Kinetics of CD8⁺ T-cell mediated immune responses specific for sub-dominant epitopes in C57BL/6 mice.

<p>Groups of C57BL/6 mice were challenged or not i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. At the indicated days, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide TsKb-18 or TsKb-20 was determined as described in the Methods Section. The results represent the mean of 4 mice±SD per group. Asterisks denote statistically significant differences when we compared <i>T. cruzi</i> challenged with control mice (<i>P</i><0.05). ND = Not done. Results are representative of two or more independent experiments.</p

Kinetics of specific CD8⁺ T-cell mediated immune responses following challenge with <i>T. cruzi.</i>

<p>Groups of C57BL/6 mice were challenged or not i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Panels A to E - At the indicated days, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide VNHRFTLV was determined as described in the Methods Section. The results represent the mean of 4 mice±SD per group. Asterisks denote statistically significant differences when we compared <i>T. cruzi</i> challenged with control mice (<i>P</i><0.05). Panel F- At the indicated days, IFN-γ producing spleen cells specific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of peptide-specific spot forming cells (SFC) per 10⁶ splenocytes±SD (n = 4). Results are representative of two or more independent experiments.</p

Specific cytotoxicity in C57BL/6 mice challenged with irradiated or non-irradiated trypomastigotes of <i>T. cruzi.</i>

<p>Groups of C57BL/6 mice were challenged or not i.p. with 10³ or 10⁴ irradiated or non-irradiated bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. A) Fifteen days after challenge, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of 4 mice±SD per group. B) Fifteen days after challenge, IFN-γ producing spleen cells specific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of SFC per 10⁶ splenocytes±SD (n = 4). Asterisks denote statistically significant differences (<i>P</i><0.05) when we compared mice challenged with irradiated or non-irradiated trypomastigotes of <i>T. cruzi</i>. Results are representative of two independent experiments.</p

Trypomastigote-induced parasitemia in C57BL/6 mice challenged with different doses of trypomastigotes of <i>T. cruzi.</i>

<p>C57BL/6 mice were infected i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Parasitemia was followed daily from days 0 to 14 after challenge. The results represent the mean of 5–6 mice±SD. At the peak of infection, the parasitemia of mice infected with each different dose was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) 10²×10³, Non-Significant (NS); ii) 10²×10⁴, <i>P</i><0.01; iii) 10²×10⁵, <i>P</i><0.01; iv) 10³×10⁴, <i>P</i><0.05; v) 10³×10⁵, NS; vi) 10⁴×10⁵, NS. Results are representative of two independent experiments.</p

Specific cytotoxicity in WT or genetically deficient mice challenged with <i>T. cruzi.</i>

<p>Groups of WT C57BL/6 (n = 4), WT 129 mice (n = 4), MHC-II KO (n = 4), perforin KO (n = 8), CD4 KO (n = 4), IL-12 KO (n = 4), and IFN-I receptor KO (n = 4) were challenged or not i.p. with 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Ten days after challenge, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of the above indicated number of mice±SD per group. The <i>in vivo</i> cytotoxicity was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) WT C57BL/6×MHC-II KO (<i>P</i><0.01); ii) WT C57BL/6×Perforin KO (<i>P</i><0.01); iii) WT C57BL/6×CD4 KO (<i>P</i><0.01); iv) WT C57BL/6×IL-12 KO (NS); v) WT 129×IFN-I receptor KO (NS). Results are representative of two or more independent experiments.</p

Immunization with the hybrid DEC-ASP2 mAb induces IFN-γ production and CD4⁺ T cell proliferation.

<p>BALB/c mice were immunized with two doses of the hybrid mAbs or the rec. ASP2 protein as described in materials and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of rec. ASP2 or ovalbumin, as a control. (A) The number of IFN-γ producing cells was detected by ELISPOT and each bar represents the mean ± SD of triplicates of pooled splenocytes. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. (B) The percentage of CD3⁺CD4⁺ T cells that proliferated after 5 days of re-stimulation with the rec. ASP2 or ovalbumin is shown. Bars represent the mean ± SD of pooled animals performed in triplicates. Percentages of proliferating cells in the absence of any stimulus were subtracted from the stimulated samples. Experiment was analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graph is representative of 2 (A) or 3 (B) independent experiments.</p

Mapping the specific T-cell epitope responsible for the response to the ASP-2 protein.

<p>BALB/c mice were immunized with two doses of the hybrid mAbs or the rec. ASP2 protein as described in materials and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of the peptides FESRDMGKTWTEAIGTLSGV or WVMSQPGVRLYKIFRVGALIT. (A) The number of IFN-γ producing cells was detected by ELISPOT and the bars represent the mean ± SD of pooled animals performed in triplicates. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. (B) The percentage of CD3⁺CD4⁺ T cells that proliferated after 5 days of re-stimulation with each peptide is shown. Bars represent the mean ± SD of pooled animals performed in duplicates. Percentages of proliferating cells in the absence of any stimulus were subtracted from the stimulated samples. Experiments were analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graphs are representative of 2 experiments.</p

Screening of the peptide pools comprising ASP-2 amino acids 261–380.

<p>BALB/c mice were immunized with two doses of the αDEC-ASP2 or Iso-ASP2 mAbs as described in material and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of the three peptide pools (5 μg/mL) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117778#pone.0117778.t001" target="_blank">Table 1</a>. The number of IFN-γ producing cells was detected by ELISPOT and the bars represent the mean ± SD of pooled animals performed in triplicates. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. Experiment was analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graph is representative of 2 experiments.</p

Production and characterization of hybrid mAbs.

<p>(A) Schematic representation of the full length ASP-2 protein and of the portion fused to the αDEC205 and Iso mAbs. (B) Approximately 1 μg of each mAb or recombinant protein were separated under reduced conditions. Gel was stained with Coomassie Blue dye. The heavy (HC) and light (LC) chains of αDEC-ASP2, Iso-ASP2 or αDEC-CS (used as control) and rec. ASP2 protein are shown. (C) Western blotting using the same concentration as in (B) and the anti-ASP-2 mAb K₂2. MW, molecular weight marker in kDa.</p

Aminoacid sequences and localization within the ASP-2 protein of the peptide pools used in this study.

<p>Aminoacid sequences and localization within the ASP-2 protein of the peptide pools used in this study.</p