Identificação e caracterização imunológica de antígenos de Leishmania (Leishmania) chagasi para o desenvolvimento de vacinas e alternativas emdiagnóstico da Leishmaniose visceral

Abstract

Visceral leishmaniasis (VL) is a severe infectious disease caused by a protozoan of the genus Leishmania. In the Americas, the dog is the main reservoir of disease, mainly in urban areas where the disease is increasing. VL is fatal if it is not adequately treated and/or if it is not diagnosed early, however the efficacy of diagnostic methods andvaccines available remain partial, therefore it is necessary to develop new strategies. Several studies have demonstrated the potential of A2 protein, an amastigote stagespecific protein family in Leishmania, as vaccine and diagnosis. In our studies, using this antigen administered as DNA vaccine, we observed protection in mice against infection by L. (L.) chagasi and L. (L.) amazonensis. A2 antigen also induces protectionin phase II studies with beagle dogs vaccinated with A2 protein associated with the saponin adjuvant (constituting the vaccine Leish-tec.) against infection by L. (L.) chagasi. The partial protection induced by A2 antigen in vaccine studies led us to search for new proteins for further vaccine tests and future associations with the A2 antigen. Regarding the diagnosis, we found that the peptides derived from in silico analysis of the A2 protein, associated with peptides from other proteins such as NH, LACK and K39 showed high sensitivity and specificity in tests with human and canine sera. However, the search for vaccine and diagnosis antigens of VL is incessant, and the current stage of technological development with the possibility of using proteomic approach to identify hundreds of immunogenic antigens simultaneously is attractive. Thus, we chose aproteomic approach combined with in silico analyzes to identify new antigens of L. (L.) chagasi. Our proteomic analysis were the first to use amastigotes purified from hamster spleen and, furthermore, is the first study to map the antigenicity of infection by L. (L.) chagasi using sera from dogs of acute and chronic phase. All the selected protein bywestern blot-2DE (WB-2DE) were mapped to B cell epitopes by the BepiPred software and subjected to immunoassay through cellulose membranes resulting in the selection of 25 peptide with high potential for serological diagnosis of VL. In parallel, we used the technique of DIGE to compare the differential expression of proteins between the amastigote and promastigote forms of L. (L.) chagasi and 40 proteins were identified, 25 increased in promastigotes, 5 increased in amastigotes and 10 with similar expression in both forms. Moreover, all the proteins identified in the work by both DIGE as by WB 2DE have been mapped to T cell epitopes using the software NetCTL allowing the selection of potential candidates for vaccine development. Finally, to complete the analysis prediction for B cells, the same immunogenic proteins identified by WB-2DE were mapped using others two programs to predict epitopes (ABCPred and BCPred) and after additional analysis of immunoassays, more 23 peptides were identified as potential diagnostic antigens in the LVC. These new peptides, together with 25 previously identified, were subjected to immuno-enzymatic assays that resulted in 10 antigens withhigh sensitivity and specificity, even greater than that found by the EIE-LVC kit, which is recommended by the Ministry of Health, which also failed to detect asymptomatic dogs. Therefore, a large-scale analysis of L. (L.) chagasi through proteome was performed to identify B and T cell epitopes potentially useful in developing diagnostic tests and vaccines.