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Highlights: [•] A monoclonal antibody (LiD1mAb16) against a recombinant SMase D isoform was produced. [•] LiD1mAb16 was cross-reactive with protein spots from different Loxosceles species. [•] LiD1mAb16 recognizes a linear epitope in the catalytic loop of SMases D. [•] LiD1mAb16 showed neutralizing potential. [•] This mAb is a candidate for therapeutic serum development or immunodiagnostic assays. [ABSTRACT FROM AUTHOR]

Abstract: The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom. [Copyright &y& Elsevier]

Abstract: Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope (25NLGANSIETDVSFDDNANPEYTYHGIP51) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings. [Copyright &y& Elsevier]

Abstract: This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25–35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv. [Copyright &y& Elsevier]

Highlights: [•] A protein consisting of epitopes of a spider venom protein was engineered. [•] The chimeric protein generated did not show toxic activities. [•] Antibodies anti-chimeric protein recognized three different antigenic regions. [•] Antibodies anti-chimeric protein showed neutralizing potential. [•] This protein is a candidate for therapeutic serum development. [ABSTRACT FROM AUTHOR]