Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

In vitro assessment and phase I randomized clinical trial of anfibatide a snake venom derived anti-thrombotic agent targeting human platelet GPIbα.

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Toxin-resolved antivenomics-guided assessment of the immunorecognition landscape of antivenoms.

Snakebite envenoming represents a major issue in rural areas of tropical and subtropical regions across sub-Saharan Africa, South to Southeast Asia, Latin America and Oceania. Antivenoms constitute the only scientifically validated therapy for snakebite envenomings, provided they are safe, effective, affordable, accessible and administered appropriately. However, the lack of financial incentives in a technology that has remained relatively unchanged for more than a century, has contributed to some manufacturers leaving the market and others downscaling production or increasing the prices, leading to a decline in the availability and accessibility for these life-saving antidotes to millions of rural poor most at risk from snakebites in low income countries. The shortage of antivenoms can be significantly alleviated by optimizing the use of current antivenoms (through the assessment of their specific and paraspecific efficacy against the different medically relevant homologous and heterologous snake venoms) and by generating novel polyspecific antivenoms exhibiting broad clinical spectrum and wide geographic distribution range. Research on venoms has been continuously enhanced by advances in technology. Particularly, the last decade has witnessed the development of omics strategies for unravelling the toxin composition of venoms ("venomics") and to assess the immunorecognition profile of antivenoms ("antivenomics"). Here, we review recent developments and reflect on near future innovations that promise to revolutionize the mutually enlightening relationship between evolutionary and translational venomics.

Assessment of the potential toxicological hazard of the Green Parrot Snake (Leptophis ahaetulla marginatus): Characterization of its venom and venom-delivery system.

Snakes are the major group of venomous vertebrates, and the rear-fanged snakes represent the vast majority of species and occur worldwide; however, relatively few studies have characterized their venoms and evaluated their potential hazards for humans. Herein we explore the protein composition and properties of the venom of the rear-fanged Green Parrot Snake, Leptophis ahaetulla marginatus, the most common snake found in the Iguazu National Park (Argentina), as well as the main features of its venom delivery system. This species has venom reminiscent of elapid venoms, composed mainly of components such as 3FTxs, CRiSPs and AChE, but it shows low toxicity toward mammals (LD50 > 20 µg/g mouse). The histology of its Duvernoy's venom gland is similar to that of other colubrids, with serous secretory cells arranged in densely packed secretory tubules. The posterior end of its maxilla exhibits 1-3 blade-shaped and slightly recurved fangs but without grooves. This study provides an initial analysis of the biological role of venom in Leptophis, with implications for potential symptoms that might be anticipated from bites by this species.

Multipurpose HTS Coagulation Analysis: Assay Development and Assessment of Coagulopathic Snake Venoms.

Coagulation assays currently employed are often low throughput, require specialized equipment and/or require large blood/plasma samples. This study describes the development, optimization and early application of a generic low-volume and high-throughput screening (HTS) assay for coagulation activity. The assay is a time-course spectrophotometric measurement which kinetically measures the clotting profile of bovine or human plasma incubated with Ca2+ and a test compound. The HTS assay can be a valuable new tool for coagulation diagnostics in hospitals, for research in coagulation disorders, for drug discovery and for venom research. A major effect following envenomation by many venomous snakes is perturbation of blood coagulation caused by haemotoxic compounds present in the venom. These compounds, such as anticoagulants, are potential leads in drug discovery for cardiovascular diseases. The assay was implemented in an integrated analytical approach consisting of reversed-phase liquid chromatography (LC) for separation of crude venom components in combination with parallel post-column coagulation screening and mass spectrometry (MS). The approach was applied for the rapid assessment and identification of profiles of haemotoxic compounds in snake venoms. Procoagulant and anticoagulant activities were correlated with accurate masses from the parallel MS measurements, facilitating the detection of peptides showing strong anticoagulant activity.

At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.

Prospective assessment of the false positive rate of the Australian snake venom detection kit in healthy human samples.

The Snake Venom Detection Kit (SVDK; bioCSL Pty Ltd, Australia) distinguishes venom from the five most medically significant snake immunotypes found in Australia. This study assesses the rate of false positives that, by definition, refers to a positive assay finding in a sample from someone who has not been bitten by a venomous snake. Control unbroken skin swabs, simulated bite swabs and urine specimens were collected from 61 healthy adult volunteers [33 males and 28 females] for assessment. In all controls, simulated bite site and urine samples [a total of 183 tests], the positive control well reacted strongly within one minute and no test wells reacted during the ten minute incubation period. However, in two urine tests, the negative control well gave a positive reaction (indicating an uninterpretable test). A 95% confidence interval for the false positive rate, on a per-patient rate, derived from the findings of this study, would extend from 0% to 6% and, on a per-test basis, it would be 0-2%. It appears to be a very low incidence (0-6%) of intrinsic true false positives for the SVDK. The clinical impresssion of a high SVDK false positive rate may be mostly related to operator error.

Comparative venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado: Identification of a novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®.

Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America. BIOLOGICAL SIGNIFICANCE: Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms.

Omics meets biology: application to the design and preclinical assessment of antivenoms.

Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxins.

Biochemical and biological analysis of Philodryas baroni (Baron's green racer; Dipsadidae) venom: relevance to the findings of human risk assessment.

Philodryas baroni--an attractively colored snake--has become readily available through the exotic pet trade. Most people consider this species harmless; however, it has already caused human envenomation. As little is known about the venom from this South American opisthoglyphous "colubrid" snake, herein, we studied its protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as its effects on the hemostatic system. Both reducing and nonreducing SDS-PAGE analysis demonstrated that the venom exhibits greatest complexity in the range of 50-80 kDa. The venom displayed proteolytic activity toward azocollagen, with a specific activity of 75.5 U mg⁻¹, and rapidly hydrolyzed the Aα-chain of fibrinogen, exhibiting lower activity toward the Bß- and γ-chains. The venom from P. baroni showed no platelet proaggregating activity per se, but it inhibited collagen- and thrombin-induced platelet aggregation. Prominent hemorrhage developed in mouse skin after intradermal injection of the crude venom, and its minimum hemorrhagic dose was 13.9 µg. When injected intramuscularly into the gastrocnemius of mice, the venom induced local effects such as hemorrhage, myonecrosis, edema, and leucocyte infiltration. Due to its venom toxicity shown herein, P. baroni should be considered dangerous to humans and any medically significant bite should be promptly reviewed by a qualified health professional.

Thrombolytic effects of the snake venom disintegrin saxatilin determined by novel assessment methods: a FeCl3-induced thrombosis model in mice.

Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bß3 (GP IIb/IIIa), α5ß1, αvß3, αvß1, and αvß5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.

Cilengitide: an RGD pentapeptide ανß3 and ανß5 integrin inhibitor in development for glioblastoma and other malignancies.

Cilengitide, a cyclicized arginine-glycine-aspartic acid-containing pentapeptide, potently blocks ανß3 and ανß5 integrin activation. Integrins are upregulated in many malignancies and mediate a wide variety of tumor-stroma interactions. Cilengitide and other integrin-targeting therapeutics have preclinical activity against many cancer subtypes including glioblastoma (GBM), the most common and deadliest CNS tumor. Cilengitide is active against orthotopic GBM xenografts and can augment radiotherapy and chemotherapy in these models. In Phase I and II GBM trials, cilengitide and the combination of cilengitide with standard temozolomide and radiation demonstrate consistent antitumor activity and a favorable safety profile. Cilengitide is currently under evaluation in a pivotal, randomized Phase III study (Cilengitide in Combination With Temozolomide and Radiotherapy in Newly Diagnosed Glioblastoma Phase III Randomized Clinical Trial [CENTRIC]) for newly diagnosed GBM. In addition, randomized controlled Phase II studies with cilengitide are ongoing for non-small-cell lung cancer and squamous cell carcinoma of the head and neck. Cilengitide is the first integrin inhibitor in clinical Phase III development for oncology.

A new Pan African polyspecific antivenom developed in response to the antivenom crisis in Africa.

Currently there is a crisis in the supply of antivenom for treatment of snake bite in sub-Saharan Africa. Commercial pressures have resulted in the reduction or even cessation of production of antivenom by European manufacturers while continued production of antivenom in Africa has been threatened by the privatisation of the only remaining company based in Africa. As a consequence, there has been an increase in snake bite morbidity and mortality in many African countries. Two Latin American antivenom manufacturers have agreed to produce antivenom suitable for Africa, using venoms from the species which are of the greatest medical importance in sub-Saharan Africa. Preclinical in vivo assays of neutralising potency demonstrated that a new Pan African antivenom produced in Colombia compared favourably with the existing commercial monospecific and polyspecific antivenoms. This new antivenom, and a similar product being manufactured in Costa Rica, are now candidates for clinical testing at an appropriate site in Africa.

Modeling the third loop of short-chain snake venom neurotoxins: roles of the short-range and long-range interactions.

The influence of long-range interactions on local structures is an important issue in understanding protein folding process and protein structure stability. Using short-chain snake venom neurotoxin as a model system, we have studied the conformational properties of eight different loop III sequences either in the environment of one of the short-chain neurotoxin, erabutoxin b (PDB ID 1nxb), or in free state by Monte Carlo simulated annealing method. The surrounding protein structure was found to be crucial in stabilizing the loop conformation. Although all the eight peptides prefer type V beta turn in solution, three of them (KPGI, KPGV, KSGI) turn to type II beta turn and the other five (KKGI, KKGV, KNGI, KQGI, and KRGV) are confined to more rigid type V beta turn conformation in the protein structure. Using flexible tetra-glycine-peptide to screen the backbone conformational space in the protein environment also validates the results. This study shows that long-range interactions do contribute to the stability and the types of conformation for a surface loop in protein, while short-range interactions may only provide candidate conformations, which then have to be filtered by the long-range interactions further.