 2.6%). For these venoms high lethality was recorded in mice, the most potent being that of C. lannomi (LD50 of 0.99 µg/g body weight), followed by C. ericsmithi (1.30 µg/g) and finally C. stejnegeri (1.79 µg/g). The antivenoms Antivipmyn® from SILANES and Fabotherapic polyvalent antiviperin® from BIRMEX neutralized the lethal activity of the three venoms. Although this group of snakes is phylogenetically related to the C. viridis group, no neurotoxic components (crotoxin or crotoxin-like proteins) common in rattlesnakes were found in their venoms. This study expands current knowledge on the venoms of understudied snake species of the Mexican herpetofauna.

Neutralization of black widow spider (Latrodectus mactans) venom with rabbit polyclonal serum hyperimmunized with recombinant alpha-latrotoxin fragments.

Alpha-latrotoxin (ɑLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits. ɑLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ɑLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans. The hyperimmune sera obtained using the complete ɑLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 µL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.

Cloud serpent coagulotoxicity: The biochemical mechanisms underpinning the anticoagulant actions of Mixcoatlus and Ophryacus venoms.

Mexico is home to an extreme diversity of herpetofauna, with venomous snakes imposing a significant burden upon public health. However, little is known about the pathophysiological venom actions of a number of potentially medically important species, including those from the genera Mixcoatlus and Ophryacus. Our study aimed to fill this knowledge gap by ascertaining the effects of Mixcoatlus melanurus, Ophryacus smaragdinus and Ophryacus sphenophrys venoms upon the coagulation cascade utilising a series of well-validated coagulation assays. While M. melanurus venom exhibited no significant coagulotoxic activities, both O. smaragdinus and O. sphenophrys venoms exerted multiple coagulotoxic activities upon the coagulation cascade which would be contributing towards a net anticoagulant venom activity. O. sphenophrys significantly inhibited the spontaneous clotting of plasma but O. smaragdinus did not. They differed in that O. sphenophrys inhibited the clotting enzymes factor IXa and factor XIa. However, O. smaragdinus was able to inhibit factor Xa in isolation-assays. Both O. smaragdinus and O. sphenophrys degraded fibrinogen, with O. smaragdinus venom causing a significantly weaker fibrinogen clot than O. sphenophrys. In vitro antivenom efficacy assays were undertaken to ascertain the efficacy of Antivipmyn-Tri antivenom (which is made using Bothrops, Crotalus, and Lachesis venoms). This antivenom was chosen due to the phylogenetic uncertain position of the Ophryacus, but with some molecular genetics' studies placing it as sister to Lachesis. Despite the complexity of the antivenom immunising mixture, the anticoagulant activity of O. sphenophrys venom was relatively poorly neutralised by the antivenom. This work contributes to the understanding of the functional activity of Mixcoatlus and Ophryacus venoms, laying a foundation for future work investigating the coagulotoxins present within Ophryacus venoms in addition to providing data useful for the evidence-based design of clinical management strategies for the envenomed patient.

Characterization of the venom and external morphology of a natural hybrid between Crotalus atrox and Crotalus mictlantecuhtli.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production.

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization.

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30-35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve-hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

Cross-reactivity of some Micrurus venoms against experimental and therapeutic anti-Micrurus antivenoms.

We developed experimental equine polyvalent and monovalent antivenoms against the venoms of Micrurus (M.) fulvius, M. nigrocinctus and M. surinamensis and studied their immunochemical reactivity on the venoms used as immunogens and on M. pyrrhocryptus, M altirostris and M. balyocoriphus venoms. Assessment of the neutralizing capacity of the polyvalent experimental antivenom was based on inhibition of lethality (preincubation and rescue assay experiments in mice) and indirect hemolytic and phospholipase activities. The immunochemical reactivity and neutralizing capacity were compared with those of two therapeutic antivenoms used for the treatment of coral snake envenomation in North America and in Argentina. In general, the experimental antivenom conferred a comparable level of neutralization against the venoms used as immunogens when compared to the therapeutic antivenoms and a certain level of cross-neutralization against the other venoms. The results suggest the need for additional venoms in the immunogenic mixture used, in order to obtain a broad spectrum anti-Micrurus antivenom with a good neutralizing potency. Paraspecific neutralization of South American coral snake venoms, although present at a higher level than the neutralization conferred by available nonspecific Micrurus therapeutic antivenoms, was rather low in relation to the specific neutralizing capacity.

BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

A Clot Twist: Extreme Variation in Coagulotoxicity Mechanisms in Mexican Neotropical Rattlesnake Venoms.

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus, and C. tzabcan. We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus. This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments.

Neutralization of crotamine by polyclonal antibodies generated against two whole rattlesnake venoms and a novel recombinant fusion protein.

