Glicosilfosfatidilinositol como alvo terapêutico e vacinal contra doenças parasitárias intracelulares

Abstract

Glycosylphosphatidylinositol (GPI) is a glycolipid complex that anchors several proteins and glycoproteins to the cell membrane, considered an essential molecule for the interaction of several protozoa with their hosts. Parasite resistance is attributed to individuals with high anti-GPI antibody titers. Thus, the objectives of this study were to develop antibodies of single-chain variable fragments (scFv) selected by Phage Display against GPI of Plasmodium falciparum and to evaluate their ability to decrease the mortality of mice with cerebral malaria, as well as to neutralize parasitic cell invasion of broad intracellular protozoa. The success of the primary goal also led us to the development of scFv-binding peptides with greater affinity for GPI. The selection of P. falciparum GPI-binding scFv was performed from a Phage Display library containing an approximate diversity of 2x106 scFv combinatorial sequences. The expression and specificity of clones were analyzed by ELISA. The DNA extracted from the highest reactivity clone was subjected to sequencing and subsequent analysis of bioinformatics, with molecular modeling to predict the three-dimensional structure, as well as the complementarity determining regions (CDRs) and frameworks (FWRs). C57BL / 6 mice infected with P. berghei-ANKA were treated with the scFv clone and analyzes of parasitemia and mortality curve of the animals were performed. The reactivity of scFv with extracts of P. falciparum, Toxoplasma gondii and Trypanosoma cruzi was also evaluated, as well as its ability to prevent cell invasion. Finally, a new biopanning process was performed by Phage Display to select mimetic peptides to the GPI molecule of P. falciparum. The reactivity of phage-fused peptide clones with the selected scFv was evaluated by Phage-Elisa, and the most reactive clones were validated in the presence of serum from mice with cerebral malaria. Among selected scFv clones, eight (G1-G8) presented different sequences, and the expressed scFv G7 had the highest affinity to the P. falciparum GPI. G7 decreased the parasitemia of the animals and was able to protect animals from symptoms of cerebral malaria, significantly impacting survival. The G7 clone also presented high affinity for T. cruzi and T. gondii extracts, besides not being toxic to cells or to the parasites. After in vitro treatment, we have observed a reduction of parasites internalization when compared to the control group. Regarding the selection of mimetic peptides to GPI, 11 clones (F1, E1, F2, D2, C4, E4, D5, H5, A6, H6 and F7) presented different sequences, four of which (F1, E1, F2 and A6) were more reactive to G7, of which two (E1 and F1) distinguished serum from mice with malaria from the healthy ones. In summary, the broad spectrum GPI scFv G7 antibody presents great potential as a promising immunobiological drug that may be applied to the treatment of parasitic diseases, such as malaria, toxoplasmosis and Chagas disease. In addition, its binding peptides, mimetic to GPI, may also have theranostic potential, by acting in the near future both in diagnosis and as vaccine antigens.