CD8+-T-cell-dependent control of Trypanosoma cruzi infection in a highly susceptible mouse strain after immunization with recombinant proteins based on amastigote surface protein 2

Abstract

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8(+) T cells, but not CD4(+) T cells, and was associated with the presence of CD8(+) T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8(+)-T-cell-dependent protective immunity against T. cruzi infection.

FIG. 1.

Schematic representation and SDS-PAGE analysis of the recombinant proteins based on the ASP-2 antigen. (A) Schematic representation of the recombinant proteins. (B) SDS-PAGE of recombinant proteins stained with Coomassie blue. The amount of protein loaded was approximately 1 μg per lane.

FIG. 2.

Trypomastigote-induced parasitemia in A/Sn mice immunized with recombinant protein His-65kDa, GST-P1-P3, GST-P4-P7, or His-25kDa. Groups of six mice were immunized at 0, 2, and 4 weeks with His-65kDa, GST-P1-P3, GST-P4-P7, His-25kDa, or adjuvant alone. Each mouse received i.p. 25 μg of recombinant protein adsorbed to alum in the presence of 10 μg of CpG ODN 1826. Two weeks after the last dose was administered, mice were challenged i.p. with 250 bloodstream trypomastigotes. The parasitemia for each individual mouse is represented. The peak parasitemias of mice immunized with His-65kDa or GST-P4-P7 were lower than the parasitemias of animals immunized with adjuvant only, GST-P1-P3, or His-25kDa (P < 0.01 in all cases by Tukey's HSD test). No significant difference was observed when we compared the peak parasitemias of mice immunized with His-65kDa or GST-P4-P7 (P > 0.05). Also, no difference was found when we compared the peak parasitemias of mice immunized with GST-P1-P3 or His-25kDa or adjuvant alone (P > 0.05).

FIG. 3.

Trypomastigote-induced mortality in A/Sn mice immunized with recombinant proteins His-65kDa, GST-P1-P3, GST-P4-P7, or His-25kDa. The graph shows the Kaplan-Meier curves for the survival of 14 to 16 mice per group. Statistical analysis revealed significant differences in the survival of mice immunized with GST-P4-P7 compared to that of mice immunized with other recombinant proteins or adjuvant only (P < 0.05 in all cases by the log rank test). Mice immunized with His-65kDa displayed a survival rate significantly higher than that for other mouse groups with the exception of the animals that received GST-P4-P7 (P < 0.05 in all cases). Mice immunized with His-25kDa had a survival rate higher than that for the control animals that received adjuvant only (P < 0.05). In contrast, the survival of mice injected with GST-P1-P3 or adjuvant only was not statistically different (P > 0.05).

FIG. 4.

Trypomastigote-induced parasitemia and mortality in mice immunized with GST-P4-P7 and treated with anti-CD4 or anti-CD8 antibodies. Groups of five or six mice were immunized as described in the legend to Fig. 2 with the recombinant protein GST-P4-P7 or adjuvant alone. Mice immunized with the recombinant protein were treated with rat IgG, anti-CD4, or anti-CD8 as described in Materials and Methods. (A) The peak parasitemias in animals immunized with GST-P4-P7 and treated with rat IgG or anti-CD4 were significantly lower than the parasitemias of immunized animals treated with anti-CD8 (P < 0.01 in all cases by Tukey's HSD test). The values of parasitemia of mice immunized with GST-P4-P7 and treated with anti-CD8 were not different from those of control mice injected with adjuvant only (P > 0.05). (B) The graph shows Kaplan-Meier curves for the survival of 10 to 14 mice per group. Animals immunized with GST-P4-P7 and treated with rat IgG or anti-CD4 survived longer than mice treated with anti-CD8 (P < 0.0001 by the log rank test). There was no significant statistical difference between the survival curves of mice vaccinated with the recombinant protein GST-P4-P7 and treated with anti-CD8 or control animals injected with adjuvant alone (P > 0.05).