Produtos de excreção/secreção de strongyloides venezuelensis: aplicação ao diagnóstico da estrongiloidíase e caracterização de proteínas imunorreativas

Abstract

Strongyloides venezuelensis EXCRETORY/SECRETORY PRODUCTS: APPLICATION TO STRONGYLOIDIASIS DIAGNOSIS AND CHARACTERIZATION OF IMMUNOREACTIVE PROTEINS.

The Human strongyloidiasis is an intestinal parasitosis caused by the nematode Strongyloides stercoralis with worldwide distribution and predominance in tropical and subtropical areas. Parasitological diagnosis presents low sensitivity due to intermittent elimination of larvae in the faeces and approximately 50% of the affected individuals are asymptomatic. In the serological diagnosis, although more sensitive, cross reactivity still occurs with other parasitoses. The search for antigens that present greater specificity, if necessary, and the use of experimental models may aid in the production of heterologous antigens. Strongyloides venezuelensis is often used to evaluate host-parasite interaction, molecular aspects during infection, efficacy of new therapies, and other immunological characteristics of strongyloidiasis. Thus, use of excretory/secretory products (E/S) of filaroid larvae of S. venezuelensis in the diagnosis of strongyloidiasis presents itself as a promising tool. The objective of the research was to obtain E/S products of filaroid larvae of S. venezuelensis, apply them in the diagnosis of strongyloidiasis and to identify and partially characterize the immunoreactive proteins. ELISA tests for anti-Strongyloid IgG were carried out using samples of rat serum experimentally infected with S. venezuelensis or human serum using total saline extract (TS) and E/S products (RPMI E/S and PBS E/S). Immunoblotting was performed to detect the immunoreactive protein fractions and were later analyzed by mass spectrometry and bioinformatics. All preparations detected anti-Strongyloides IgG with emphasis on RPMI E/S (Se = 83.3% and Sp = 100%) on analysis with mouse serum and PBS E/S (Se = 81.7% and Sp = 88.9%) in human serum samples. In immunoblotting, specific protein bands with approximately 17 kDa were identified in the three preparations. In the bioinformatics analysis, proteins containing CAP domain belonging to the superfamily CRISP (A0A0K0FJA1) were identified in the three preparations, in TS a calcium binding protein, RPMI E/S two proteins containing CAP domain and PBS E/S an uncharacterized protein of the family ADF/Cofilin. Most of the identified proteins are E/S products, presenting predicted sequences as B cell epitopes. The E/S products presented superior diagnostic performance to TS, both in experimental and human strongyloidiasis. Protein bands of 17 kDa specific for Strongyloides, presented in their composition, proteins containing CAP domain. The construction of the three-dimensional models of the proteins and their validations were satisfactory and allowed to understand how these proteins present their conformation. Prediction of B-cell epitopes by the Bepipred 2.0 program identified sequences as B-cell epitopes and could be used as possible diagnostic tools as well as in vaccine trials.