Persistent hypercoagulability in dogs envenomated by the European adder (Vipera berus berus).

BACKGROUND: Envenomation by the European adder, Vipera berus berus (Vbb), is a medical emergency. The overall in vivo haemostatic effects of pro- and anticoagulant components in Vbb venom, and the downstream effects of cellular injury and systemic inflammation, are unclear. OBJECTIVES: To longitudinally describe the global coagulation status of dogs after Vbb envenomation and compare to healthy controls. A secondary aim was to investigate differences between dogs treated with and without antivenom. METHODS: Citrated plasma was collected at presentation, 12 hours (h), 24 h, 36 h and 15 days after bite from 28 dogs envenomated by Vbb, and from 28 healthy controls at a single timepoint. Thrombin generation (initiated with and without exogenous phospholipids and tissue factor), thrombin-antithrombin (TAT)-complexes and the procoagulant activity of phosphatidylserine (PS)-expressing extracellular vesicles (EVs), expressed as PS-equivalents, were measured. RESULTS: At presentation the envenomated dogs were hypercoagulable compared to controls, measured as increased thrombin generation, TAT-complexes and PS-equivalents. The hypercoagulability decreased gradually but compared to controls thrombin generation and PS-equivalents were still increased at day 15. The discrepancy in peak thrombin between envenomated dogs and controls was greater when the measurement was phospholipid-dependent, indicating that PS-positive EVs contribute to hypercoagulability. Lag time was shorter in non-antivenom treated dogs, compared to antivenom treated dogs <24 h after envenomation. CONCLUSIONS: Hypercoagulability was measured in dogs up to 15 days after Vbb envenomation. Dogs treated with antivenom may be less hypercoagulable than their non-antivenom treated counterparts. Thrombin generation is a promising diagnostic and monitoring tool for Vbb envenomation.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms.

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Snake Venom Proteomics, Immunoreactivity and Toxicity Neutralization Studies for the Asiatic Mountain Pit Vipers, Ovophis convictus, Ovophis tonkinensis, and Hime Habu, Ovophis okinavensis.

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.

Intravenous Vipera berus Venom-Specific Fab Fragments and Intramuscular Vipera ammodytes Venom-Specific F(ab')2 Fragments in Vipera ammodytes-Envenomed Patients.

Vipera ammodytes (V. ammodytes) is the most venomous European viper. The aim of this study was to compare the clinical efficacy and pharmacokinetic values of intravenous Vipera berus venom-specific (paraspecific) Fab fragments (ViperaTAb) and intramuscular V. ammodytes venom-specific F(ab')2 fragments (European viper venom antiserum, also called "Zagreb" antivenom) in V.ammodytes-envenomed patients. This was a prospective study of V.ammodytes-envenomed patients that were treated intravenously with ViperaTAb or intramuscularly with European viper venom antiserum that was feasible only due to the unique situation of an antivenom shortage. The highest venom concentration, survival, length of hospital stay and adverse reactions did not differ between the groups. Patients treated with intravenous Fab fragments were sicker, with significantly more rhabdomyolysis and neurotoxicity. The kinetics of Fab fragments after one or more intravenous applications matched better with the venom concentration in the early phase of envenomation compared to F(ab')2 fragments that were given intramuscularly only on admission. F(ab')2 fragments given intramuscularly had 25-fold longer apparent total body clearance and 14-fold longer elimination half-time compared to Fab fragments given intravenously (2 weeks vs. 24 h, respectively). In V.ammodytes-envenomed patients, the intramuscular use of specific F(ab')2 fragments resulted in a slow rise of antivenom serum concentration that demanded their early administration but without the need for additional doses for complete resolution of all clinical signs of envenomation. Intravenous use of paraspecific Fab fragments resulted in the immediate rise of antivenom serum concentration that enabled their use according to the clinical progress, but multiple doses might be needed for efficient therapy of thrombocytopenia due to venom recurrence, while the progression of rhabdomyolysis and neurotoxic effects of the venom could not be prevented.

