Estudo de repertório sorológico de anticorpos de cavalo contra veneno de aranhas do gênero Loxosceles de importância médica no Brasil

Abstract

The accidents caused by spiders of the Loxosceles genus (also called brown-spiders) are rising in incidence in Brazil and may cause severe dermonecrotic lesions, renal failure and death. Serotherapy is the only specific treatment available and is based on antibodies from horses hyperimmunized with the spiders’ venom. The use of this therapy may cause adverse reactions and its production relies on the extraction of the venom from the spiders for the immunization of the horses, a process that impacts negatively on the life of the inoculated animals. Many attempts to overcome these limitations have been explored, but the lack of knowledge about the characteristics of horses’ immunoglobulins limits the search for new solutions. In this work, the repertoire of serological antibodies of four horses hyperimmunized with venom from L. intermedia, L. gaucho and L. laeta was characterized relative to its classes and subclasses and to the sequences that compose them. Equine plasmas collected before and after the immunization were used for the quantification of immunoglobulin classes and IgG subclasses and, after that, for isolation of IgG, obtention of F(ab’)2, selection of F(ab’)2 fractions specific against the pool of venoms or against rLiD1 and trypsin digestion. Specific F(ab')2 tryptic peptides were analyzed by LC-MS/MS and mapped to the immunoglobulin heavy chain sequences of the transcriptome of PBMCs of each animal, using CDRH3 as a parameter for identification. As for the immunoglobulin classes, a median concentration of 0.63 for IgM and of 241.42 mg/mL for IgG were found in hyperimmune plasmas. Before isolation using protein G, the IgG(T) subclass was found with greater abundance in plasmas and after isolation, IgGb predominated. From the LC-MS/MS data, 132 heavy chain variable region sequences specific against the pool of venoms were identified, as well as 19 against rLiD1 and 12 with both specificities. In these sequences, the IGHV4-29, IGHD2-26 and IGHJ6 gene segments were more used, the CDRH3 had a higher frequency of charged residues (Asp and Arg) compared to the nonspecific repertoire and had a median length of 17 amino acids. Calculation of abundance from the area under the curve of the peptides revealed a polarized response with more than 80% of the abundance concentrated in the top 3 and top 5 clones of specific immunoglobulins against the pool of venoms and rLiD1, respectively. The prediction of paratopes against rLiD1 indicates high convergence, with four possible epitopes recognized by the 31 sequences. To our knowledge, this is the first work to use Ig-Seq for the characterization of equine antibodies. The discovery of sequences of the variable region of the heavy chain of immunoglobulins and its characteristics may be useful in the production and optimization of synthetic antibodies designed for therapeutical applications.