Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring.

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 µg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights.

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 μg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Neutralization of Centruroides tecomanus scorpion venom by the use of two human recombinant antibody fragments.

The first toxic component identified against mammals in the venom from Centruroides tecomanus scorpion from Colima, Mexico was Ct1a toxin, which was neutralized by human single chain variable fragment (scFv) RAS27. Venom characterization from these scorpions collected on the Pacific coast of Colima, enabled the identification of a second component of medical importance named Ct71 toxin. Amino acid sequence of Ct71 shares a high identity with Chui5 toxin from C. huichol scorpion, which was neutralized by scFv HV. For this reason, the kinetic parameters of interaction between Ct71 toxin and scFv HV were determined by surface plasmon resonance. Results showed a significantly higher affinity for Ct71 as compared to Chui5. As expected, this toxin was neutralized by scFv HV. The injection of a mixture of scFvs HV and RAS27, resulted in the neutralization of C. tecomanus venom, corroborating that human recombinant antibody fragments can efficiently contribute to the neutralization of medically important toxins and their respective venoms from Mexican scorpions.

Insight into the Toxicological and Pathophysiological Effects of Moroccan Vipers' Venom: Assessing the Efficacy of Commercial Antivenom for Neutralization.

Morocco is one of the richest countries in biodiversity in the Mediterranean region, especially in its ophidian fauna. In total, there are eight species of venomous snakes, with seven belonging to the Viperidae family, responsible for 67.2% of severe envenomation cases in the country. Cerastes cerastes, Daboia mauritanica and Bitis arietans are considered among the most venomous vipers whose bites cause high levels of morbidity, disability or mortality. Despite their wide distribution in the kingdom, the incidence of these snakebites remains poorly understood and largely underestimated. Moreover, intraspecific variations in the venom composition significantly affect the effectiveness of antivenoms. Due to the unavailability of locally produced antivenoms, we evaluated the efficacy of Inoserp-MENA, the only available antivenom in Morocco, against C. cerastes, D. mauritanica and B. arietans. First, we conducted a comprehensive characterization of these venoms, including an LD50 test to examine their toxicity and SDS-PAGE as a technique to analyze the enzymes responsible for biological activities, such as hemorrhagic and edematous activities and myotoxicity, which generate physiopathological effects in the skin, paws and muscles of envenomed mice. Then, we assessed the ability of Inoserp-MENA antivenom to neutralize the toxic activities of Moroccan vipers. Our results indicate that the venom of C. cerastes, D. mauritanica and B. arietans are toxic, causing severe alterations such as edema, myotoxicity, myonecrosis and significant hemorrhages with the formation of hemorrhagic foci. C. cerastes venom is more dangerous in terms of lethality and hemorrhages, while B. arietans venom is more edematous. The effects of C. cerastes venom were effectively neutralized, but Inoserp-MENA antivenom failed to protect mice against the toxic effects induced by B. arietans and D. mauritanica venom. The study reveals alarming shortcomings in the effectiveness of the current commercially available antivenom's dosage and neutralization capabilities, highlighting the urgent need to develop a region-specific viper envenomation therapy.

Expression in Pichia pastoris of human antibody fragments that neutralize venoms of Mexican scorpions.

The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.

The potential of Brazilian native plant species used in the therapy for snakebites: A literature review.

Snakebite envenoming is a potentially fatal disease categorized as a neglected public health issue for not receiving the appropriate attention from national and international health authorities. The most affected people by this problem usually live in poor rural communities, where medical resources are often sparse and, in some instances, there is even a scarcity of serum therapy. The administration of the appropriate antivenom is the only specific treatment available, however it has limited efficacy against venom-induced local effects. In this scenario, various plant species are used as local first aid for the treatment of snakebite accidents in Brazil, and some of them can effectively inhibit lethality, neurotoxicity, hemorrhage, and venom enzymes activities. This review compiles a list of plants used in the treatment of snakebites in Brazil, focusing on the native Brazilian species registered in the databases Pubmed, Scielo, Scopus and Google Scholar. All these searches were limited to peer-reviewed journals written in English, with the exception of a few articles written in Portuguese. The most cited native plant species were Casearia sylvestris Sw., Eclipta prostrata (L.) L., Mikania glomerata Spreng., Schizolobium parahyba (Vell.) S.F.Blake and Dipteryx alata Vogel, all used to decrease the severity of toxic signs, inhibit proteolytic and hemorrhagic activities, thus increasing survival time and neutralizing myotoxicity effects. Different active compounds showing important activity against the snake venoms and their toxins include flavonoids, alkaloids and tannins. Although some limitations to the experimental studies with medicinal plants were observed, including lack of comparison with control drugs and unknown active extracts compounds, species with anti-venom characteristics are effective and considered as candidates for the development of adjuvants in the treatment of snake envenomation. Further studies on the chemistry and pharmacology of traditionally used plant species will help to understand the role that snakebite herbal remedies may display in local medical health systems. It might also contribute to the development of alternative or complementary treatments to reduce the number of severe disabilities and deaths.

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom.

The monocled cobra (Naja kaouthia) is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments. Here, we describe the discovery and subsequent optimization of a recombinant human monoclonal immunoglobulin G antibody against α-cobratoxin using phage display technology. Affinity maturation by light chain-shuffling resulted in a significant increase in in vitro neutralization potency and in vivo efficacy. The optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.

Synthesis, characterization and utilization of a new series of 1,2,3-triazole derivatives to neutralize some toxic activities of Bothrops jararaca snake venom

Abstract Snake envenomation is a public health problem, and while serum therapy prevents death, the local effects of venoms can lead to amputations or morbidities. Thus, alternative treatments deserve attention. In this study, we tested eight derivatives of 1,2,3-triazole against some toxic activities of Bothrops jararaca venom. The derivatives were synthesized, and their structures analyzed by infrared and nuclear magnetic resonance. After that, the ability of compounds to inhibit hemolysis, coagulation, proteolysis, hemorrhaging, edema, and lethal activities of B. jararaca venom was investigated. The derivatives were incubated with B. jararaca venom (incubation protocol), administered before (prevention protocol) or after (treatment protocol) injecting venom into the mice. Then, hemorrhaging assay occurred. As a result, most of the derivatives inhibited the activities, even if they were incubated, injected before or after B. jararaca venom. However, the derivatives TRI 07 and TRI 18 seemed to be the most efficient in impairing hemorrhaging. The derivatives showed a low drug score of toxicity based on an in silico technique. Therefore, the derivatives fulfilled physicochemical and biological requirements to become drugs, and they may be a brand new initiative for designing antivenom molecules to complement antivenom therapy to efficiently block tissue necrosis or any other local effects.

Full Neutralization of Centruroidessculpturatus Scorpion Venom by Combining Two Human Antibody Fragments.

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10-9 and 6.1 × 10-11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.

The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments.

Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.

Synthesis, characterization & evaluation of venom neutralization potential of silver nanoparticles mediated Alstonia scholaris Linn bark extract.

OBJECTIVE: The venom neutralization potential of silver nanoparticle(AgNP-AS) mediated bark extract of Alstonia scholaris Linn R.Br was investigated in the study. METHODS & MATERIALS: AgNP-AS was synthesized with respect to optimal temperature, pH of extract. UV-vis, FT-IR, XRD, TEM, SEM studies were used to characterize silver nanoparticles of Alstonia scholaris Linn(AgNP-AS). The potential of AgNP-AS in neutralization of venom lethality, rise in myotoxicity markers(LDH) and proinflammatory cytokines(IL6, TNFα) were evaluated in animal models. RESULTS: AgNP-AS was synthesized optimally with AgNO3 (2 mM); extract concentration, 0.2 gm/l (1% w/v); extract (pH 9) and optimal temperature (40 °C). The colour change and synthesis of AgNP-AS was validated by UV-vis analysis at 432 nm. Transmission electron microscopy of AgNP-AS showed that the particle size for AgNP-AS was 14 nm-20 nm. FT-IR revealed peaks at 3445 cm-1, 1646 cm-1, 1346 cm-1 and 1108 cm-1. From the dynamic light scattering studies the hydrodynamic diameter (115.87 nm) and zeta potential(-29.8 mV) were estimated. The EDAX exhibited a peak for silver validating that the synthesized silver was pure. The biosynthesized (AgNP-AS) could significantly neutralize Viper russelli venom(VRV) induced rise in serum lactate dehydrogenase(LDH) and proinflammatory cytokines(IL6, TNFα) in animal models. CONCLUSION: The culmination of nanotechnology with herbal medicine might endow with a really constructive tool in coming up with future drugs with fewer toxicity.

In vivo neutralization of bee venom lethality by IgY antibodies.

Bee venom is a complex mixture of molecules, among which melittin and phospholipase A2 (PLA2) are the toxic components involved in envenoming accidents with multiple honeybee stings. Traditionally, the treatment of envenomings has been based on the administration of specific antibodies to neutralize the deleterious effects of toxins. An alternative to mammalian polyclonal antibodies is the use of egg yolk immunoglobulins (IgY) due to their advantages regarding animal welfare and lower costs of production as compared to the conventional production methods. In this work, a novel composition containing specific IgY antibodies was developed. After four immunizations, IgY extracted from the egg yolks was able to recognize several components of the bee venom, including melittin and PLA2. The performance of IgY to neutralize the lethal activity was evaluated in a mouse model by using one median lethal dose (LD50) of the bee venom. The effective dose of the IgY extract was determined as 30.66 µg/mg. These results demonstrate the feasibility to produce IgY-based antivenoms to treat envenomings by multiple bee stings.

Biochemical, electrophysiological and immunological characterization of the venom from Centruroides baergi, a new scorpion species of medical importance in Mexico.

In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na+ channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized.

Proteomic Analysis of Sri Lanka Echis carinatus Venom: Immunological Cross-Reactivity and Enzyme Neutralization Potency of Indian Polyantivenom.

The saw-scaled viper (Echis carinatus carinatus) is a major venomous snake in Sri Lanka (SL) responsible for massive numbers of snakebites on the island; nevertheless, its venom proteome composition has never been explored. The proteome composition of SL E. c. carinatus venom (SL ECV), revealed by tandem mass spectrometry analysis, showed that it is composed of 42 enzymatic and nonenzymatic proteins belonging to 12 snake venom protein families. Snake venom metalloproteases (SVMP) and snaclec comprised the most abundant enzymatic and nonenzymatic proteins, respectively. When the composition of SL ECV was compared to the previously determined venom composition of Southern India ECV (SI ECV), 16 proteins were found in common. The SL ECV proteome composition was correlated with the clinical manifestations and pathophysiology of E. c. carinatus envenomation in SL. Polyvalent antivenom (PAV) raised in equine against the "Big Four" venomous snakes of India is typically exported to SL for snakebite treatment; however, the poor immunological cross-reactivity, partial in vitro neutralization of enzymatic activities, and some pharmacological properties, mostly shown by low molecular mass toxins (25 kDa) of SL ECV by Indian PAVs are major concerns for the effective treatment of ECV envenomation in SL.

Mass spectrometric analysis to unravel the venom proteome composition of Indian snakes: opening new avenues in clinical research.

INTRODUCTION: The 'Big Four' venomous snakes - Daboia russelii, Naja naja, Bungarus caeruleus, and Echis carinatus - are primarily responsible for the majority of snake envenomation in India. Several other lesser-known venomous snake species also inflict severe envenomation in the country. AREAS COVERED: A comprehensive analysis of the venom proteome composition of the 'Big Four' and other medically important venomous snakes of India and the effect of regional variation in venom composition on immunorecognition and/or neutralization by commercial antivenom was undertaken by searching the literature (from 1985 to date) available in large public databases. Further, mass spectrometric identification of poorly immunogenic toxins of snake venom (against which commercial polyvalent antivenom contains a significantly lower proportion of antibodies) and its impact on antivenom therapy against snakebite are discussed. The application of mass spectrometry to identify protein (toxin) complexes as well as drug prototypes from Indian snake venoms and the clinical importance of such studies are also highlighted. EXPERT OPINION: Further detailed clinical and proteomic research is warranted to better understand the effects of regional snake venom composition on the clinical manifestation of envenomation and antivenom therapy and to improve the production of antibodies against poorly immunogenic venom components.

Quantitative proteomics to reveal the composition of Southern India spectacled cobra (Naja naja) venom and its immunological cross-reactivity towards commercial antivenom.

Indian cobra (Naja naja) envenomation is frequently reported across Indian subcontinent. Geographical differences in the venom composition of a particular species of snake often leads to inconsistencies in the antivenom neutralization. Consequently, determining the venom proteome from every locale is necessary for the production of effective antivenom. In this study, we deciphered the proteome composition of N. naja venom (NnV) from southern India (SI) by label-free quantitative proteomics that identified 45 proteins (toxins) belonging to 14 venom protein families when searched against Elapidae (taxid: 8602) protein entries in the non-redundant NCBI database. Low molecular mass (<15 kDa) toxins such as PLA2 (18.2%) and 3FTx (37.4%) are the most abundant enzymatic and non-enzymatic proteins, respectively, in SI NnV. Nevertheless, the relative abundance of 3FTxs in SI NnV was found to be lower than the relative abundance of these toxins in previously determined eastern and western India NnV samples. Immuno-recognition and in vitro neutralization of some enzymatic activities and pharmacological properties of SI NnV by commercial polyvalent antivenom evidently demonstrated poor recognition of the most abundant low molecular mass toxins of SI NnV. This finding points to the need for new strategies for antivenom production for the successful treatment of cobra bite.

Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka.

Russell's viper (Daboia russelii) is, together with Naja naja, Bungarus caeruleus and Echis carinatus, a member of the medically important 'Big Four' species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the "Big Four" venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. BIOLOGICAL SIGNIFICANCE: Russell's viper (Daboia russelii), the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), and the saw-scaled viper (Echis carinatus) constitute the 'Big Four' snake species responsible for most snakebite envenomings and deaths in the Indian subcontinent. Despite the medical relevance of Daboia russelii, and the well documented variations in the clinical manifestations of envenomings by this wide distributed species, which are doubtless functionally related to differences in venom composition of its geographic variants, antivenoms for the clinical treatment of envenomings by D. russelii across the Indian subcontinent are invariably raised using venom sourced mainly from the southeastern Indian state of Tamil Nadu. We have applied a phylovenomics approach to compare the venom proteomes of Russell's vipers from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and South India/Sri Lanka, and have assessed the in vitro (third-generation antivenomics) and in vivo preclinical efficacy of a panel of homologous antivenoms. The identification of two dispersal routes of ancestral D. russelii into the Indian subcontinent provides the ground for rationalizing the variability in composition and immunoreactivity of the venoms of extant geographic variants of Russell's viper. Such knowledge is relevant for envisioning strategies to improve the clinical coverage of anti- D. russelii antivenoms.

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy.

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies - IgY-technology - by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20-40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 µg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.