Production of Neutralizing Anti-Loxoscelic Horse Sera Using Recombinant Sphingomyelinases As Immunogens

¹Fernandes-Pedrosa, M.F.; ¹Ferracini, M.; ²Guidolin, R., ²Higashi, H.G. ²Marcelino, J.R. and ¹Tambourgi D.V.

1 Laboratório de Imunoquímica, 2 Divisão de Desenvolvimento Tecnológico e Produção, Instituto Butantan, Brazil.

Bites by Loxoscelesspiders can produce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, hemolysis, and persistent inflammation. The causative factor is a sphingomyelinase D (SMaseD) that cleaves sphingomyelin into choline and ceramide 1-phosphate and has intrinsic lysophospholipase D activity toward LPC. We have cloned and expressed the fully active sphingomyelinase recombinants from L. laeta (Smase I) and L. intermedia (recP1 and recP2) spider venoms endowed with all biological properties ascribed for the whole venoms including dermonecrotic and complement-dependent haemolytic activities and the ability of hydrolysing sphingomyelin. Considering the difficulty in obtaining large amounts of spider venom for antiserum production, we are currently immunizing horses in order to analyse the possibility of using recombinant sphingomyelinases as antigens for the production of anti-loxoscelic serum by Butantan Institute. Sera obtained from horses immunized with small amounts of the recombinants proteins showed to contain high antibody titters against the recombinants and native toxins. Western blot analysis and ELISA showed a strong sera cross reactivity against venoms from Loxosceles species of medical importance in Brazil, e.g., L. intermedia, L. gaucho and L. laeta. Neutralization tests revealed that the antisera produced were able to neutralize the dermonecrotic reaction, the sphingomyelinase activity and the complement mediated lysis induced by Loxosceles venoms, indicating that SMases could be used as immunogens for anti-loxoscelic serum production for human serum therapy.

Supported by: The Wellcome Trust and FAPESP

Correspondence to: mpedrosa@butantan.gov.br