Combination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis

Abstract

The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals.

Immunogenicity of Potential CD4+ and CD8+ T Cell Epitopes Derived From the Proteome of Leishmania braziliensis

“…Molecules corresponding to the previously described top 10 15-mer peptides (23) were commercially synthesized (Genome Biotechnology, Brazil). Linear peptides were purified through high-performance liquid chromatography (HPLC), with a final purity >95%.…”

“…Cells from AD, PT [data derived from E Silva et al (23)], SC, and the control groups were labeled with CFDA-SE (carboxyfluorescein diacetate succinimidyl ester, Invitrogen, USA) in order to assess the level of cell proliferation induced by the peptides. The PBMCs (4 × 10 6 ) were resuspended in 1 ml of PBS supplemented with 2 µM of CFDA-SE and incubated at 37 • C for 10 min (the CFDA-SE concentration was previously titrated in order to prevent inhibition of cell proliferation or cell death).…”

“…It is estimated that each DC can express 10 6 -10 7 MHC Class II (MHC II) and 10 5 MHC Class I (MHC I) proteins (22). Considering the potential of L. braziliensis antigens and the lack of successful vaccines and approaches for their development, we have previously described the use of a combination of in silico methods in the search for potentially robust CD4 + and CD8 + T cell epitopes in the proteome of L. braziliensis (23). We have described a set of 15-mer peptides with an intrinsic potential to bind MHC complexes from major alleles present in human populations.…”

High-throughput identification of MHC class I binding peptides using an ultradense peptide array

“…However, these methods exploit our understanding of anchor residues and rely on previously generated peptide binding datasets for pattern recognition, and so are limited to MHC molecules whose binding repertoires have already been well studied, creating a self-fulfilling prophecy of sorts (6, 7). A different computational technique, molecular docking, uses the three-dimensional crystal structure of MHC molecules to predict peptides likely to bind (8, 9). While this method has the benefit of working for both MHC class I and II molecules, it still cannot serve as a substitute for in vitro binding assays.…”

Not All Antigens Are Created Equally: Progress, Challenges, and Lessons Associated with Developing a Vaccine for Leishmaniasis

“…A wide variety of adjuvants have been used in Leishmania research programs, and the importance of adjuvants within vaccines has been the subject of numerous review articles (13)(14)(15)(16). Simultaneous with the progress in adjuvant technologies, advances in bioinformatics and molecular techniques have expanded the number of potential Leishmania-specific targets for use within a vaccine (17)(18)(19). A considerable number of Leishmania antigens have been tested as subunit protein or DNA vaccine candidates against various Leishmania species, but variable results have meant that very few antigens have advanced to human or canine clinical trials (20).…”