Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency.

Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2-10 µL/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC50 = 58.95 µg/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.

Determination of hyaluronidase activity in Tityus spp. Scorpion venoms and its inhibition by Brazilian antivenoms.

Hyaluronidases (HYALs) are enzymes ubiquitously found in venoms from diverse animals and seem to be related to venom spreading. HYAL activity might be important to Tityus spp. envenoming, since anti-Tityus serrulatus HYAL (TsHYAL) rabbit antibodies neutralize T. serrulatus venom (TsV) lethality. The present work aimed to verify and compare HYAL activity of venoms from other Brazilian Tityus spp. (Tityus bahiensis, Tityus stigmurus and Tityus obscurus) and to test whether anti-TsHYAL antibodies and Brazilian horse therapeutic scorpion antivenom (produced by Fundação Ezequiel Dias (FUNED), Butantan and Vital Brazil Institutes) can recognize and inhibit HYAL activity from these venoms. In ELISA assays, anti-TsHYAL and scorpion antivenoms recognized T. serrulatus, T. bahiensis and T. stigmurus venoms, however, they demonstrated weaker reaction with T. obscurus, which was also observed in Western blotting assay. Epitope mapping by SPOT assay revealed different binding patterns for each antivenom. The assay showed a weaker binding of scorpion antivenom produced by FUNED to peptides recognized by anti-TsHYAL antibodies. Anti-TsHYAL antibodies and antivenoms produced by Butantan and Vital Brazil institutes inhibited HYAL activity of all tested venoms in vitro, whereas FUNED antivenom did not show the same property. These results call attention to the importance of hyaluronidase inhibition, that can aid the improvement of antivenom production.