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Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.

Frontiers in immunology (2018-04-19)
Sabrina de Almeida Lima, Clara Guerra-Duarte, Fernanda Costal-Oliveira, Thais Melo Mendes, Luís F M Figueiredo, Daysiane Oliveira, Ricardo A Machado de Avila, Valéria Pereira Ferrer, Dilza Trevisan-Silva, Silvio S Veiga, João C Minozzo, Evanguedes Kalapothakis, Carlos Chávez-Olórtegui
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.

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Sigma-Aldrich
Anti-Horse IgG (whole molecule)−Peroxidase antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution