Immunization with the Amastigote Surface Protein-2 (ASP-2) andTrans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages ofTrypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge withT. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels ofT. cruzi-specific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+T cell response. Immunization with either vaccine protected dogs against challenge withT. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.