Comparison of Preclinical Properties of Several Available Antivenoms in the Search for Effective Treatment of Vipera ammodytes and Vipera berus Envenoming.

Snakebites are a relatively rare medical emergency in Europe. In more than half of the annual cases caused by Vipera ammodytes, Vipera berus, and Vipera aspis, immunotherapy with animal-derived antivenom is indicated. Among eight products recently identified as available against European medically relevant species, only Zagreb antivenom, Viperfav, and ViperaTAb have been used almost exclusively for decades. Zagreb antivenom comprises V. ammodytes-specific F(ab')2 fragments. Viperfav is a polyspecific preparation based on F(ab')2 fragments against V. aspis, V. berus, and V. ammodytes venoms. ViperaTAb contains Fab fragments against the venom of V. berus. In 2014 the production of Zagreb antivenom was discontinued. Additionally, in the period of 2017 to 2018 a shortage of Viperfav occurred. Due to a lack of the product indicated for the treatment of V. ammodytes bites, other antivenoms were implemented into clinical practice without comparative assessment of their eligibility. The aim of our work was to identify a high-quality antivenom that might ensure the successful treatment of V. ammodytes and V. berus bites at the preclinical level. Differentiation between bites from these two species is difficult and unreliable in clinical practice, so the availability of a unique antivenom applicable in the treatment of envenoming caused by both species would be the most advantageous for Southeastern Europe. Zagreb antivenom, Viperfav, and ViperaTAb, as well as Viper venom antitoxin for V. berus envenoming and the in-development Inoserp Europe, which was designed to treat envenoming caused by all medically important European snakes, were comparatively tested for the first time. Emphasis was placed on their physicochemical properties, primarily purity and aggregate content, as well as their in vivo protective efficacies. As Zagreb antivenom is no longer available on the European market, Viperfav is the highest-quality product currently available and the only antivenom whose neutralisation potency against V. ammodytes and V. berus venoms was above regulatory requirements.

In Vitro Immunological Cross-Reactivity of Thai Polyvalent and Monovalent Antivenoms with Asian Viper Venoms.

The intravenous administration of polyclonal antibodies known as antivenom is the only effective treatment for snakebite envenomed victims, but because of inter-specific variation in the toxic components of snake venoms, these therapies have variable efficacies against different snake species and/or different populations of the same species. In this study, we sought to characterize the in vitro venom binding capability and in vitro cross-neutralizing activity of antivenom, specifically the Hemato Polyvalent antivenom (HPAV; The Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) and three monovalent antivenoms (QSMI) specific to Daboia siamensis, Calloselasma rhodostoma, and Trimeresurus albolabris venoms, against a variety of South Asian and Southeast Asian viper venoms (Calloselasma rhodostoma, Daboia russelii, Hypnale hypnale, Trimeresurus albolabris, Trimeresurus purpureomaculatus, Trimeresurus hageni, and Trimeresurus fucatus). Using ELISA and immunoblotting approaches, we find that the majority of protein components in the viper venoms were recognized and bound by the HPAV polyvalent antivenom, while the monospecific antivenom made against T.albolabris extensively recognized toxins present in the venom of related species, T. purpureomaculatus, T. hageni, and T. fucatus. In vitro coagulation assays using bovine plasma revealed similar findings, with HPAV antivenom significantly inhibiting the coagulopathic activities of all tested viper venoms and T. albolabris antivenom inhibiting the venoms from Malaysian arboreal pit vipers. We also show that the monovalent C. rhodostoma antivenom exhibits highly comparable levels of immunological binding and in vitro venom neutralization to venom from both Thailand and Malaysia, despite previous reports of considerable intraspecific venom variation. Our findings suggest that Thai antivenoms from QSMI may by useful therapeutics for managing snake envenomings caused by a number of Southeast Asian viper species and populations for which no specific antivenom currently exists and thus should be explored further to assess their clinical utility in treating snakebite victims.

