DNA sequences encoding CD4+ and CD8+ T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene - PubMed (original) (raw)

DNA sequences encoding CD4+ and CD8+ T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene

A E Fujimura et al. Infect Immun. 2001 Sep.

Abstract

Immunization of BALB/c mice with a plasmid containing the gene for Trypanosoma cruzi trans-sialidase (TS) induced antibodies that inhibited TS enzymatic activity, CD4+ Th1 and CD8+ Tc1 cells, and protective immunity against infection. We used this model to obtain basic information on the requirement of CD4 or CD8 or B-cell epitopes for an effective DNA-induced immunity against T. cruzi infection. For that purpose, mice were immunized with plasmids containing DNA sequences encoding (i) the entire TS protein, (ii) the TS enzymatic domain, (iii) the TS CD4+ T-cell epitopes, (iv) the TS CD8+ T-cell epitope, or (v) TS CD4+ and CD8+ T-cell epitopes. Plasmids expressing the entire TS or its enzymatic domain elicited similar levels of TS-inhibitory antibodies, gamma interferon (IFN-gamma)-producing T cells, and protective immunity against infection. Although the plasmid expressing TS CD4 epitopes was immunogenic, its protective efficacy against experimental infection was limited. The plasmid expressing the CD8 epitope was poorly immunogenic and provided little protective immunity. The reason for the limited priming of CD8+ T cells was due to a requirement for CD4+ T cells. To circumvent this problem, a plasmid expressing both CD4+ and CD8+ T-cell epitopes was produced. This plasmid generated levels of IFN-gamma-producing T cells and protective immunity comparable to that of the plasmid expressing the entire catalytic domain of TS. Our observations suggest that plasmids expressing epitopes recognized by CD4+ and CD8+ T cells may have a better protective potential against infection with T. cruzi.

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Figures

FIG. 1

FIG. 1

Schematic view of the primary structure of T. cruzi TS.

FIG. 2

FIG. 2

Immune responses of mice immunized with pcDNA3-TS and p154/13. BALB/c mice were immunized as described in detail in Materials and Methods with pcDNA3-TS (▴), p154/13 (■), or pcDNA3 (●). Fourteen days after the last immunization, blood samples were collected and the sera were assayed for the presence of antibodies to TS by ELISA using polystyrene wells coated with recombinant TS-cat (A), or for the presence of TS-inhibitory antibodies (B). The results represent the mean values obtained from eight mice ± the standard deviations (SD). Pooled spleen cells obtained from three mice immunized with pcDNA3-TS, p154/13, or pcDNA3 were expanded for 6 days in the presence of irradiated A20J-TS cells. (C) The expanded cells were restimulated in the presence of A20J-pcDNA3, A20J-TS, A20J cells, or A20J cells coated with 1 μM TS359-367 peptide (A20J-TS359–367). IFN-γ was estimated in supernatants collected after 18 h. Results are expressed as averages of triplicate cultures ± SD.

FIG. 3

FIG. 3

Trypomastigote-induced parasitemia and mortality in mice immunized with pcDNA3-TS, p154/13, and pcDNA3. BALB/c mice were immunized with pcDNA3-TS, p154/13, or pcDNA3. Three weeks after the last immunization, mice were challenged i.p. with 6,500 bloodstream trypomastigotes. (A) Course of parasitemia, estimated as described in Materials and Methods. The results represent the mean values obtained from eight mice ± the standard deviations. At the peak of infection (day 7), the parasitemia of mice immunized with pcDNA3-TS or p154/13 was significantly lower than the parasitemia of control animals injected with pcDNA3 (p<0.001). (B) Kaplan-Meier curves for survival of eight mice immunized with the indicated plasmid(s). Statistically significant survival was observed in mice immunized with pcDNA3-TS or p154/13 compared to animals that received pcDNA3 (P < 0.05).

FIG. 4

FIG. 4

Immune responses of mice immunized with p154/13 and pΔ154/13. BALB/c mice were immunized with p154/13, pΔ154/13, or pcDNA3. Fourteen days after the last immunization, blood samples were collected and the sera were assayed for the presence of antibodies to recombinant TS-cat by ELISA (A) or for the presence of TS-inhibitory antibodies (B). The results represent the mean value obtained from eight mice ± the standard deviations (SD). Pooled spleen cells obtained from three mice immunized with p154/13, pΔ154/13, or pcDNA3 were expanded for 6 days in the presence of irradiated A20J-TS cells. (C) The expanded cells were restimulated in the presence of A20J-pcDNA3, A20J-TS, A20J cells, or A20J cells coated with 1 μM TS359-367 peptide (A20J-TS359-367). IFN-γ was estimated in supernatants collected after 18 h. Results are expressed as averages of triplicate cultures ± SD.

FIG. 5

FIG. 5

Trypomastigote-induced parasitemia and mortality in mice immunized with p154/13, pΔ154/13, and pcDNA3. BALB/c mice were immunized with p154/13, pΔ154/13, or pcDNA3. Three weeks after the last immunization, mice were challenged i.p., with 6,500 bloodstream trypomastigotes. (A) Course of infection, with the mean values obtained from eight mice ± the standard deviations. At the peak of infection (day 8), the parasitemia of mice immunized with each plasmid were compared. The results were as follows: (i) p154/13 versus pcDNA3, P < 0.001; (ii) p154/13 versus pΔ154/13, _P_ = 0.011; (iii) pΔ154/13 versus pcDNA3, _P_ > 0.05. (B) Kaplan-Meier curves for survival of eight mice immunized with the indicated plasmid. Statistically significant survival was observed in mice immunized with p154/13 compared to animals that received pΔ154/13 or pcDNA3 (P < 0.05). The number of mice immunized with pΔ154/13 or pcDNA3 that survived infection was not significantly different (_P_ > 0.05).

FIG. 6

FIG. 6

Immune response, trypomastigote-induced parasitemia, and mortality of mice immunized with p154/13, pCD8-epitope, and pcDNA3. BALB/c mice were immunized with p154/13, pCD8-epitope, or pcDNA3. Fourteen days after the last immunization, pooled spleen cells obtained from three mice immunized with each plasmid were expanded for 6 days in the presence of irradiated A20J-TS cells. (A) The expanded cells were restimulated in the presence of A20J-pcDNA3, A20J-TS, A20J cells, or A20J cells coated with 1 μM peptide TS359-367 (A20J-TS359-367). IFN-γ was estimated in supernatants collected after 18 h. Results are expressed as averages of triplicate cultures ± the standards of deviation (SD). Three weeks after the last immunization, mice were challenged i.p. with 6,500 bloodstream trypomastigotes. (B) Course of infection, with mean values obtained from eight mice ± SD. At the peak of infection (day 7), the parasitemia of mice immunized with each plasmid was compared. The results were as follows: (i) p154/13 versus pcDNA3, P < 0.001; (ii) p154/13 versus pCD8-epitope, _P_ < 0.01; (iii) pCD8-epitope versus pcDNA3, _P_ < 0.01. (C) Kaplan-Meier curves for survival of eight mice immunized with the indicated plasmid. Statistically significant survival was observed in mice immunized with p154/13 compared to animals that received pCD8-epitope or pcDNA3 (_P_ < 0.05). The number of mice immunized with pCD8-epitope or pcDNA3 that survived infection was not significantly different (_P_ > 0.05).

FIG. 7

FIG. 7

Depletion of CD4+ T cells significantly reduces TS-specific antibodies and IFN-γ-secreting CD4+ and CD8+ T cells induced by immunization with p154/13. BALB/c mice were treated for three consecutive days with anti-CD4, anti-CD8, or rat-IgG. One and 22 days later, these mice were immunized i.m. with 100 μg of p154/13. In parallel, control mice received 100 μg of pcDNA3. (A) Fourteen days after the last immunization, blood samples were collected and the sera were assayed for the presence of antibodies to recombinant TS-cat by ELISA The results represent the mean values obtained from four mice ± the standard deviations (SD). (B) Pooled spleen cells obtained from two mice immunized with p154/13 and treated with anti-CD4, anti-CD8, or rat-IgG were expanded for 6 days in the presence of irradiated A20J-TS cells. The expanded cells were restimulated in the presence of A20J-pcDNA3, A20J-TS, A20J cells, or A20J cells coated with 1 μM peptide TS359-367 (A20J-TS359-367). IFN-γ was estimated in supernatants collected after 18 h. Results are expressed as the average of triplicate cultures ± SD.

FIG. 8

FIG. 8

Immune responses of mice immunized with p154/13 and pΔ154/13-CD8. BALB/c mice were immunized with p154/13, pΔ154/13-CD8, or pcDNA3. Fourteen days after the last immunization, blood samples were collected and the sera were assayed for the presence of antibodies to recombinant TS-cat by ELISA (A) or for the presence of TS-inhibitory antibodies (B). Results represent the mean values obtained from eight mice ± the standard deviations (SD). (C) Pooled spleen cells obtained from three mice immunized with p154/13, pΔ154/13-CD8, or pcDNA3 were expanded for 6 days in the presence of irradiated A20J-TS cells. The expanded cells were restimulated in the presence of A20J-pcDNA3, A20J-TS, A20J cells, or A20J cells coated with 1 μM TS359-367 peptide (A20J-TS359-367). IFN-γ was estimated in supernatants collected after 18 h. Results are expressed as the average of triplicate cultures ± SD.

FIG. 9

FIG. 9

Trypomastigote-induced parasitemia and mortality in mice immunized with p154/13, pΔ154/13-CD8, and pcDNA3. BALB/c mice were immunized with p154/13, pΔ154/13-CD8, or pcDNA3. Three weeks after the last immunization, mice were challenged i.p. with 6,500 bloodstream trypomastigotes. (A) Course of infection and the mean values obtained from eight mice ± standard deviations. At the peak of infection (day 8). the parasitemia of mice immunized with each plasmid was compared. The results were as follows: (i) p154/13 versus pcDNA3, P < 0.001; (ii) p154/13 versus pΔ154/13-CD8, _P_ > 0.05; (iii) pΔ154/13-CD8 versus pcDNA3, P < 0.001. (B) Kaplan-Meier curves for survival of eight mice immunized with the indicated plasmid. Statistically significant survival was observed in mice immunized with p154/13 or pΔ154/13-CD8 compared to animals that received pcDNA3 (P < 0.01).

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