Trans-sialidase-based vaccine candidate protects against Trypanosoma cruzi infection, not only inducing an effector immune response but also affecting cells with regulatory/suppressor phenotype

Abstract

Prophylactic and/or therapeutic vaccines have an important potential to control Trypanosoma cruzi (T. cruzi)infection. The involvement of regulatory/suppressor immune cells after an immunization treatment and T. cruzi infection has never been addressed. Here we show that a new trans-sialidase-based immunogen (TSf) was able to confer protection, correlating not only with beneficial changes in effector immune parameters, but also influencing populations of cells related to immune control. Regarding the effector response, mice immunized with TSf showed a TS-specific antibody response, significant delayed-type hypersensitivity (DTH) reactivity and increased production of IFN-γ by CD8+ splenocytes. After a challenge with T. cruzi, TSf-immunized mice showed 90% survival and low parasitemia as compared with 40% survival and high parasitemia in PBS-immunized mice. In relation to the regulatory/suppressor arm of the immune system, after T. cruzi infection TSf-immunized mice showed an increase in spleen CD4+ Foxp3+ regulatory T cells (Treg) as compared to PBS-inoculated and infected mice. Moreover, although T. cruzi infection elicited a notable increase in myeloid derived suppressor cells (MDSC) in the spleen of PBS-inoculated mice, TSf-immunized mice showed a significantly lower increase of MDSC. Results presented herein highlight the need of studying the immune response as a whole when a vaccine candidate is rationally tested.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; Trypanosoma cruzi; foxp3 regulatory T cells; myeloid-derived suppressor cells; trans-sialidase; vaccine.

Figure 1. Development of the TSf fragment

A. The TSf fragment obtained showed 90% aminoacidic identity as aligned with the TS indexed in the GenBank AJ276679 [15]. B. A representative picture of the TSf fragment as compared to the full-length TS protein. C. Assessment of TSf protein purification on SDS polyacrylamide gel electrophoresis: lane 1 non-induced colony, lane 2: TSf protein that was purified in Ni-NTA resin-based affinity chromatography and dialyzed against urea 0,5M; lane 3: TSf protein expressed but not purified, lane 4: low molecular weight marker.

Figure 2. Immunological parameters of the immune response elicited by TSf-ISPA immunization

A. BALB/c mice were immunized with TSf-ISPA, PBS, ISPA-alone or TSf-alone and plasma samples were analyzed for TSf-specific IgG2a and IgG1 antibodies titers by ELISA. Antibodies titers were negligible in plasma from PBS, TSf-alone or ISPA-alone immunized mice. B. Trypomastigote lysis assay was performed using serum from TSf-ISPA, ISPA-alone, TSf-alone and PBS-inoculated mice. C. DTH response in immunized mice: Footpad thickness was measured before and 48 h after inoculation of 5 μg of TSf seven days after completion of immunization schedule. Results are expressed as “delta mm” which was the difference between the values obtained after and before inoculation. D. Representative dot plots from CD8+ splenocytes from TSf-ISPA immunized and PBS-inoculated mice cultured in the presence of T. cruzi homogenate. E. Splenocytes from TSf-ISPA immunized mice showed higher production of IFN-γ within CD8+ cultured cells. F. Splenocytes from TSf-ISPA immunized mice showed an increase in the mean fluorescence intensity (MFI) of IFN-γ within CD8+ cultured cells. Data are expressed as means + standard deviations. Results shown are representative of 2-3 independent experiments (n = 4-10 mice per group), *p < 0.05, Mann-whitney test.

Figure 3. Changes in Foxp3+ CD4+ T cell populations and CD11b+ GR-1+ cells in the spleen of Balb/c mice, seven days after the last immunization

A. FACS analysis of the expression of intracellular Foxp3+ within CD4+ cell in control (PBS-inoculated), TSf-alone, ISPA-alone and TSf-ISPA inoculated mice B. Absolute number of CD4+Foxp3+ cells (x10⁶) in the spleen of inoculated mice C. FACS analysis of the expression of CD11b+ GR-1+ MDSC cells in control (PBS-inoculated), TSf-alone, ISPA-alone and TSf-ISPA inoculated mice D. Absolute number of CD11b+ GR-1+ cells (x10⁶) in the spleen of inoculated mice. Data are expressed as means + standard deviations (n = 4 per group). The results are representative of two independent experiments, p < 0,05, Mann-whitney test.

