B10.A mice were infected with 10⁴ T. cruzi blood parasites. A/S mice were immunized with pIgSPCl.9 followed by AdASP-2 and injected i.m. at 0 and 3 weeks, respectively. Control mice were naïve or injected with pcDNA3 followed by pcDNA3/Adβ-gal. A- Two weeks after infection or the final immunizing dose, splenic cells were re-stimulated in vitro in the presence of medium or the indicated peptides at a final concentration of 10 µM. The number of splenic IFN-γ spot-forming cells (SFC) was estimated by ex vivo ELISPOT assay. B- In vivo cytotoxic activity was estimated by injecting each mouse with syngeneic CFSE-labeled splenic cells coated with or without 2 µM of the indicated peptide. Results are expressed as means ± SD of 4 mice per group and are representative of experiments performed at least twice with similar results. Asterisks denote that the number of SFC or in vivo cytotoxicity were significantly higher when compared to naïve or pcDNA3/Adβ-gal injected mice (P<0.01, one-way ANOVA).

A/Sn mice were immunized with pcDNA3, pIgSPCl.9, pIgSPTAWETGQA or simultaneously with the last two (pIgSPCl.9 and pIgSPTAWETGQA). Immunization consisted of 3 doses of 100 µg of DNA each given by the i.m. route in the tibialis anterioris three weeks apart. A- Two weeks after the final immunizing dose, mice were challenged i.p. with 150 bloodstream trypomastigotes. Two weeks after infection, splenic cells were re-stimulated in vitro in the presence of medium only or the indicated peptides at a final concentration of 10 µM. The number of splenic IFN-γ spot-forming cells (SFC) was estimated by ex vivo ELISPOT assay. B- In vivo cytotoxic activity was estimated by injecting each mouse with syngeneic CFSE-labeled splenic cells coated with or without 2 µM of the indicated peptide. Results are expressed as means ± SD of 4 mice per group and are representative of experiments performed at least twice with similar results. Asterisks denote that the number of SFC, or the in vivo cytotoxicity, were significantly higher when compared to SFC found in naïve or pcDNA3/Adβ-gal-injected mice (P<0.01). C- Parasitemia for each mouse group is represented as mean ± SD (n = 5–7). Asterisks denote that mice from groups immunized with pIgSPCl.9 or pIgSPTAWETGQA or both had significantly lower parasitemia (P<0.01) than animals injected with pcDNA3. The curves of parasitemia of animals immunized with pIgSPCl.9 (squares) or pIgSPTAWETGQA (triangles) are superimposed. D- Kaplan-Meier curves for the survival of mouse groups immunized and challenged as described above (n = 5–7). Mice from groups immunized with pIgSPCl.9 or pIgSPCl.9/pIgSPTAWETGQA survived significantly longer than animals injected with pcDNA3 or pIgSPTAWETGQA (P<0.05, in all cases, LogRank test). Mice immunized with pIgSPTAWETGQA also survived longer than animals injected with pcDNA3 (P<0.05). No animals died after the 30^th day until they were euthanized. Results are representative of two independent experiments.

A/Sn mice were immunized as depicted in the legend of Fig. 5. Two weeks after the final immunizing dose, mice were challenged i.p. (Panels A and B) or s.c. (Panels C and D) with 150 bloodstream trypomastigotes. Parasitemia for each mouse group is represented as mean ± SD (n = 10 or 11). Asterisks denote that mice from groups immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWTEGQA had significantly lower parasitemia (P<0.01) than pcDNA3/Adβ-gal-injected animals. Panels B and D represent Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (n = 10 or 11). Mice immunized with pIgSPCl.9/AdASP-2 survived significantly longer than animals immunized with pIgSPTAWETGQA/AdTAWETGQA or pcDNA3/Adβ-gal (P = 0.01 or P<0.01, respectively). Mice immunized with pIgSPTAWETGQA/AdTAWETGQA survived significantly longer than pcDNA3/Adβ-gal-injected animals (P<0.01). Results are representative of two pooled experiments. No animals died after the 40^th day.

A- Schematic view of the primary structure of T. cruzi ASP-2. B- HeLa cells were infected for 48 h with trypomastigotes of the Y strain. After fixation, indirect immunofluorescence or DAPI staining were performed as described using MAb K22 and imaged using fluorescence microscopy [39]. Bar, 14 µM.

