Desenvolvimento e avaliação de vacina contra tripanossomatídeos composta por sequências peptídicas selecionadas por phage display

Abstract

Leishmaniasis and Chagas disease are among the most important neglected tropical diseases in the world, affecting millions of people. Although control through vaccination is promising, no effective vaccine is available. In this work, the selection of vaccine candidates was performed using the phage display technique. This technique consists on fusing peptides to the surface of a bacteriophage capsid, producing libraries able to display a wide repertoire of linear and conformational random epitopes, which can be selected through their affinity to different types of ligands, such as antibodies. Thus, three infections were performed in BALB/c mice using a strain of Leishmania donovani genetically deficient for the centrin gene (LdCEN-/-), while C57BL/6 mice were infected twice with the Y strain of Trypanosoma cruzi. Increase in antibody reactivity was evaluated through ELISA assay, and anti-LdCEN-/- and anti-T. cruzi class G immunoglobulins were precipitated and purified. To identify specifically binding peptides, selection cycles were performed using peptide libraries with 12, 15 and 17 amino acids. The most reactive phage clones had their DNA sequenced and translated into amino acids. These peptides were synthesized in cellulose membrane using the "SPOT Synthesis" technique and also in a soluble form, in order to investigate their reactivity against Leishmania spp. and T. cruzi sera. Phage clones expressing the selected peptides were used to immunize BALB/c mice, which had their post- vaccination cellular immune response profile evaluated by flow cytometry. After challenge infection by L. infantum, L. amazonensis and T. cruzi, parasite load was quantified and possible histopathological changes were evaluated. Sequencing identified ten different peptides, of which seven were selected by anti-LdCEN-/- IgG and three by anti-T. cruzi IgG. All peptides synthesized both in membrane and in soluble form were recognized by at least one sera pool evaluated, demonstrating its potential as a vaccine component. The immunization scheme induced increasing and specific antibody production, in addition to stimulating a cellular memory profile, IFN-ɣ increase and IL-10 reduction. A reduction in parasite load was observed in mice challenged by L. infantum, L. amazonensis and T. cruzi, combined with greater morphological preservation of all analyzed organs. As future perspective, the selected peptide sequences will be inserted into the genome of a genetically deficient and protective Leishmania major strain (LmCEN-/-). Validating the use of a live vaccine as an immunogenic peptide carrier could then allow the insertion of other genes of interest. This could lead to the development of an effective multicompetent vaccine not only against leishmaniasis and Chagas disease, but also several other pathologies, both parasitic and non-parasitic.