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

A symphony of destruction: Dynamic differential fibrinogenolytic toxicity by rattlesnake (Crotalus and Sistrurus) venoms.

What factors influence the evolution of a heavily selected functional trait in a diverse clade? This study adopts rattlesnakes as a model group to investigate the evolutionary history of venom coagulotoxicity in the wider context of phylogenetics, natural history, and biology. Venom-induced clotting of human plasma and fibrinogen was determined and mapped onto the rattlesnake phylogenetic tree to reconstruct the evolution of coagulotoxicity across the group. Our results indicate that venom phenotype is often independent of phylogenetic relationships in rattlesnakes, suggesting the importance of diet and/or other environmental variables in driving venom evolution. Moreover, the striking inter- and intraspecific variability in venom activity on human blood highlights the considerable variability faced by physicians treating envenomation. This study is the most comprehensive effort to date to describe and characterize the evolutionary and biological aspects of coagulotoxins in rattlesnake venom. Further research at finer taxonomic levels is recommended to elucidate patterns of variation within species and lineages.

Pan-American Lancehead Pit-Vipers: Coagulotoxic Venom Effects and Antivenom Neutralisation of Bothrops asper and B. atrox Geographical Variants.

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species' geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting.

Assessment of neutralization of Micrurus venoms with a blend of anti-Micrurus tener and anti-ScNtx antibodies.

BACKGROUND: Micrurus venoms contain two main groups of toxic protein components: short-chain α-neurotoxins (SNtx) and phospholipases type A2 (PLA2). In North America, generally, the Micrurus venoms have low abundance of SNtx compared to that of PLA2s; however, both are highly toxic to mammals, and consequently both can play a major role in the envenomation processes. Concerning the commercial horse-derived antivenoms against Micrurus from the North America region, they contain a relatively large amount of antibodies against PLA2s, and a low content of antibodies against short chain α-neurotoxins. This is mainly due to the lower relative abundance of SNtxs, and also to its poor immunogenicity due to their size and nature. Hence, Micrurus antivenoms made in North America usually present low neutralizing capacity towards Micrurus venoms whose lethality depend largely on short chain α-neurotoxins, such as South American Micrurus species. METHODS: Horses were hyperimmunized with either the venom of M. tener (PLA2-predominant) or a recombinant short-chain consensus α-neurotoxin (ScNtx). Then, the combination of the two monospecific horse antibodies (anti-M. tener and anti-ScNtx) was used to test their efficacy against eleven Micrurus venoms. RESULTS: The blend of anti-M. tener and anti-ScNtx antibodies had a better capacity to neutralize the lethality of diverse species from North, Central and South American Micrurus venoms. The antibodies combination neutralized both the ScNtx and ten out of eleven Micrurus venom tested, and particularly, it neutralized the venoms of M. distans and M. laticollaris that were neither neutralized by monospecific anti-M. tener nor anti-ScNtx. CONCLUSIONS: These results provide a proof-of-principle for using recombinant immunogens to enrich poor or even non-neutralizing antisera against elapid venoms containing short chain α-neurotoxins to develop antivenoms with higher effectiveness and broader neutralizing capacity.

Protein composition and biochemical characterization of venom from Sonoran Coral Snakes (Micruroides euryxanthus).

The elapid genus, Micruroides, is considered the sister clade of all New World coral snakes (Genus Micrurus), is monotypic, and is represented by Sonoran Coral Snakes, Micruroides euryxanthus. Coral snakes of the genus Micrurus have been reported to have venoms that are predominantly composed of phospholipases A2 (PLA2) or three finger toxins (3FTx), but the venoms of the genus Micruroides are almost completely unstudied. Here, we present the first description of the venom of M. euryxanthus including identification of some proteins as well as transcriptomic, and biological activity assays. The most abundant components within M. euryxanthus venom are 3FTxs (62.3%) and there was relatively low proportion of PLA2s (14.2%). The venom phenotype supports the hypothesis that the common ancestor of Micrurus and Micruroides had a 3FTx-dominated venom. Within the venom, there were two nearly identical α-neurotoxins (α-Ntx), one of which was designated Eurytoxin, that account for approximately 60% of the venom's lethality to mice. Eurytoxin was cloned, expressed in a soluble and active form, and used to produce rabbit hyperimmune serum. This allowed the analysis of its immunochemical properties, showing them to be different from the recombinant αNTx D.H., present in the venoms of some species of Micrurus. Finally, we observed that the commercial antivenom produced in Mexico for coral snake envenomation is unable to neutralize the lethality from M. euryxanthus venom. This work allowed the classification of Micruroides venom into the 3FTx-predominant group and identified the main components responsible for toxicity to mice.