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.

Snake venomics across genus Lachesis. Ontogenetic changes in the venom composition of Lachesis stenophrys and comparative proteomics of the venoms of adult Lachesis melanocephala and Lachesis acrochorda.

We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.

Impact of regional variation in Bothrops asper snake venom on the design of antivenoms: integrating antivenomics and neutralization approaches.

Intraspecific snake venom variations have implications in the preparation of venom pools for the generation of antivenoms. The impact of such variation in the cross-reactivity of antivenoms against Bothrops asper venom was assessed by comparing two commercial and four experimental antivenoms. All antivenoms showed similar immunorecognition pattern toward the venoms from adult and neonate specimens. They completely immunodepleted most P-III snake venom metalloproteinases (SVMPs), l-amino acid oxidases, serine proteinases, DC fragments, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like proteins, and partially immunodepleted medium-sized disintegrins, phospholipases A(2) (PLA(2)s), some serine proteinases, and P-I SVMPs. Although all antivenoms abrogated the lethal, hemorrhagic, coagulant, proteinase, and PLA(2) venoms activities, monospecific experimental antivenoms were more effective than the polyspecific experimental antivenom. In addition, the commercial antivenoms, produced in horses subjected to repeated immunization cycles, showed higher neutralization than experimental polyspecific antivenom, produced by a single round of immunization. Overall, a conspicuous pattern of cross-neutralization was evident for all effects by all antivenoms, and monospecific antivenoms raised against venom from the Caribbean population were effective against venom from the Pacific population, indicating that geographic variations in venom proteomes of B. asper from Costa Rica do not result in overt variations in immunological cross-reactivity between antivenoms.

Studies on the venom proteome of Bothrops asper: perspectives and applications.

Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and several studies have demonstrated that specific toxic and enzymatic activities of its venom vary with the geographic origin and age of the specimens. Variability in venom proteins and enzymes between specimens from the Caribbean and the Pacific versants of Costa Rica has been reported since 1964. Recently, we performed a comparative proteomic characterization of the venoms from one population of each versant. Proteins belonging to several families, including disintegrin, phospholipases A(2), serine proteinases, C-type lectins, CRISP, l-amino acid oxidase, and Zn(2+)-dependent metalloproteinases show a variable degree of relative occurrence in the venoms of both populations. The occurrence of prominent differences in the protein profile between venoms from adults and newborns, and among venom samples from individual specimens of the same region or developmental stage, further demonstrated the existence of geographic, ontogenetic and individual variability in the venom proteome of this species. These findings provide new insights towards understanding the biology of B. asper, contribute to a deeper understanding of the pathology induced by its venom and underscore the importance of the use of venoms pooled from specimens from both regions for producing antivenom exhibiting the broadest cross-reactivity. Furthermore, knowledge of the protein composition of B. asper venom paves the way for detailed future structure-function studies of individual toxins as well as for the development of new protocols to study the reactivity of therapeutic antivenoms.

Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations.

We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2, serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical location and age has long been appreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production.