An analysis of preclinical efficacy testing of antivenoms for sub-Saharan Africa: Inadequate independent scrutiny and poor-quality reporting are barriers to improving snakebite treatment and management.

BACKGROUND: The World Health Organization's strategy to halve snakebite mortality and morbidity by 2030 includes an emphasis on a risk-benefit process assessing the preclinical efficacy of antivenoms manufactured for sub-Saharan Africa. To assist this process, we systematically collected, standardised and analysed all publicly available data on the preclinical efficacy of antivenoms designed for sub-Saharan Africa. METHODOLOGY/PRINCIPAL FINDINGS: Using a systematic search of publication databases, we focused on publicly available preclinical reports of the efficacy of 16 antivenom products available in sub Saharan Africa. Publications since 1999 reporting the industry standard intravenous pre-incubation method of murine in vivo neutralisation of venom lethality (median effective dose [ED50]) were included. Eighteen publications met the criteria. To permit comparison of the several different reported ED50 values, it was necessary to standardise these to microlitre of antivenom resulting in 50% survival of mice challenged per milligram of venom (µl/mg). We were unable to identify publicly available preclinical data on four antivenoms, whilst data for six polyspecific antivenoms were restricted to a small number of venoms. Only four antivenoms were tested against a wide range of venoms. Examination of these studies for the reporting of key metrics required for interpreting antivenom ED50s were highly variable, as evidenced by eight different units being used for the described ED50 values. CONCLUSIONS/SIGNIFICANCE: There is a disturbing lack of (i) preclinical efficacy testing of antivenom for sub Saharan Africa, (ii) publicly available reports and (iii) independent scrutiny of this medically important data. Where reports do exist, the methods and metrics used are highly variable. This prevents comprehensive meta-analysis of antivenom preclinical efficacy, and severely reduces the utility of antivenom ED50 results in the decision making of physicians treating patients and of national and international health agencies. Here, we propose the use of a standardised result reporting checklist to resolve this issue. Implementation of these straightforward steps will deliver uniform evaluation of products across laboratories, facilitate meta-analyses, and contribute vital information for designing the clinical trials needed to achieve the WHO target of halving snakebite morbidity and mortality by 2030.

Evaluation of the geographical utility of Eastern Russell's viper (Daboia siamensis) antivenom from Thailand and an assessment of its protective effects against venom-induced nephrotoxicity.

BACKGROUND: Daboia siamensis (Eastern Russell's viper) is a medically important snake species found widely distributed across Southeast Asia. Envenomings by this species can result in systemic coagulopathy, local tissue injury and/or renal failure. While administration of specific antivenom is an effective treatment for Russell's viper envenomings, the availability of, and access to, geographically-appropriate antivenom remains problematic in many rural areas. In this study, we determined the binding and neutralizing capability of antivenoms manufactured by the Thai Red Cross in Thailand against D. siamensis venoms from four geographical locales: Myanmar, Taiwan, China and Thailand. METHODOLOGY/PRINCIPLE FINDINGS: The D. siamensis monovalent antivenom displayed extensive recognition and binding to proteins found in D. siamensis venom, irrespective of the geographical origin of those venoms. Similar immunological characteristics were observed with the Hemato Polyvalent antivenom, which also uses D. siamensis venom as an immunogen, but binding levels were dramatically reduced when using comparator monovalent antivenoms manufactured against different snake species. A similar pattern was observed when investigating neutralization of coagulopathy, with the procoagulant action of all four geographical venom variants neutralized by both the D. siamensis monovalent and the Hemato Polyvalent antivenoms, while the comparator monovalent antivenoms were ineffective. These in vitro findings translated into therapeutic efficacy in vivo, as the D. siamensis monovalent antivenom was found to effectively protect against the lethal effects of all four geographical venom variants preclinically. Assessments of in vivo nephrotoxicity revealed that D. siamensis venom (700 µg/kg) significantly increased plasma creatinine and blood urea nitrogen levels in anaesthetised rats. The intravenous administration of D. siamensis monovalent antivenom at three times higher than the recommended scaled therapeutic dose, prior to and 1 h after the injection of venom, resulted in reduced levels of markers of nephrotoxicity and prevented renal morphological changes, although lower doses had no therapeutic effect. CONCLUSIONS/SIGNIFICANCE: This study highlights the potential broad geographical utility of the Thai D. siamensis monovalent antivenom for treating envenomings by the Eastern Russell's viper. However, only the early delivery of high antivenom doses appears to be capable of preventing venom-induced nephrotoxicity.

A hemorrhagic metalloprotease of Egyptian Cerastes vipera venom: Biochemical and immunological properties.

A hemorrhagic metalloprotease (CVHT1) was isolated from Cerastes vipera (CV) viper venom and characterized in a set of biochemical and immunological assays. A simple two-step purification procedure included gel filtration and ion-exchange increase the purity of enzyme 39-fold with specific activity of 20,200 Umg-1 compared to 520 Umg-1 for crude venom. CVHT1 is a dimer enzyme with two subunits of ~60 kDa. The LC-MS/MS analysis of CVHT1 revealed that the identified peptides show high homology to other P-III snake venom zinc-metalloproteases. The activity of CVHT1 showed stability at pH (6.5-8.5) and temperature (30-60 °C) with optima at pH 8.5 and 60 °C. Activators for CVHT1 included Mg+2, Zn+2, Ca+2, K+, Ba+2 and Na+, while the full inhibition was given by other tested ions, SH-group reagents and metalloproteinase inhibitors. The CVHT1 potentially digested gelatin, fibrinogen, fibronectin and inhibited the platelet aggregation. The hemorrhagic and proteolytic activities of medically important Egyptian viper venoms were highly cross-neutralized by anti-CVHT1. The anti-CVHT1 increased the survival time of mice injected with lethal dose of CV venom to 23 ±â€¯2.5 h compared to the mice injected with venom alone 0.52 ±â€¯0. 05 h. This study could be useful for production of safer and more efficient therapeutic anti-venom.

Use of irradiated elapid and viperid venoms for antivenom production in small and large animals.

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD50 of 1.25 mg/kg in mice, and the elapid mixture had an LD50 of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LD50s of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.

Proteomic analysis reveals geographic variation in venom composition of Russell's Viper in the Indian subcontinent: implications for clinical manifestations post-envenomation and antivenom treatment.

INTRODUCTION: The Russell's Viper (RV) (Daboia russelii), a category I medically important snake, is responsible for a significant level of morbidity and mortality in the Indian sub-continent. Areas covered: The current review highlights the variation in RV venom (RVV) composition from different geographical locales on the Indian sub-continent, as revealed by biochemical and proteomic analyses. A comparison of these RVV proteomes revealed significant differences in the number of toxin isoforms and relative toxin abundances, highlighting the impact of geographic location on RVV composition. Antivenom efficacy studies have shown differential neutralization of toxicity and enzymatic activity of different RVV samples from the Indian sub-continent by commercial polyvalent antivenom (PAV). The proteome analysis has provided deeper insights into the variation of RVV composition leading to differences in antivenom efficacy and severity of clinical manifestations post RV-envenomation across the Indian sub-continent. Expert commentary: Variation in RVV antigenicity due to geographical differences and poor recognition of low molecular mass (<20 kDa) RVV toxins by PAV are serious concerns for effective antivenom treatment against RV envenomation. Improvements in immunization protocols that take into account the poorly immunogenic components and geographic variation in RVV composition, can lead to better hospital management of RV bite patients.

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.

Development of an ELISA assay to determine neutralising capacity of horse serum following immunisation with Daboia siamensis venom in Myanmar.

