Linear B-cell epitopes in BthTX-I, BthTX-II and BthA-I, phospholipase A_2's from Bothrops jararacussu snake venom, recognized by therapeutically neutralizing commercial horse antivenom

Background

The benefits from treatment with antivenom sera are indubitable. However, the mechanism for toxin neutralization has not been completely elucidated. A mixture of anti-bothropic and anti-crotalic horse antivenom has been reported to be more effective in neutralizing the effects of B. jararacussu snake venom than anti-bothropic antivenom alone. This study determined which regions in the three PLA₂s from B.jararacussu snake venom are bound by antibodies in tetravalent anti-bothropic and monovalent anti-crotalic commercial horse antivenom.

Methods

The epitopes recognized by therapeutic horse antivenom sera in the three major PLA₂s present in the venom of B. jararacussu, BthTX-I, BthTX-II and BthA-I, were mapped using the parallel Spot-synthesis strategy. Two peptide libraries were designed to more precisely define the epitopes recognized by anti-bothropic and/or anti-crotalic horse antivenom. Each consisted of 69 peptide sequences of fourteen amino acids each that overlapped by nine amino acids and covered the entire protein sequences of the three PLA₂s. The oligomeric structure of PLA_2S proteins were solved by X-ray crystallography and are available in the protein data bank. The sequences of fifty PLA₂s were selected and grouped into three sub-groups. Shared amino acids sequence from the 12 epitopes recognized by the reaction between the B. jararacussu PLA₂s and anti-crotalic/anti-bothropic horse antivenom were analyzed by a multiple sequence alignment.

Results and conclusions

Mapping experiments of BthTX-I, BthTX-II and BthA-I using two small libraries of 69 peptides each revealed six major IgG-binding epitopes that were recognized by both anti-bothropic and anti-crotalic horse antivenom. Two epitopes in BthTX-I were only recognized by the anti-bothropic horse antivenom, while anti-crotalic horse antivenom recognized four unique epitopes across the three PLA₂s. Our studies suggest that the harmful activities of the PLA₂s present in the venom of B. jararacussu are neutralized by the combinatorial treatment with both antivenom sera through their complementary binding sites, which provides a wide coverage on the PLA₂s. In conclusion, the peptide arrays formed directly onto cellulose membranes allowed the identification of the major antigenic determinants in the three most important PLA₂s (BthTX-I, BthTX-II and BthA-I) isolated from B. jararacussu snake venom recognized by commercial anti-bothropic and anti-crotalic horse antivenom. The cross-reactive epitopes located in the Lys49-PLA₂, the major protein of this venom, recognized two specific epitopes located in a region of the enzyme responsible for the myotoxic action, which contributes to the deleterious effects of snake venom. In addition, the ability of the anti-crotalic horse antivenom to neutralize the anticoagulant activity was most likely associated with the acidic Asp49-PLA₂. This study provides proof that the mixture of anti-crotalic and anti-bothropic horse antivenom is qualitatively more effective in neutralizing the effects unleashed of B. jararacussu snakebite. Regions recognized by the protective antivenom sera are prime candidates for improved venom cocktails or a chimeric protein encoding the multiple epitopes to immunize animals as well as for designing future synthetic vaccines.