For stimulation of spleen cells and maintenance of T-cell clones, we added to the medium 2-mercaptoethanol, sodium pyruvate, nonessential amino acid solutions, vitamin, 10% fetal calf serum (FCS; Hyclone, Logan, Utah), and 30 U of recombinant human interleukin-2 (IL-2; kindly provided by Hoffmann-LaRoche) per ml. peptides, we determined a CD8 epitope recognized by several clones as being represented by BIO-acetoxime amino acids IYNVGQVSI. The antiparasitic activity of a CD4 Th1 and a CD8 Tc1 clone was assessed in vitro. CD4 or CD8 T cells significantly inhibited development in infected macrophages or fibroblasts, respectively. We concluded that DNA vaccine efficiently generates potentially protective CD4 Th1 and CD8 Tc1 cells specific for a antigen, therefore reinforcing the possibility of using this strategy for developing a preventive or therapeutic vaccine against Chagas disease. Despite the significant reduction in transmission observed in the last 20 years, Chagas disease (American trypanosomiasis) is still a major problem for most Latin American countries, afflicting between 16 and 18 million individuals. After contact with trypomastigotes of infection in mice or humans, parasite-specific CD4 BIO-acetoxime and CD8 T cells are activated. These T cells are important factors for host resistance, as genetically modified mice that do not have either CD4 or CD8 cells are significantly more susceptible to infection than wild-type animals (34, 43; reviewed in references 11 and 42). Several bodies of evidence strongly suggest that immunoprotection is mediated by CD4 Th1 and CD8 Tc1 cells (1, 27, 48). Their antiparasitic effect is mediated in part by their ability to secrete gamma interferon (IFN-), a cytokine that inhibits the development of infection. Initial reports indicated that the administration of recombinant IFN- to mice reduced the parasitemia and prevented death due to infection (29). IFN- participates in naturally acquired immunity, as treatment with neutralizing monoclonal antibodies BIO-acetoxime (MAbs) significantly reduced the resistance to acute infection (40). IFN- increases host resistance by contributing to the production of nitric oxide (NO), a potent inhibitor of intracellular development of (26, 39). The critical role for IFN- in the induction of NO production during experimental infection was confirmed in studies using genetically modified mice that lack the IFN- receptor. This mouse strain fails to produce NO during the course of the disease and was extremely susceptible to infection (17). Recently, we and others reported that immunization BIO-acetoxime with plasmids containing genes encoding for antigens expressed on the surface of infective trypomastigotes of induced protective immunity against experimental Chagas disease (6, 49). CD14 We used the gene encoding for the catalytic domain of an enzyme called infection (6). In view of data suggesting an important role of IFN–producing T cells in resistance to experimental infection, we considered it important to evaluate whether DNA vaccination with the TS gene could efficiently generate these potentially protective CD4 Th1 and CD8 T cytotoxic type 1 (Tc1) cells. For this purpose, we performed a detailed analysis of the type of T-cell immune response in animals immunized with BIO-acetoxime the TS gene. MATERIALS AND METHODS Parasites and animals. Trypomastigotes of the Y strain were obtained from tissue culture as described in reference 38. Female 5- to 8-week-old BALB/c mice used in this study were purchased from University of S?o Paulo. Plasmid generation, purification, and immunization. Plasmid 154/13 was created as described by Costa et al. (6). It contains the nucleotide sequence coding for amino acids 1 to 678 of TS inserted into the commercially available plasmid pcDNA3 (Invitrogen, San Diego, Calif.). As control, we used plasmid pcDNA3 alone. Plasmids were produced in DH5 and purified on cesium chloride density gradients according to standard protocols (35). DNA concentration was estimated at 260 nm. DNA was diluted in saline at a concentration of 1 1 mg/ml, and 50 l was injected per leg. BALB/c mice were immunized according to the protocol described by.
For stimulation of spleen cells and maintenance of T-cell clones, we added to the medium 2-mercaptoethanol, sodium pyruvate, nonessential amino acid solutions, vitamin, 10% fetal calf serum (FCS; Hyclone, Logan, Utah), and 30 U of recombinant human interleukin-2 (IL-2; kindly provided by Hoffmann-LaRoche) per ml