Figure 4. Parasitemia and survival rates in immunized mice after T. cruzi challenge

Mice immunized with TSf-ISPA, TSf, ISPA and PBS were challenged with 1000 trypomastigotes of the Tulahuen strain 14 days after the last immunization. A. Parasitemia from PBS-inoculated (PBS, white bar), TSf-ISPA immunized (TSf-ISPA, black bar), TSf-immunized (dark gray bar) and ISPA-immunized (gray bar) mice at day 21 post infection. B. Survival rates are shown for TSf-ISPA immunized and infected mice (TSf-ISPA), PBS-inoculated and infected mice (PBS), TSf-immunized and infected mice (TSf), ISPA-immunized and infected mice (ISPA). The results are representative of two independent experiments (n = 6-10 mice per group), *p < 0,05, Mann-Whitney test was used for parasitemia analysis, Mantel-Cox Long rank test was used for survival analysis between TSf-ISPA vs PBS mice.

Figure 5. Production of IFN-γ within CD4+ and CD8+ in vitro

Splenocytes from control non-infected (control), PBS-inoculated and infected mice (PBS Tc) and TSf-ISPA immunized and infected mice (TSf-ISPA Tc) were cultured with T. cruzi homogenate during 40h. PMA and monesin were added to the culture four hours before flow cytometry staining with anti-CD4, anti-CD8 and anti-IFN-γ antibodies. A. Percentage of IFN-γ production within splenic CD4+ T cells B. MFI of IFN-γ within CD4+ cells. C. Percentage of IFN-γ production within splenic CD8+ T cells, D. MFI of IFN-γ within CD8+ cells (n = 4/group). Data shown are representative of two independent experiments, *p < 0.05, Mann-whitney test.

Figure 6. Changes in spleen CD4+ Foxp3+ cells 21 days post T. cruzi infection

Expression of CD4 and intracellular Foxp3 was analyzed by FACS. A. the Figure shows representative dot plots used for selecting the CD4 region based on side-sideward scatter (SSC) in the Y-axis and CD4 expression in the X axis. B. Foxp3+ expression was assessed in the CD4 region in non-infected (control), PBS-inoculated and T. cruzi infected (PBS Tc), and TSf-ISPA immunized and T. cruzi infected mice (TSf-ISPA Tc). C. Percentage of Foxp3+ expression within CD4+ cells among groups D. Absolute number of CD4+Foxp3+ cells (10⁶). Data are expressed as means + standard deviations (n = 4 per group). *, p < 0.05, Mann-whitney test. Data shown are representative of two independent experiments.

Figure 7. Changes in spleen CD11b+ GR-1+ cells at day 21 post T. cruzi infection

Expression of CD11b+ and GR-1+ was analyzed by FACS in control non-infected (control), PBS-inoculated and infected mice (PBS Tc) and TSf-ISPA immunized and infected mice (TSf-ISPA Tc). A. percentage of CD11b+ GR-1+ cells. B. Absolute number of CD11b+ GR-1+ cells (x10⁶). Data are expressed as means + standard deviations (n = 4 per group). *, p < 0.05, Mann-whitney test. Data shown are representative of two independent experiments.

Figure 8. Changes in spleen CD11b+ Ly6G+/- cells at day 21 post T. cruzi infection

Splenocyte suspensions were prepared and expression of Ly6C-FITC, Ly6G-PE and CD11b-PerCP-Cy5.5 expression was analyzed by flow cytometry in control non-infected mice (control), PBS-inoculated and infected mice (PBS Tc) and TSf-ISPA immunized and infected mice (TSf-ISPA Tc). A. Representative dot plots showing alterations in the percentage of spleen CD11b+ cells among groups. B. Representative dot plots showing alterations in the percentage of CD11b+Ly6G+ (G-MDSC) and CD11b+Ly6G- (M-MDSC) cells among groups. C. Percentage of G-MDSC CD11b+Ly6G+ cells, D. Absolute number of G-MDSC CD11b+Ly6G+ cells (x10⁶). E. Percentage of M-MDSC CD11b+ Ly6G- cells. F. Absolute number of M-MDSC CD11b+Ly6G- cells (x10⁶). Data are expressed as means + standard deviations (n = 4 per group). *, p < 0.05, Mann-whitney test. Data shown are representative of three independent experiments.

Figure 9. Tissue pathology

Picrosirius red staining of hearts after five months of infection. Arrows indicate examples of red staining considered as collagen fibers. Magnification 100x. (n = 3-4 mice analyzed per group). *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).