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Abstract: We recently described that vaccination of mice with a gluthatione S transferase fusion protein representing amino acids 261–500 of the Amastigote Surface Protein-2 efficiently cross-primed protective CD8+ T cells against a lethal challenge with the human protozoan parasite Trypanosoma cruzi. In this study, we initially established that this protective immunity was long lived. Subsequently, we studied the importance of TLR9 agonist CpG ODN 1826, TLR4 and CD4+ T cells for the generation of these protective CD8+ T cells. We found that: (i) the TLR9 agonist CpG ODN 1826 improved the efficiency of protective immunity; (ii) TLR4 is not relevant for priming of specific CD8+ T cells; (iii) CD4+ T cells are critical for priming of memory/protective CD8+ T cells. [Copyright &y& Elsevier]