Chagas disease is a parasitic disease caused by the protozoan Trypanosoma cruzi (T. cruzi) which is prevalent in all the American continent including the US, afflicting humans as well as canines. The prognosis for infected dogs is poor due to the lack of effective treatment. In addition, they are a domestic reservoir and can increase the risk of human infection. We have developed a vaccine formulation for the immunotherapy of T. cruzi infection, based on two T. cruzi antigens, Tc24.C4 and TSA-1.C4 with a TLR4 adjuvant. This therapeutic vaccine was safe and effective in a pilot field study in dogs with natural infection with T. cruzi, leading to a strong decrease parasitemia, and at least delaying cardiac disease progression. Therefore, we aimed to evaluate a therapeutic formulation based on Tc24.C4 and TSA-1.C4 recombinant proteins in a larger clinical field trial to prevent chagasic cardiomyopathy in chronic naturally T. cruzi-infected dogs, as well as elucidated the protective immune response induced by the vaccine. The therapeutic efficacy will be measured with the blood parasite burden, and analysis of the cardiac structure and function by transthoracic echocardiographic and electrocardiographic (ECG) recordings, respectively. We will also characterize the humoral and cellular immune response by measuring antigen-specific IgG antibodies in plasma and antigen-specific CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMC). Transcriptomic analysis of PBMC stimulated with the antigens will be a key step for understanding the protective immune response and specific genes involved in the efficacy of the immunotherapeutic vaccine.