Fator de elongação 1α (EF-1α) de Leishmania amazonensis: investigação do seu papel na interação parasito-hospedeiro e do seu potencial vacinal

Abstract

The elongation factor 1 alpha (eEF1A or EF-1α) is a protein canonically involved in protein translation in all eukaryotic cells, for which non-canonical functions have been described in different species. In Leishmania spp., EF-1α is exported in microvesicles through non-classical pathways and acts as a virulence factor by activating SHP-1 phosphatase activity in macrophages, inducing inactivated phenotypes and contributing to the infection's progression. Like other virulence factors, Leishmania spp.'s EF-1α is recognized by antibodies from recovered patients and stimulates peripheral blood mononuclear cells of these patients to produce pro-inflammatory cytokines. Additionally, its potential for use as a vaccine has been demonstrated for L. donovani. However, several aspects of the non-canonical functions of EF-1α from Leishmania spp. remain to be elucidated, and there are currently no published data on EF-1α from L. amazonensis. In this study, the presence of 7 copies of EF-1α in the genome of the PH8 strain of L. amazonensis was demonstrated. In silico analysis of the protein's primary structure revealed an interaction motif with SH2 domains unique to Leishmania (160-VMYAQS-165), as well as consensus sequences recognized by SH3 domains of other signaling molecules, which could be potential therapeutic targets. Knocking down EF-1α encoding genes via CRISPR-Cas9 generated parasites with reduced gene copy number, transcription levels, and protein expression, but did not result in changes in morphology or cell growth. Knockdown parasites exhibited reduced capacity to infect macrophages and multiply in the amastigote form, but the mechanisms underlying these phenotypes still need to be investigated. Recombinant EF-1α from L. amazonensis produced in E. coli associated with adjuvants such as CpG + alum, Hiltonol®, CTVad1®, and Hiltonol® + CTVad1® induced both humoral and cellular immune responses and significantly reduced tissue parasitism in animals challenged with L. infantum, demonstrating the protein's vaccine potential for cross-protection against other species. The combination of adjuvants Hiltonol® + CTVad1® was also effective in combination with the antigen DTL10, indicating that using more than one adjuvant yields promising results in leishmaniasis protection. Consequently, a new antigen combining EF-1α and DTL10 proteins was proposed. In conclusion, this study demonstrated for the first time the importance of EF-1α as a virulence factor for L. amazonensis and provided valuable tools to advance understanding its function in the parasite-host relationship. The results also indicate that it’s promising to develop a vaccine by combining these antigens, in the form of an antigenic chimera, with new adjuvants as a strategy to obtain an optimized vaccine formulation in terms of efficacy and safety.