B10.A or A/Sn mice were infected or immunized as described in the legend of Fig. 2. Control mice were either naive mice or mice immunized with pCDNA3/Adβ-gal. Twenty one or fourteen days after infection or immunization, respectively, these mice had their splenic cells cultured in the presence of anti-CD107a and anti-CD28, with or without the peptides TEWETGQI, PETLGHEI or YEIVAGYI. After 12 h, cells were stained for CD8, IFN-γ, TNF-α, IL-2 and IL-10. Representative analyses (medians) are shown from four mice performed per experiment. A) Example of splenic CD8⁺ cells from B10.A naive mice cultivated in vitro in the presence of medium alone (Medium) or with the indicated peptides and stained for expression of IFN-γ and TNF-α. B, C and D) Examples of splenic CD8⁺ cells from B10.A infected mice cultivated in vitro in the presence of medium alone (Medium) or with the indicated peptides and stained for expression of: B) IFN-γ and TNF-α; C) IFN-γ and IL-2; D) IFN-γ and IL-10. E and F) Examples of splenic CD8⁺ cells from mice immunized with pcDNA3/Adβ-gal cultivated in vitro in the presence of medium alone (Medium) or with the indicated peptides and stained for surface mobilization of CD107a and expression IFN-γ (panel E) or expression of IFN-γ and TNF-α (panel F). G and H) Examples of splenic CD8⁺ cells from mice immunized with pIgSPCl.9/AdASP-2 cultivated in vitro in the presence of medium alone (Medium) or with the indicated peptides and stained for surface mobilization of CD107a and expression IFN-γ (panel G) and expression IFN-γ and TNF-α (panel H). I and J) Determination of multifunctional CD8⁺ cells from mice immunized with pIgSPCl.9/AdASP-2 cultivated in vitro in the presence of the indicated peptides and stained for surface mobilization of CD107a and expression IFN-γ and TNF-α.

A/Sn mice were primed i.m. with 100 µg of plasmids pcDNA3, pIgSPCl.9 or pIgSPTAWETGQA. Three weeks later, these mice were boosted i.m. with 2×10⁸ pfu Adβ-gal, AdASP-2 or AdTAWETGQA. A- Two weeks after the last dose, splenic cells were re-stimulated in vitro in the presence of medium only or the indicated peptides at a final concentration of 10 µM. The number of splenic IFN-γ spot forming cells (SFC) was estimated by ex vivo ELISPOT assay. B- In vivo cytotoxic activity was estimated by injecting each mouse with syngeneic CFSE-labeled splenic cells coated with or without 2 µM of the indicated peptide. Results are expressed as mean ± SD of 4 mice per group and are representative of experiments performed at least twice with similar results. Asterisks denote that the number of SFC or in vivo cytotoxicity were significantly higher when compared to SFC found in naïve or pcDNA3/Adβ-gal injected mice (P<0.01). C- Fourteen days after the last dose, these mice had their splenic cells cultured in the presence of anti-CD28 and Medium or the indicated peptides. After 12 h, cells were stained for CD8, IFN-γ and TNF-α. Examples of splenic CD8⁺ cells from immunized mice. Representative analyses (medians) are shown from four mice performed per experiment.

A/Sn mice were immunized as described in the legend of Fig. 5. pIgSPTAWETGQA/AdTAWTEGQA had significantly lower parasitemia (P<0.01) than pcDNA3/Adβ-gal-injected animals. Before and after challenge, mice were treated as described in Methods section with rat IgG (control) or anti-CD8 MAb. The parasitemia for each mouse group is represented as mean ± SD (n = 6). Asterisks denote that mice from groups immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWTEGQA and treated with Rat IgG had significantly lower parasitemia (P<0.01) than vaccinated mice treated with anti-CD8 (Panels A and B). Panels C and D represent Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (n = 6). Mice immunized with pIgSPCl.9/AdASP-2 or pIgSPTAWETGQA/AdTAWETGQA and treated with Rat IgG survived significantly longer than vaccinated animals treated with anti-CD8 (P<0.01 in both cases).