Authors

S Teixeira¹; G Burle-Caldas¹; RS Fernandes¹; JT Castro¹; I Vieira¹; AF Braz¹; NS Hojo-Souza¹; NA Santos¹; AP Fernandes¹; RT Gazzinelli¹;  ¹ Departamento de Bioquimica e Imunologia and Centro de Tecnologia de Vacinas, Universidade Federal de Minas Gerais, Brazil

Discussion

Genomic studies allowed us to investigate virulence factors that are essential for survival of intracellular parasites, among them, several surface proteins from Trypanosoma cruzi and Leishmania spp. Using RNAi, we have shown that amastins are essential factors involved in amastigote interaction with the parasitophorus vacuole (PV) within infected macrophage. Using CRISPR-Cas9, we generated T. cruzi knockout (KO) mutants in which genes encoding active Trans-sialidases (TS) were disrupted and showed that the transfer of sialic acid from the host to the parasite surface is essential for parasite survival in mice. TS are encoded by the largest T. cruzi gene family with more than 1,000 genes, but only 16 of them encode proteins with an active catalytic site. Disruption of active TS genes does not affect epimastigote growth in the insect vector or the parasite's capacity to invade cells but resulted in impaired differentiation of intracellular amastigotes into trypomastigotes and parasite egress. In vitro infection of HeLa cells with TS KO mutants resulted in significantly lower levels of IL-1-β, IL-1-α and IL-6 compared to cells infected with WT parasites. Importantly, when inoculated into mice, TS KO parasites were unable to establish infection even in immunodeficient animals. Like other T. cruzi surface proteins expressed in the mammalian stage, TS are anchored at the parasite membrane through a GPI anchor that contains inositol-phosphorylceramide (IPC). Like TS mutants, parasites with disrupted IPC synthase gene have no growth defect but have impaired capacity to differentiate into metacyclic trypomastigotes and are unable to infect mice. Mice immunized with these attenuated T. cruzi strains are fully protected against a challenge infection with a virulent strain. Besides using genetically attenuated parasites as vaccinal strains we showed that mice can be also protected against infection with T. cruzi and L. amazonensis if they are immunized with recombinant proteins containing sequences from TS genes or from a conserved Leishmania antigen. Finally, we have also tested lipid nanoparticle-encapsulated RNAs encoding TS as well as RNA encoding a L. amazonenis antigen and showed that the LNP-RNA immunization protocol represents a promising vaccine strategy for preventing leishmaniasis and Chagas disease. Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Fundação de Apoio a Pesquisa do Estado de Minas Gerais-FAPEMIG and Instituto Nacional de Ciencia e Tecnologia de Vacinas-INCTV (Brasil) and The global Challenges Research Fund-GCRF (UK)