Abstract
Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.
Highlights
Loxoscelism is the most important form of araneism in South America
We report the localization of linear B-cell epitopes, using the SPOT method of multiple peptide synthesis [25, 26] on astacin-like protease (LALP-1) and hyaluronidase (LiHYAL) toxins from L. intermedia venom
Binding between anti-Loxosceles astacin-like proteases (LALPs)-1r sera antibodies and membrane-bound peptides seems to be specific since neither PI serum or alkaline phosphatase labeled anti-rabbit antibody showed reactivity with the membrane
Summary
Loxoscelism is the most important form of araneism in South America. It constitutes the first cause of accidents by venomous animals in Peru and Chile [1, 2]. In Brazil, more than 7,000 human cases of loxoscelism occur annually [(3)—SINAN]. Clinical manifestations of loxoscelism include local cutaneous and systemic forms. Local effects are commonly observed near the bite site, which is characterized by skin necrosis and slow progression ulceration. Systemic envenoming occurs in approximately 10% of the cases, depending on the species involved. Systemic symptoms include acute renal failure, intravascular hemolysis, thrombocytopenia, and disseminated intravascular coagulation [4]
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