Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation

Abstract

Background: Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.

Methodology/principal findings: We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.

Conclusion: Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

Fig 1. Specific CD8⁺ T cell-mediated immune response in L. braziliensis-infected BALB/c mice.

Target cells were pulsed with L. braziliensis peptides (A) AYLASCDFI, WYLATHSLI, AYIDGHVTI and SYMGYFQNI and (B) GYVGIVVAL, KYQHSTEML, IYVSYADLY and VYLSFGFRL. Target cells were then transferred to L. braziliensis infected mice. In vivo cytoxoxicity was analyzed in spleen (white squares) and in draining lymph nodes (red squares) of recipient mice, by flow cytometry, as described in Materials and Methods. Data are shown individually.