Knockout of the dhfr-ts gene in Trypanosoma cruzi generates attenuated parasites that induce specific CD8+ T cells in infected mice and confer long term protection against a virulent challenge

PÉREZ BRANDAN C, PADILLA AM, XU D, CARDOZO R, TARLETON RL, BASOMBRÍO MA

Ciudad de Santa Fe – Argentina

Otro; XXIII Reunión Científica Anual, Sociedad Argentina de de Protozoología; 2009

Sociedad Argentina de Protozoología

It has been shown that the naturally attenuated TCC strain of Trypanosoma cruzi, confers partial protection against a virulent challenge. However, the factors involved in the attenuation of this strain are still unknown and persistence of these parasites in immunocompetent mice has been observed. We generated monoallelic mutant parasites for the dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) in the TCC strain based on the rationale that these mutant parasites would became auxotrophic for thymidine and would not persist in the infected host. We have previously reported the impairment in growth of dhfr-ts-/+ epimastigotes and the induction of a lower number of specific CD8+ T cell response in C57BL/6 infected mice when compared with TCC wild type infected ones, as demonstrated by staining with MHC class I tetramers containing the T. cruzi specific peptide TSKB20. Here we report the results of immunization/challenge assays performed in Balb/c and C57BL/6 mice. In two independent short term assays we observed no remarkable differences in the protective effect of wild type versus mutant parasites measured by parasitemia. Skeletal muscle samples were collected to assess histological damage after challenge of vaccinated and non vaccinated animals. We then carried out a long term immunization assay where mice where challenge 370 days after immunization. At 300 days post vaccination, CD8+ T cells specific for T. cruzi expressed the CD127 memory marker, suggesting the presence of memory T cells in these infected mice. We then challenged these mice in the footpad with fluorescent parasites and measured fluorescence intensity at the site of infection for 13 days. The results indicate that, a year after the immunization, these animals are able to control the challenging parasite load at the site of infection. This study suggests that it is possible to generate genetically attenuated T. cruzi parasites that generate CD8+ memory cells and confer protection against further infections with T. cruzi. Supported by FONCYT grant to MAB and NIH grant to RLT.