Proteomic analysis of the mandibular glands from the Chinese crocodile lizard, Shinisaurus crocodilurus - Another venomous lizard?

Based on its phylogenetic relationship to monitor lizards (Varanidae), Gila monsters (Heloderma spp.), and the earless monitor Lanthanotus borneesis, the Chinese crocodile lizard, Shinisaurus crocodilurus, has been assigned to the Toxicofera clade, which comprises venomous reptiles. However, no data about composition and biological activities of its oral secretion have been reported. In the present study, a proteomic analysis of the mandibular gland of S. crocodilurus and, for comparison, of the herbivorous Solomon Island skink Corucia zebrata, was performed. Scanning electron microscopy (SEM) of the teeth from S. crocodilurus revealed a sharp ridge on the anterior surface, but no grooves, whereas those of C. zebrata possess a flattened crown with a pointed cusp. Proteomic analysis of their gland extracts provided no evidence of venom-derived peptides or proteins, strongly supporting the non-venomous character of these lizards. Data are available via ProteomeXchange with identifier PXD039424.

Half a century of research on Bothrops asper venom variation: biological and biomedical implications.

Snake venoms are a complex biological mixture of proteins with or without enzymatic activity, peptides, and nucleotides, among other components. It is produced in specialized secretory glands located in the maxillary region, being the result of millions of years of evolution and whose biological functions are defense, immobilization, and digestion of prey. Venoms present intraspecific (i.e., individual, ontogenetic, geographical) and interspecific (i.e., between sympatric and allopatric species) variation, and the study of this variability has become the focus of toxinological research. Bothrops asper is responsible for highest incidence, morbimortality and severe cases of envenoming in Mesoamerica and northern South America. Given its clinical importance, its venom has been characterized and compared qualitatively and quantitatively across the species range. More than 50 years of research show that B. asper venom is endowed with an interesting intraspecific variability. Knowing this variation has allowed advances in the elucidation of the biological role of the venom, a better understanding of the clinical signs and symptoms in patients envenomed by B asper, the immunological implications in the context of antivenoms production, and the generation of new ideas that could be useful to solve different biological and evolutionary questions of one of the venomous snakes with the greatest distribution and strongest public health impact in Latin America.

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-µHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-µHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Comparative venomics and preclinical efficacy evaluation of a monospecific Hemachatus antivenom towards sub-Saharan Africa cobra venoms.

Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.

Antivenomics and in vivo preclinical efficacy of six Latin American antivenoms towards south-western Colombian Bothrops asper lineage venoms.

BACKGROUND: Bothrops asper represents the clinically most important snake species in Central America and Northern South America, where it is responsible for an estimated 50-80% of snakebites. Compositional variability among the venom proteomes of B. asper lineages across its wide range mirrors clinical differences in their envenomings. Bothropic antivenoms generated in a number of Latin American countries commonly exhibit a certain degree of paraspecific effectiveness in the neutralization of congeneric venoms. Defining the phylogeographic boundaries of an antivenom's effectivity has implications for optimizing its clinical use. However, the molecular bases and impact of venom compositions on the immune recognition and neutralization of the toxic activities of across geographically disparate populations of B. asper lineages has not been comprehensively studied. METHODOLOGY/PRINCIPAL FINDINGS: Third-generation antivenomics was applied to quantify the cross-immunorecognizing capacity against the individual components of venoms of three B. asper lineages (B. asper (sensu stricto), B. ayerbei and B. rhombeatus) distributed in south-western (SW) Colombia, of six Latin American antivenoms, produced against homologous (Colombia, INS-COL and PROBIOL) and Costa Rica (ICP)), and heterologous (Argentina (BIOL), Perú (INS-PERU) and Venezuela (UCV)) bothropic venoms. In vivo neutralization assays of the lethal, hemorrhagic, coagulant, defibrinogenating, myotoxic, edematogenic, indirect hemolytic, and proteolytic activities of the three SW Colombian B. asper lineage venoms were carried to compare the preclinical efficacy of three (Colombian INS-COL and PROBIOL, and Costa Rican ICP) antivenoms frequently used in Colombia. Antivenomics showed that all the six antivenom affinity matrices efficiently immunoretained most of the B. asper lineages venom proteins and exhibited impaired binding towards the venoms' peptidomes. The neutralization profile of the INS-COL, PROBIOL and ICP antivenoms towards the biological activities of the venoms of SW Colombian B. asper (sensu stricto), B. ayerbei and B. rhombeatus lineages was coherent with the antivenomics outcome. In addition, the combination of in vitro (antivenomics) and in vivo neutralization results allowed us to determine their toxin-specific and venom neutralizing antibody content. Noteworthy, heterologous INS-PERU, BIOL, and UCV bothropic antivenoms had equal or higher binding capacity towards the venoms components of SW Colombian B. asper lineages that the homologous Colombian and Costa Rican antivenoms. CONCLUSIONS/SIGNIFICANCE: The combined in vitro and in vivo preclinical outcome showed that antivenoms manufactured in Colombia and Costa Rica effectively neutralize the major toxic activities of SW Colombian B. asper lineage venoms. The antivenomics profiles of the heterologous antivenoms manufactured in Argentina, Venezuela, and Perú strongly suggests their (pre)clinical adequacy for the treatment of B. asper lineage envenomings in SW Colombia. However, their recommendation in the clinical setting is pending on in vivo neutralization testing and clinical testing in humans. Bothrops asper is a highly adaptable snake species complex, which is considered the most dangerous snake throughout much of its distribution range from the Atlantic lowland of eastern México to northwestern Perú. Antivenoms are the only scientifically validated treatment of snakebite envenomings. Venom variation is particularly common in wide ranging species, such as B. asper, and may result in variable clinical presentations of envenomings, as is the case for the B. asper species complex, potentially undermining the efficacy of snakebite treatments depending on the immunization mixture used in the generation of the antivenom. Conversely, phylogenetic conservation of antigenic determinants confers an unpredictable degree of paraspecificity to homologous antivenoms produced for a geographic area, but also to heterologous congeneric antivenoms, towards the venom components of allopatric conspecific populations. This work aimed at comparing the preclinical profile of a panel of Latin American homologous and heterologous antivenoms against the venoms of B. asper lineages distributed in SW Colombia. The outcome of this study strongly suggests the suitability of considering the heterologous antivenoms BIOL (Argentina), UCV (Venezuela) and INS-PERU (Perú) as alternatives to homologous Colombian INS-COL and PROBIOL and Costa Rican ICP antivenoms for the treatment of envenomings by B. asper (sensu stricto) in W Colombia and Ecuador, B. ayerbei in Cauca and Nariño (Colombia), and B. rhombeatus in Cauca river valley, SW Colombia.

Venom variation in Bothrops asper lineages from North-Western South America.

Bothrops asper is a venomous pitviper that is widely distributed and of clinical importance in Mesoamerica and northern South America, where it is responsible for 50-80% of all envenomations by Viperidae species. Previous work suggests that B. asper has a complex phylogeographic structure, with the existence of multiple evolutionarily distinct lineages, particularly in the inter-Andean valleys of north South America. To explore the impact of the evolutionary history of B. asper on venom composition, we have investigated geographic variation in the venom proteome of this species from the populations from the Pacific side of Ecuador and south-western Colombia. Among the 21 classes of venom components identified, proteins from mainly four major toxin families, snake venom metalloproteases (PI- and PII-SVMP), phospholipases A2 (K49- and D49-PLA2s), serine proteinases (SVSP), and C-type lectins-like (CTL) proteins are major contributors to the geographic variability in venom. Principal component analyses demonstrate significant differences in venom composition between B. asper lineages previously identified through combination of molecular, morphological and geographical data, and provide additional insights into the selection pressures modulating venom phenotypes on a geographic scale. In particular, altitudinal zonation within the Andean mountain range stands out as a key ecological factor promoting diversification in venom. In addition, the pattern of distribution of PLA2 molecules among B. asper venoms complements phylogenetic analysis in the reconstruction of the dispersal events that account for the current biogeographic distribution of the present-day species' phylogroups. Ontogenic variation was also evident among venoms from some Ecuadorian lineages, although this age-related variation was less extreme than reported in B. asper venoms from Costa Rica. The results of our study demonstrate a significant impact of phylogenetic history on venom composition in a pitviper and show how analyses of this variation can illuminate the timing of the cladogenesis and ecological events that shaped the current distribution of B. asper lineages. BIOLOGICAL SIGNIFICANCE: Bothrops asper, called "the ultimate pitviper" due to its defensive behavior, large body size, and medical importance, represents a species complex that is widely distributed from southern México southwards across north-western South America to north-western Perú. This work reports the characterization of the venom proteomes of B. asper lineages from the Pacific sides of Ecuador and south-western Colombia. Multivariate analyses indicate that variability in venom composition among the B. asper lineages is driven by proteins from four major toxin families, presumably in response to selection pressures created by recent and historical ecological conditions created by geological and climatic events from the Pliocene-Pleistocene to the present along the Central and South American Continental Divide. The emerging biogeographic pattern of venom variation, interpreted in the context of the current phylogenetic hypotheses, support and complement previously proposed evolutionary Plio-Pleistocene dispersal events that shaped the present-day distribution range of B. asper lineages. In addition, our venomics data indicate the occurrence of genetic exchange between Colombian and Pacific Costa Rican populations, which may have occurred during the second wave of B. asper migration into Mesoamerica. Our work represents a foundation for a future broader sampling and more complete "-omics" analyses to deepen our understanding of the patterns and causes of venom variation in this medically important pitviper.

Dagestan blunt-nosed viper, Macrovipera lebetina obtusa (Dwigubsky, 1832), venom. Venomics, antivenomics, and neutralization assays of the lethal and toxic venom activities by anti-Macrovipera lebetina turanica and anti-Vipera berus berus antivenoms.

We have applied a combination of venomics, in vivo neutralization assays, and in vitro third-generation antivenomics analysis to assess the preclinical efficacy of the monospecific anti-Macrovipera lebetina turanica (anti-Mlt) antivenom manufactured by Uzbiopharm® (Uzbekistan) and the monospecific anti-Vipera berus berus antivenom from Microgen® (Russia) against the venom of Dagestan blunt-nosed viper, Macrovipera lebetina obtusa (Mlo). Despite their low content of homologous (anti-Mlt, 5-10%) or para-specific (anti-Vbb, 4-9%) F(ab')2 antibody fragments against M. l. obtusa venom toxins, both antivenoms efficiently recognized most components of the complex venom proteome's arsenal, which is made up of toxins derived from 11 different gene families and neutralized, albeit at different doses, key toxic effects of M. l. obtusa venom, i.e., in vivo lethal and hemorrhagic effects in a murine model, and in vitro phospholipase A2, proteolytic and coagulant activities. The calculated lethality neutralization potencies for Uzbiopharm® anti-Mlt and anti-Vbb Microgen® antivenoms were 1.46 and 1.77 mg/mL, indicating that 1 mL of Uzbiopharm® and Microgen® antivenoms may protect mice from 41 to 50 LD50s of Mlo venom, respectively. The remarkable degree of conservation of immunogenic determinants between species of the clades of European and Oriental viper, which evolved geographically segregated since the early Miocene, suggests an eventual window of opportunity for the treatment of envenomings by Eurasian snakes. Clearly, the rational use of heterologous antivenoms requires establishing their para-specificity landscapes. This paper illustrates the analytical power of combining in vitro and in vivo preclinical quantitative assays toward this goal.

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.

Vipera berus berus Venom from Russia: Venomics, Bioactivities and Preclinical Assessment of Microgen Antivenom.

The common European adder, Vipera berus berus, is a medically relevant species, which is widely distributed in Russia and thus, is responsible for most snakebite accidents in Russia. We have investigated the toxic and enzymatic activities and have determined the proteomic composition of its venom. Phospholipases A2 (PLA2, 25.3% of the venom proteome), serine proteinases (SVSP, 16.2%), metalloproteinases (SVMP, 17.2%), vasoactive peptides (bradykinin-potentiating peptides (BPPs), 9.5% and C-type natriuretic peptides (C-NAP, 7.8%), cysteine-rich secretory protein (CRISP, 8%) and L-amino acid oxidase (LAO, 7.3%) represent the major toxin classes found in V. b. berus (Russia) venom. This study was also designed to assess the in vivo and in vitro preclinical efficacy of the Russian Microgen antivenom in neutralizing the main effects of V. b. berus venom. The results show that this antivenom is capable of neutralizing the lethal, hemorrhagic and PLA2 activities. Third-generation antivenomics was applied to quantify the toxin-recognition landscape and the maximal binding capacity of the antivenom for each component of the venom. The antivenomics analysis revealed that 6.24% of the anti-V. b. berus F(ab')2 molecules fraction are toxin-binding antibodies, 60% of which represent clinically relevant antivenom molecules.

Third-generation antivenomics analysis of the preclinical efficacy of Bothrofav® antivenom towards Bothrops lanceolatus venom.

Bothrops lanceolatus inflicts severe envenomings in the Lesser Caribbean island of Martinique. Bothrofav®, a monospecific antivenom against B. lanceolatus venom, has proven highly effective at the preclinical and clinical levels. Here, we report a detailed third-generation antivenomics quantitative analysis of Bothrofav®. With the exception of poorly-immunogenic peptides, Bothrofav® immunocaptured all the major protein components. These results, along with previous preclinical and clinical observations, underscore the high neutralizing efficacy of the antivenom against B. lanceolatus venom.

Defining the pathogenic threat of envenoming by South African shield-nosed and coral snakes (genus Aspidelaps), and revealing the likely efficacy of available antivenom.

While envenoming by the southern African shield-nosed or coral snakes (genus Aspidelaps) has caused fatalities, bites are uncommon. Consequently, this venom is not used in the mixture of snake venoms used to immunise horses for the manufacture of regional SAIMR (South African Institute for Medical Research) polyvalent antivenom. Aspidelaps species are even excluded from the manufacturer's list of venomous snakes that can be treated by this highly effective product. This leaves clinicians, albeit rarely, in a therapeutic vacuum when treating envenoming by these snakes. This is a significantly understudied small group of nocturnal snakes and little is known about their venom compositions and toxicities. Using a murine preclinical model, this study determined that the paralysing toxicity of venoms from Aspidelaps scutatus intermedius, A. lubricus cowlesi and A. l. lubricus approached that of venoms from highly neurotoxic African cobras and mambas. This finding was consistent with the cross-genus dominance of venom three-finger toxins, including numerous isoforms which showed extensive interspecific variation. Our comprehensive analysis of venom proteomes showed that the three Aspidelaps species possess highly similar venom proteomic compositions. We also revealed that the SAIMR polyvalent antivenom cross-reacted extensively in vitro with venom proteins of the three Aspidelaps. Importantly, this cross-genus venom-IgG binding translated to preclinical (in a murine model) neutralisation of A. s. intermedius venom-induced lethality by the SAIMR polyvalent antivenom, at doses comparable with those that neutralise venom from the cape cobra (Naja nivea), which the antivenom is directed against. Our results suggest a wider than anticipated clinical utility of the SAIMR polyvalent antivenom, and here we seek to inform southern African clinicians that this readily available antivenom is likely to prove effective for victims of Aspidelaps envenoming. BIOLOGICAL SIGNIFICANCE: Coral and shield-nosed snakes (genus Aspidelaps) comprise two species and several subspecies of potentially medically important venomous snakes distributed in Namibia, Botswana, Zimbabwe, Mozambique and South Africa. Documented human fatalities, although rare, have occurred from both A. lubricus and A. scutatus. However, their venom proteomes and the pathological effects of envenomings by this understudied group of nocturnal snakes remain uncharacterised. Furthermore, no commercial antivenom is made using venom from species of the genus Aspidelaps. To fill this gap, we have conducted a transcriptomics-guided comparative proteomics analysis of the venoms of the intermediate shield-nose snake (A. s. intermedius), southern coral snake (A. l. lubricus), and Cowle's shield snake (A. l. cowlesi); investigated the mechanism of action underpinning lethality by A. s. intermedius in the murine model; and assessed the in vitro immunoreactivity of the SAIMR polyvalent antivenom towards the venom toxins of A. l. lubricus and A. l. cowlesi, and the in vivo capability of this antivenom at neutralising the lethal effect of A. s. intermedius venom. Our data revealed a high degree of conservation of the global composition of the three Aspidelaps venom proteomes, all characterised by the overwhelming predominance of neurotoxic 3FTxs, which induced classical signs of systemic neurotoxicity in mice. The SAIMR polyvalent antivenom extensively binds to Aspidelaps venom toxins and neutralised, with a potency of 0.235 mg venom/mL antivenom, the lethal effect of A. s. intermedius venom. Our data suggest that the SAIMR antivenom could be a useful therapeutic tool for treating human envenomings by Aspidelaps species.

The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms.

Snake envenoming causes several potentially lethal pathologies. The specific pathology is dictated by the toxin composition of venom, which varies by species, geography and ontogeny. This variation severely restricts the paraspecific efficacy of antivenoms used to treat snakebite victims. With a view to devising pathology-specific snakebite treatments, we assessed the procoagulant activity of 57 snake venoms and investigated the efficacy of various antivenoms. We find that procoagulant venoms act differentially on key steps of the coagulation cascade, and that certain monospecific antivenoms work in a previously unrecognised paraspecific manner to neutralise this activity, despite conventional assumptions of congener-restricted efficacy. Moreover, we demonstrate that the metal chelator EDTA is also capable of neutralising venom-induced lethality in vivo. This study illustrates the exciting potential of developing new, broad-spectrum, toxin-targeting antivenoms capable of treating key snakebite pathologies, and advocates a thorough re-examination of enzyme inhibiting compounds as alternative therapies for treating snakebite victims.

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.

Transcriptomics-guided bottom-up and top-down venomics of neonate and adult specimens of the arboreal rear-fanged Brown Treesnake, Boiga irregularis, from Guam.

The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake native to northern and eastern regions of Australia, Papua New Guinea and the Solomon Islands. It was inadvertently introduced onto the island of Guam during the late 1940's to early 1950's, and it has caused massive declines and extirpations of the native bird, lizard, and mammal populations. In the current study, we report the characterization of the venom proteome of an adult and a neonate B. irregularis specimens from Guam by a combination of venom gland transcriptomic and venomic analyses. Venom gland transcriptomic analysis of an adult individual identified toxins belonging to 18 protein families, with three-finger toxin isoforms being the most abundantly expressed transcripts, comprising 94% of all venom protein transcript reads. Transcripts for PIII-metalloproteinases, C-type lectins, cysteine-rich secretory proteins, acetylcholinesterases, natriuretic peptides, ficolins, phospholipase A2 (PLA2) inhibitors, PLA2s, vascular endothelial growth factors, Kunitz-type protease inhibitors, cystatins, phospholipase Bs, cobra venom factors, waprins, SVMP inhibitors, matrix metalloproteinases, and hyaluronidases were also identified, albeit, at very low abundances ranging from 0.05% to 1.7% of the transcriptome. The venom proteomes of neonate and adult B. irregularis were also both overwhelmingly (78 and 84%, respectively) dominated by monomeric and dimeric 3FTxs, followed by moderately abundant (21% (N) and 13% (A)) CRISPs, low abundance (1% (N), 3% (A)) PIII-SVMPs, and very low abundance (<0.01%) PLA2 and SVMP inhibitors. The differences in relative toxin abundances identified between neonate and adult snakes likely correlates to shifts in prey preference between the two age classes, from nearly-exclusively lizards to lizards, birds and small mammals. Immunoaffinity antivenomics with experimentally designed rabbit anti-Brown Treesnake (anti-BTS) venom IgGs against homologous venom from adult snakes demonstrated that CRISPs, PIII-SVMPs, and 60-70% of 3FTxs were effectively immunocaptured. Western blot analysis showed that all venom proteins were recognized by anti-BTS IgGs, and cross-reactivity with other rear-fanged snake venoms was also observed. Incubation of anti-BTS venom IgGs with crude B. irregularis venom resulted in a significant decrease in proteolytic (SVMP) activity against azocasein. These results provide the first comparative venomic and anti-venomic analysis of neonate and adult B. irregularis from Guam, further highlighting evolutionary trends in venom composition among rear-fanged venomous snakes. SIGNIFICANCE PARAGRAPH: The Brown Treesnake (Boiga irregularis) has caused extensive ecological and economic damage to the island of Guam where it has become a classic example of the negative impacts of invasive species. In the current study, we report the first combined transcriptomic and proteomic analysis of B. irregularis venom of Guam origin. The transcriptome of an adult snake contained toxin sequences belonging to 18 protein families, with three-finger toxin (3FTx) isoforms being the most abundant and representing 94% of all venom protein transcript reads. Our bottom-up and top-down venomic analyses confirmed that 3FTxs are the major components of B. irregularis venom, and a comparative analysis of neonate and adult venoms demonstrate a clear ontogenetic shift in toxin abundance, likely driven by dietary variation between the two age classes. Second-generation antivenomics and Western blot analysis using purified anti-Brown Treesnake rabbit serum IgGs (anti-BTS IgGs) showed strong immunoreactivity toward B. irregularis venom. Interestingly, our anti-BTS IgGs did not cross-react with 3FTxs found in several other rear-fanged snake venoms, or against 3FTxs in the venom of the elapid Ophiophagus hannah, indicating that epitopes in these 3FTx molecules are quite distinct.

A synthetic biology approach for consistent production of plant-made recombinant polyclonal antibodies against snake venom toxins.

Antivenoms developed from the plasma of hyperimmunized animals are the only effective treatment available against snakebite envenomation but shortage of supply contributes to the high morbidity and mortality toll of this tropical disease. We describe a synthetic biology approach to affordable and cost-effective antivenom production based on plant-made recombinant polyclonal antibodies (termed pluribodies). The strategy takes advantage of virus superinfection exclusion to induce the formation of somatic expression mosaics in agroinfiltrated plants, which enables the expression of complex antibody repertoires in a highly reproducible manner. Pluribodies developed using toxin-binding genetic information captured from peripheral blood lymphocytes of hyperimmunized camels recapitulated the overall binding activity of the immune response. Furthermore, an improved plant-made antivenom (plantivenom) was formulated using an in vitro selected pluribody against Bothrops asper snake venom toxins and has been shown to neutralize a wide range of toxin activities and provide protection against lethal venom doses in mice.

Cross-reactivity, antivenomics, and neutralization of toxic activities of Lachesis venoms by polyspecific and monospecific antivenoms.

BACKGROUND: Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. METHODS: To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. RESULTS: A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. CONCLUSIONS: Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. GENERAL SIGNIFICANCE: This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.

Drought Tolerance in Pinus halepensis Seed Sources As Identified by Distinctive Physiological and Molecular Markers.

Drought is one of the main constraints determining forest species growth, survival and productivity, and therefore one of the main limitations for reforestation or afforestation. The aim of this study is to characterize the drought response at the physiological and molecular level of different Pinus halepensis (common name Aleppo pine) seed sources, previously characterized in field trials as drought-sensitive or drought-tolerant. This approach aims to identify different traits capable of predicting the ability of formerly uncharacterized seedlings to cope with drought stress. Gas-exchange, water potential, photosynthetic pigments, soluble sugars, free amino acids, glutathione and proteomic analyses were carried out on control and drought-stressed seedlings in greenhouse conditions. Gas-exchange determinations were also assessed in field-planted seedlings in order to validate the greenhouse experimental conditions. Drought-tolerant seed sources presented higher values of photosynthetic rates, water use efficiency, photosynthetic pigments and soluble carbohydrates concentrations. We observed the same pattern of variation of photosynthesis rate and maximal efficiency of PSII in field. Interestingly drought-tolerant seed sources exhibited increased levels of glutathione, methionine and cysteine. The proteomic profile of drought tolerant seedlings identified two heat shock proteins and an enzyme related to methionine biosynthesis that were not present in drought sensitive seedlings, pointing to the synthesis of sulfur amino acids as a limiting factor for drought tolerance in Pinus halepensis. Our results established physiological and molecular traits useful as distinctive markers to predict drought tolerance in Pinus halepensis provenances that could be reliably used in reforestation programs in drought prone areas.

Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.

Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of "antivenomics", which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms.

Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms.

What killed Karl Patterson Schmidt? Combined venom gland transcriptomic, venomic and antivenomic analysis of the South African green tree snake (the boomslang), Dispholidus typus.

BACKGROUND: Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. METHODS: A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. RESULTS: Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A2 (PLA2); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. CONCLUSIONS: The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. GENERAL SIGNIFICANCE: This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt.