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.

Figure 1. Protein family distribution for the venoms of the three different snake genera, as determined using a shotgun proteomics approach.

Each venom sample was prepared in duplicate, and the MS analyses were performed in triplicate for each venom sample replicate (a total of six MS analyses per venom). The data represent the mean of the normalized total spectral count distributed as follows: 1,891 (B. atrox); 1,727 (B. jararacussu); 2,719 (B. jararaca); 2,287 (B. neuwiedi); 1,252 (R. alternatus) and 1,767 (R. cotiara). The following were identified: SVMP-I, SVMP-II and SVMP–III (snake venom metalloproteinase - classes P-I, P-II and P-III); PLA2 (phospholipase A2); SVSP (snake venom serine proteinase); CLEC (C-type lectin); CLECL (C-type lectin-like); LAAO (L-amino acid oxidase); NGF (nerve growth factor); HYALU (hyaluronidase); VEGF (vascular endothelial growth factor); CRISP (cysteine-rich secretory protein); PDIEST (phosphodiesterase 1); ECTONT (ecto-5′-nucleotidase); PLB (phospholipase B); GLUTCYC (glutaminyl cyclase) and ACTIN (actin).

Figure 2. Comparison of the elution profiles of venoms from snakes classified in different genera.

Samples containing 5 mg of crude lyophilized venom from Bothrops atrox, Bothrops jararacussu, Bothropoides jararaca, Bothropoides neuwiedi, Rhinocerophis alternatus and Rhinocerophis cotiara, species maintained at Instituto Butantan herpetarium, were applied to a Vydac C-18 column (4.6×250 mm, 10-µm particle size) coupled to an Agilent 1100 HPLC system. The fractions were eluted at 1 mL/min, with a gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, 45–70% B over 20 min and 70–100% B over 10 min). The separations were monitored at 214 nm.

Figure 3. Venom clustering according to toxin composition.

The venoms from Bothrops atrox (ATR), Bothrops jararacussu (JSU), Bothropoides jararaca (JAR), Bothropoides neuwiedi (NEU), Rhinocerophis alternatus (ALT) and Rhinocerophis cotiara (COT) were classified according to their protein composition by hierarchical clustering of the observations, including as a variable the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A) or normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The procedure used an agglomerative hierarchical method linked by the minimum Euclidean distance between an item in one cluster and an item in another cluster (nearest neighbor) using the Minitab 16 software.

Figure 4. Principal Component Analysis relative to toxin composition.

Loading (top) and score (bottom) plots of the principal components 1 and 2 of the venoms from Bothrops atrox (ATR), Bothrops jararacussu (JSU), Bothropoides jararaca (JAR), Bothropoides neuwiedi (NEU), Rhinocerophis alternatus (ALT) and Rhinocerophis cotiara (COT) according to their protein composition including as variables the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A), or the normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The Principal Component Analysis was based on the covariance matrix and all calculations were carried out in the software Minitab 16.

Figure 5. Comparison of ELISA titration curves of Bothrops antivenom with venom from snakes classified in different genera.

Samples containing 100 µL whole venom (10 µg/mL) were used to coat maxisorb microplates (Nunc), which were incubated with crescent dilutions of SAB (starting from 1∶10,000), followed by incubation with anti-horse IgG labeled with peroxidase (1∶2,000). The reactions were developed with ortho-phenylenediamine/H₂O₂ as the enzyme substrate, and the products were detected at 490 nm. The experiments were performed in duplicate in three independent experiments, and the results are expressed as the mean ± sd of the six OD values.

Figure 6. Comparison of electrophoretic profile (A) and Bothrops antivenom antigenic reactivity (B) of venoms from snakes classified in different genera.

Samples containing 10 µg Bothropoides jararaca (JAR), Bothropoides neuwiedi (NEU), Bothrops atrox (ATR), Bothrops jararacussu(JSU), Rhinocerophis alternatus (ALT) and Rhinocerophis cotiara (COT) venoms were fractionated by SDS-PAGE (12.5% acrylamide gels) under non-reducing conditions and were either stained with Coomassie blue (A) or transferred to nitrocellulose membranes, which were then incubated with SAB (1∶1,000) as the primary antibody and peroxidase-labeled goat anti-horse IgG (1∶1,000). The reactive bands were detected by incubation with 4-chloro-α-naphthol and H₂O₂ (B). The numbers at the left indicate the mobility of the molecular mass markers in kDa. These results represent three independent runs.

Figure 7. ELISA reactivity with Bothrops antivenom and MAJar-3 monoclonal antibody of fractions collected from chromatograms of venom from snakes classified in different genera.

Samples containing 100 µL 1-µg/mL fractions collected at the elution times represented in the chromatograms were used to coat maxisorb microplates (Nunc), which were incubated with SAB (1∶1,000) or a monoclonal antibody against jararhagin (class P-III SVMP) MAJar-3 (1∶50), followed by incubation with anti-horse IgG (1∶2,000) or anti-mouse IgG (1∶1,000) labeled with peroxidase. The reactions were developed with ortho-phenylenediamine/H₂O₂ as the enzyme substrate, and the products were detected at 490 nm. The ELISA reactivity was calculated as % reactivity, taking as 100% the maximal OD value obtained in each of three independent experiments performed in duplicate.

Figure 8. Neutralizing ability of Bothrops antivenom (SAB) against the major toxic activities of Bothropoides jararaca and Bothrops atrox venoms.

In the neutralization assays, the Bothrops atrox venom was pooled from 8 adult snakes collected in FLONA Tapajós, Santarém, Pará, Brazil. For the neutralization of lethality and hemorrhagic activity, doses of B. jararaca or B. atrox venoms were pre-incubated with SAB at ratios of 1, 2 or 4 times the SAB volume required to neutralize an equal amount of reference venom according to the manufacturer. To assess hemorrhage, 10 µg was incubated and injected intradermically in the dorsum of a group of 5 mice. The results show the % neutralization of the mean values, taken as 100% activity, of the data obtained after injection with venom incubated with saline. For the neutralization of lethal activity, 3 LD₅₀ doses of B. jararaca (105 µg) or B. atrox (225 µg) venom were incubated, and the mixtures were injected intraperitoneally into groups of 5 mice; lethality was recorded over a period of 48 hours. The results represent the values obtained in 3 independent experiments and are expressed as % neutralization, considering the number of live/dead mice after this period. To assess the neutralization of coagulant activity, a constant amount of venom (2 times the minimum coagulant concentrations) was incubated with several dilutions of antivenom; the mixture was added to 100 µl bovine plasma, and the clotting times were recorded using a model ST4 mechanical coagulometer (Diagnostica Stago). The neutralization was expressed as the effective dose (ED), defined as the antivenom/venom ratio at which the clotting time was increased threefold when compared to the clotting time of plasma incubated with venom alone.