Snakebite envenoming is a serious problem in Myanmar. The great majority of snakebite in this country is due to Russell's Viper (Daboia siamensis). For many years, the Burma Pharmaceutical Industry has produced a monovalent antivenom to Russell's Viper in horses. At present, the only way of determining the level of antibody against D. siamensis venom in hyperimmune horse serum is to perform venom neutralisation tests in mice. In this study, we describe the development of an in vitro ELISA assay to estimate neutralising capacity of horse serum. We found a strong correlation between the ELISA assay and the venom neutralisation test in mice (r = 0.982). The assay is robust and has sufficient sensitivity (92%) and specificity (96%) to replace the venom neutralisation test in mice during the immunisation phase in horses.

Venom proteomics and antivenom neutralization for the Chinese eastern Russell's viper, Daboia siamensis from Guangxi and Taiwan.

The eastern Russell's viper (Daboia siamensis) causes primarily hemotoxic envenomation. Applying shotgun proteomic approach, the present study unveiled the protein complexity and geographical variation of eastern D. siamensis venoms originated from Guangxi and Taiwan. The snake venoms from the two geographical locales shared comparable expression of major proteins notwithstanding variability in their toxin proteoforms. More than 90% of total venom proteins belong to the toxin families of Kunitz-type serine protease inhibitor, phospholipase A2, C-type lectin/lectin-like protein, serine protease and metalloproteinase. Daboia siamensis Monovalent Antivenom produced in Taiwan (DsMAV-Taiwan) was immunoreactive toward the Guangxi D. siamensis venom, and effectively neutralized the venom lethality at a potency of 1.41 mg venom per ml antivenom. This was corroborated by the antivenom effective neutralization against the venom procoagulant (ED = 0.044 ± 0.002 µl, 2.03 ± 0.12 mg/ml) and hemorrhagic (ED50 = 0.871 ± 0.159 µl, 7.85 ± 3.70 mg/ml) effects. The hetero-specific Chinese pit viper antivenoms i.e. Deinagkistrodon acutus Monovalent Antivenom and Gloydius brevicaudus Monovalent Antivenom showed negligible immunoreactivity and poor neutralization against the Guangxi D. siamensis venom. The findings suggest the need for improving treatment of D. siamensis envenomation in the region through the production and the use of appropriate antivenom.

Proteomic Analysis and Immuno-Profiling of Eastern India Russell's Viper ( Daboia russelii) Venom: Correlation between RVV Composition and Clinical Manifestations Post RV Bite.

The proteomes of Russell's viper venom (RVV) from Burdwan (RVV B) and Nadia (RVV N), the two districts of West Bengal, eastern India (EI), were investigated by gel-filtration chromatography (GFC) followed by tandem mass spectrometry of tryptic fragments of the fractions. A total of 73 and 69 proteins belonging to 15 snake venom protein families were identified in RVV B and RVV N, respectively, by MS/MS search against Viperidae (taxid 8689) protein entries of the nonredundant NCBI database. The minor differences in venom composition of both the EI RV were established unequivocally by their biochemical and pharmacological properties and by SDS-PAGE, gel filtration chromatography, and LC-MS/MS analyses. The composition of EI RVVs was well correlated with published reports on the pathophysiology of RV-envenomed patients from this part of the country. Venom-antivenom cross-reactivity determined by ELISA, Western blotting, and antivenomics approaches demonstrated poor recognition of low molecular mass (<20 kDa) RVV proteins by commercial polyvalent antivenoms, which was substantiated by neutralization of RVV enzymes by antivenom.

Snake venoms from Angola: Intra-specific variations and immunogenicity.

Snakebite is a public health problem in many countries of world. These accidents are considered a Neglected Tropical Disease and are responsible for a high morbidity and mortality index in the South and Southeast Asia and Sub-Saharan Africa. Angolan snake venoms are poorly investigated and no specific antivenom against them is available in the country. Thus, the aim of this study was to evaluate biochemical and immunogenic properties of male and female venoms from Naja nigricollis, Bitis arietans and Bitis gabonica snakes. These animals were collected during an expedition covering 1350 km of Angola, including the Provinces of Cuanza Sul, Benguela, Huíla and Malanje. Results showed that Angolan snake venoms present distinctive immunogenic properties and large intra-specific variations, associated to the gender and the geographic origin of the animals. Thus, it is possible to suggest that for the preparation of a therapeutic antivenom, intra-species variability should be taken into account, in order to obtain an efficient serum to neutralize the toxic effects of the Angolan snake venoms.

Contributions of the snake venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys to the paraspecificity of the Central American polyspecific antivenom (PoliVal-ICP).

PoliVal-ICP antivenom is produced from plasma of horses immunized toward the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys. The antibody response induced by these venoms confers PoliVal-ICP the capacity to neutralize the venoms of the most important Central American viperids, including not only homologous venoms (i.e., venoms used as immunogen), but many heterologous venoms (i.e., venoms not used as immunogen). In this work, the individual contributions of homologous venoms to the paraspecificity of PoliVal-ICP were inferred from the capacity of experimental monospecific antivenoms toward venoms of B. asper (anti-Ba), C. simus (anti-Cs) and L. stenophrys (anti-Ls), and an experimental polyspecific antivenom (anti-Ba/Cs/Ls) to neutralize the lethality induced by different venoms in mice. It was found that all antivenoms neutralized their corresponding homologous venoms. Moreover, the anti-Ba antivenom cross-neutralized the venoms of Agkistrodon howardgloydi, Atropoides picadoi, Bothriechis lateralis, Bothriechis supraciliaris and Porthidium ophryomegas; the anti-Cs antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, Cerrophidion sasai and Porthidium nasutum; and the anti-Ls antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, C. sasai and Lachesis melanocephala. All venoms neutralized by any monospecific antivenom were also neutralized by the anti-Ba/Cs/Ls antivenom. Venoms of Atropoides mexicanus, Bothriechis nigroviridis and Bothriechis schlegelii were not neutralized by any experimental antivenom, thus explaining the limitations of PoliVal-ICP to neutralize these venoms. Consequently, an enlargement of the neutralization scope of PoliVal-ICP could be achieved by including these venoms in the group of those used as immunogens.

Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus.

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P&lt;0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 &micro;g/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

Proteomics and antivenomics of Echis carinatus carinatus venom: Correlation with pharmacological properties and pathophysiology of envenomation.

The proteome composition of Echis carinatus carinatus venom (ECV) from India was studied for the first time by tandem mass spectrometry analysis. A total of 90, 47, and 22 distinct enzymatic and non-enzymatic proteins belonging to 15, 10, and 6 snake venom protein families were identified in ECV by searching the ESI-LC-MS/MS data against non-redundant protein databases of Viperidae (taxid 8689), Echis (taxid 8699) and Echis carinatus (taxid 40353), respectively. However, analysis of MS/MS data against the Transcriptome Shotgun Assembly sequences (87 entries) of conger E. coloratus identified only 14 proteins in ECV. Snake venom metalloproteases and snaclecs, the most abundant enzymatic and non-enzymatic proteins, respectively in ECV account for defibrinogenation and the strong in vitro pro-coagulant activity. Further, glutaminyl cyclase, aspartic protease, aminopeptidase, phospholipase B, vascular endothelial growth factor, and nerve growth factor were reported for the first time in ECV. The proteome composition of ECV was well correlated with its biochemical and pharmacological properties and clinical manifestations observed in Echis envenomed patients. Neutralization of enzymes and pharmacological properties of ECV, and immuno-cross-reactivity studies unequivocally point to the poor recognition of <20 kDa ECV proteins, such as PLA2, subunits of snaclec, and disintegrin by commercial polyvalent antivenom.

Identification of B cell recognized linear epitopes in a snake venom serine proteinase from the central American bushmaster Lachesis stenophrys.

Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein-kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms.