Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

Abstract

In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.

Comparison of venoms from wild and long-term captive Bothrops atrox snakes and characterization of Batroxrhagin, the predominant class PIII metalloproteinase from the venom of this species

“…Venom samples (4 mg) were dissolved in 500 µL 0.1% trifluoroacetic acid (TFA) and fractionated on a reverse-phase column (Column C-18 Vydac, 10 um, 4.6 mm id x 250 mm) in high-performance liquid chromatography-HPLC (Shimadzu, Japan), as previously described [20]. The parameters for elution were 2 mL/min flow, detection UV 214nm and gradient of 0.1% TFA in water (solution A) or in acetonitrile (solution B) as follows: 5% B for 5 min, followed by 5-15% B over 10 min, 15-45% B over 60 min, 45-70% B over 10 min, 70-100% B over 5 min and 100% B over 10 min.…”

“…The elution profiles of venoms from snakes from the captive and natural environment were visually compared. The protein identification in each chromatographic fraction was inferred from previous proteomic studies with the same venom [15,20].…”

“…In opposition, in venoms from snakes collected in Brazil, Ecuador and Peru, PIII-SVMPs were the most abundant toxin, corresponding to the paedomorphic phenotype [15,16,18]. In Brazilian Amazon, PIII-SVMPs were certainly the major toxins with relative concentrations in venoms ranging around 50% both in proteomic or transcriptomics studies [15,19,20].…”

“…Venom metalloproteinases, particularly PIII-SVMPs, are responsible for the local and systemic hemorrhage and are also involved in the local lesions due to their pro-inflammatory action or the ischemia at the site of the bite which arises from the reduction of blood supply following the damage of microvasculature [22,23]. Fortunately, PIII-SVMPs are highly immunogenic and are the predominant antigens recognized by commercial antivenoms for Bothrops snakes [20]. However, sequence and functional variability within PIII-SVMPs have been reported and may compromise antigen neutralization.…”

Evolution and diversification of the cnidarian venom system

“…Peptides were considered identified if they could be established at greater than 99.0% probability, and proteins were considered identified if they could be established at greater than 99.0% probability and contained at least 2 identified peptides and all matched spectra were confirmed by manual inspection. The accepted false discovery rates (FDR) for peptides and proteins were less than or equal to 1% [237]. In order compare the protein level and quantitative results across venom obtained by CDN and PDN, the protein summary of both techniques were submitted to Protein Alignment Template (V2.000p, ABSciex, Canada), where the reference protein ID and aligned protein ID were designated to PDN and CDN, respectively.…”