ASP-2/Trans-sialidase chimeric protein induces robust protective immunity in experimental models of Chagas’ disease

Figures

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  Fig. 1. Construction of the chimeric protein TRASP and immunization schedule. a The immunogenic regions of TS, ASP-2 and three SAPA-repeats were joined into a single sequence, giving rise to a chimeric protein named TRASP. b TRASP and the isolated fragments rTS and rASP-2 were expressed in E. coli and purified. Proteins’ purification was confirmed by SDS-PAGE. After that, the potential of TRASP as a vaccine was tested in murine (c) and canine (d) models.

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  Fig. 2. Analysis of prolonged protection. a, b Immunized mice were challenged with 104 blood trypomastigotes of T. cruzi Y strain at 30 (a) or 90 (b) days after the last vaccine dose, and parasitemia was followed by 15 days post-infection (DPI), mean ± SEM. Mice survival was evaluated until 25 DPI in both time points (c, d). e–h The levels of IFN-γ (e, f) and IL-10 (g, h) produced by splenocytes were evaluated after culture with specific stimuli at 30 (e, g) and 90 (f, h) days post-immunization. Additionally, anti-TRASP IgG isotypes were evaluated in mice sera at 30 (i) and 90 (j) days post-immunization. The statistical analysis of parasitemia and cytokine levels was carried out using two-way ANOVA and Tukey’s multiple comparisons test. The statistical analysis of survival was performed using the log-rank test. Data were representative of two independent experiments (n = 4–6 mice per group). *p < 0.05; ***p < 0.001; ****p < 0.0001.

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  Fig. 3. Immunophenotyping of CD8+ T lymphocytes in immunized mice. After 90 days of immunization, mice splenocytes were stimulated with TRASP for 18 h or left unstimulated (RPMI only) and were then characterized by flow cytometry. a UMAP projection of IFN-γ production by CD8+ T lymphocytes compartments, separated in Naïve (CD8+ CD44− CD62L−), effector/effector memory (CD8+ CD44+ CD62L−), and central memory (CD8+ CD44+ CD62L+). b, c The frequency of IFN-γ-producing CD8+ effector/effector memory (b) or central memory T cells (c). The statistical analysis was carried out using paired t-test. Data were representative of two independent experiments (n = 3 mice per group). ns non-significant.

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  Fig. 4. Characterization of CD4+ T and B lymphocytes elicited by vaccination. In vitro-stimulated splenocytes were immunophenotyped by flow cytometry after 90 days of immunization. a UMAP projection showing the production of IFN-γ by CD4+ Th1 cells (CD4+ CXCR3+). b Frequency of CD4+ CXCR3+ IFN-γ+ c CD4+ Tfh (CD4+ ICOS+ PD-1+ Bcl6+), and d class-switched memory B cells (CD19+ IgD− CD27+). The statistical analysis was carried out using paired t-test. Data were representative of two independent experiments (n = 3 mice per group). ns non-significant.

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  Fig. 5. Importance of T and B lymphocytes in the protection of immunized mice against T. cruzi infection. β-2m−/− (a, b), IFN-γ−/− (c, d), μMT (e), and C57BL/6 (f) mice were immunized with TRASP-pICLC and challenged with T. cruzi. Parasitemia (a, c, e, f) and survival (b, d) were measured for 9 to 20 days, depending on the model. Statistics of parasitemia was calculated by using two-way ANOVA and Tukey’s multiple comparisons test. Data were representative of two independent experiments, mean ± SEM (n = 4–6 mice per group). *p < 0.05; ***p < 0.001; ****p < 0.0001.

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  Fig. 6. Humoral and cellular immune response induced by vaccination in dogs. Elevated levels of IgG isotypes anti-ASP-2 (a–c) and anti-TS (d–f) were detected in the sera from dogs immunized with TRASP-pICLC in different time points. g–i IFN-γ production was analyzed in PBMC’s culture supernatant stimulated with TRASP at T3 (g), T4 (h), and T6 (i). j–o The proliferation of CD4+ and CD8+ T cells under specific stimuli of ASP-2 (j, k), TS (l, m), and TRASP (n, o) was evaluated at T0, T3, and T4. Intracellular staining of IFN-γ on CD4+ (p) and CD8+ (q) T lymphocytes was assessed by flow cytometry and is represented by index (PMA/Control). All the statistical analysis was carried out using two-way ANOVA and Tukey’s multiple comparisons test, except data from IFN-γ detection, which was analyzed by Kruskal–Wallis and Dunn’s multiple comparisons test. Mean ± SEM. n = 6–9 dogs per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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  Fig. 7. Evaluation of protection against T. cruzi infection. The parasite load in the heart was measured by qPCR (a) and the apex section, the most parasite-abundant, was selected to investigate the difference between the groups (b, c). It was analyzed the percentage of positive animals (b) and the number of parasites/100 ng of gDNA (c). Histopathological analysis showed an increase in inflammatory process in non-vaccinated dogs, represented by the number of cells per microscope field (d) and photomicrographs of the groups PBS (e), Poly-ICLC (f), DNA∅ + AdβGal (g), TRASP-pICLC (h), and DNA TS/ASP + Ad TS/ASP (i). a, d Mean ± SEM, c median ± range. The statistical analysis of the parasite load was assessed by Mann–Whitney test and histopathology was analyzed by one-way ANOVA and Tukey’s multiple test. n = 6–9 dogs per group. *p < 0.05; **p < 0.01.