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. 2020 Mar 23;88(4):e00908-19.
doi: 10.1128/IAI.00908-19. Print 2020 Mar 23.

Induction of Effective Immunity against Trypanosoma cruzi

Tere Williams  1 , Ignacio Guerrero-Ros  1 , Yanfen Ma  1 , Fabiane Matos Dos Santos  2 , Philipp E Scherer  3 , Ruth Gordillo  3 , Aline Horta  4 , Fernando Macian  1 , Louis M Weiss  1   5 , Huan Huang  6
Affiliations

Induction of Effective Immunity against Trypanosoma cruzi

Tere Williams et al. Infect Immun. .

Abstract

Chagas disease, caused by Trypanosoma cruzi, is a major public health issue. Limitations in immune responses to natural T. cruzi infection usually result in parasite persistence with significant complications. A safe, effective, and reliable vaccine would reduce the threat of T. cruzi infections; however, no suitable vaccine is currently available due to a lack of understanding of the requirements for induction of fully protective immunity. We established a T. cruzi strain expressing green fluorescent protein (GFP) under the control of dihydrofolate reductase degradation domain (DDD) with a hemagglutinin (HA) tag, GFP-DDDHA, which was induced by trimethoprim-lactate (TMP-lactate), which results in the death of intracellular parasites. This attenuated strain induces very strong protection against reinfection. Using this GFP-DDDHA strain, we investigated the mechanisms underlying the protective immune response in mice. Immunization with this strain led to a response that included high levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), as well as a rapid expansion of effector and memory T cells in the spleen. More CD8+ T cells differentiate to memory cells following GFP-DDDHA infection than after infection with a wild-type (WT) strain. The GFP-DDDHA strain also provides cross-protection against another T. cruzi isolate. IFN-γ is important in mediating the protection, as IFN-γ knockout (KO) mice failed to acquire protection when infected with the GFP-DDDHA strain. Immune cells demonstrated earlier and stronger protective responses in immunized mice after reinfection with T. cruzi than those in naive mice. Adoptive transfers with several types of immune cells or with serum revealed that several branches of the immune system mediated protection. A combination of serum and natural killer cells provided the most effective protection against infection in these transfer experiments.

Keywords: Trypanosoma cruzi; immunity; immunization.

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Figures

FIG 1
FIG 1
The GFP-DDDHA strain provides cross-protection and does not cause persistent infection. (A) C3H mice were inoculated with the GFP-DDDHA Tulahuen strain and then challenged with the Brazil strain. Immunized mice did not develop parasitemia, while nonimmunized mice developed high parasitemia (n = 10). (B) Immunized mice survived the challenge, while nonimmunized mice all died during the acute infection phase (n = 10). (C) Histology (hematoxylin and eosin [H&E] staining) of heart tissues at day 14 postchallenge demonstrates that nonimmunized C3H mice infected with the Brazil strain had numerous amastigotes within myocardial tissue, associated with extensive myocarditis, while GFP-DDDHA-immunized mice infected with the Brazil strain had no amastigotes and did not exhibit myocarditis. ×20 magnification. Bar, 25 μm. (D) C57BL/6 mice immunized with the GFP-DDDHA Tulahuen strain and then challenged with half a million wild-type Tulahuen strain parasites. Similarly to C3H mice, immunized mice did not develop parasitemia and survived, while nonimmunized mice developed high parasitemia and all died in the acute phase (n = 10). (E) C57BL/6 mice immunized with the GFP-DDDHA Tulahuen strain did not have persistent infection, as real-time PCR did not detect T. cruzi DNA in heart and adipose tissues (n = 5). See Materials and Methods for details. Data presented are one representative example of two separate experiments. Both experiments produced similar results.
FIG 2
FIG 2
Mice immunized with the GFP-DDDHA strain exhibited high systemic IFN-γ and TNF-α levels. C57BL/6 mice immunized with the GFP-DDDHA Tulahuen strain from 7 to 30 dpi had serum cytokines measured using a multiplex assay. Note the elevated levels of IFN-γ and TNF-α in the GFP-DDDHA mice. C, control; I, inoculated with the GFP-DDDHA strain. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired two-tailed t test between immunized and unimmunized mice; n = 5). Data presented are one representative example of two separate experiments. Both experiments produced similar results.
FIG 3
FIG 3
CD4+ and CD8+ T-cell expansion during immunization with the GFP-DDDHA Tulahuen strain. (A) C57BL/6 mice were inoculated with either the GFP-DDDHA Tulahuen strain or a WT strain and then sacrificed at the indicated time points following infection. The numbers of CD4+ and CD8+ T cells in the spleen were measured by fluorescence-activated cell sorting (FACS) and compared to those populations in uninfected mice. Mice infected with the WT strain died after 28 days postinoculation. The graph represents the average ± standard error of the mean (SEM) of data obtained from 3 mice per time point and condition. Data were analyzed by analysis of variance (ANOVA) with Tukey’s posttest (days 7 to 28) or unpaired two-tailed t test (days 75 to 120). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (infected versus uninfected mice). Data presented are one representative example of three separate experiments. All three experiments produced similar results. (B) Flow cytometry analysis show that CD8+ T cells in splenocytes from C57BL/6 mice with the GFP-DDDHA Tulahuen strain expressed much higher TNF-α levels at 21 dpi than those of uninfected mice and mice infected with the WT strain. (C) Plots from flow cytometry analysis. Data were analyzed by ANOVA with Tukey’s posttest **, P < 0.01; ***, P < 0.001 (n = 5). Data presented are one representative example of three separate experiments. All three experiments produced similar results.
FIG 4
FIG 4
The GFP-DDDHA strain favors development of T-cell memory. C57BL/6 mice were infected with wild-type (WT) T. cruzi or the GFP-DDDHA strain. Mice were then sacrificed at different time points following infection, and the distribution of naive (CD44− CD62Lhigh), effector memory (CD44+ CD62Llow), and central memory (CD44+ CD62Lhigh) CD4+ and CD8+ T cells in the spleen was measured by FACS and compared to the distribution of those populations in uninfected mice and WT strain-infected mice. (A) FACS analysis of the distribution of the populations of CD4+ T cells (left). Plots from the data (right, n = 3). (B) FACS analysis of the distribution of the populations of CD8+ T cells (left). Plots from the data (right, n = 3). Note that immunization with the GFP-DDDHA strain stimulates more CD8+ T-cell effector memory than that in WT strain infection. TEM, effector memory T cells; TCM, central memory T cells. Data were analyzed by ANOVA with Tukey’s posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data presented are one representative example of three separate experiments. All three experiments produced similar results.
FIG 5
FIG 5
Immunization with the GFP-DDDHA strain enhances IFN-γ expression during antigen reencounter. (A) C57BL/6 mice were inoculated with the GFP-DDDHA Tulahuen strain and sacrificed at the indicated time points. Single-cell suspensions of splenocytes were then left unstimulated or were stimulated with T. cruzi lysates or anti-CD3 antibodies. IFN-γ production was measured by enzyme-linked immunosorbent assay (ELISA). Graphs represent the average ± SEM of data obtained from 3 mice per time point and condition. Data were analyzed by ANOVA with Tukey’s posttest (days 7 to 14) or unpaired two-tailed t test (days 75 to 120). In summary, all uninfected cells stimulated with antibodies were significantly different (P < 0.001) from resting cells or cells stimulated from lysates, whereas in GFP-DDDHA strain-infected mice no differences were found between cells stimulated with lysates or with antibodies (except at day 120; P < 0.01), and both of them are different from the resting condition (P < 0.001) at any time point. All GFP-DDDHA strain-infected cells stimulated with lysates were different from uninfected cells stimulated with lysates (P < 0.001). **, P < 0.01; ***, P < 0.001. Data presented are one representative example of three separate experiments. All three experiments produced similar results. (B) Immunized C57BL/6 mice at 42 days postimmunization were reinfected with half a million WT Tulahuen parasites at 12 and 24 h postinfection. Splenocytes were analyzed by FACS and gated on CD4+ and CD8+ T cells, and the percentage of cells expressing IFN-γ was determined. Note that CD4+ and CD8+ T cells from immunized mice expressed much higher IFN-γ levels than those of nonimmunized mice. Data were analyzed with an unpaired two-tailed t test. **, P < 0.01; ***, P < 0.001. Data presented are one representative example of three separate experiments. All three experiments produced similar results. (C) Schematic representation of IFN-γ knockout mouse immunization, treatment with recombinant IFN-γ, and then challenge with WT parasites. Eight-week-old IFN-γ knockout mice and age-matched C57BL/6 mice were immunized with the GFP-DDDHA strain and then challenged with a WT infection. To allow protection to establish and to specifically analyze the role of IFN-γ in the secondary response to lethal infection, IFN-γ KO mice were administered recombinant IFN-γ (1.2 μg [1,000 U] per /mouse) twice weekly i.p. for 5 weeks starting 1 day postadministration of the GFP-DDDHA strain. After 5 weeks, treatment with IFN-γ was stopped, and the next day, following IFN-γ removal, the immunized IFN-γ-treated IFN-γ KO mice and the immunized C57BL/6 mice, as well as a group of nonimmunized C57BL/6 mice, were infected with 5 × 105 wild-type Tulahuen strain parasites. (D) Survival curve of IFN-γ knockout mice and control mice. Nonimmunized C57BL/6 mice and the immunized treated IFN-γ KO mice died with this lethal infection by day 17, while the immunized C57BL/6 mice all survived. This suggests that IFN-γ is important in mediating protection after immunization. Data presented are one representative example of two separate experiments. Both experiments produced similar results. (E) IFN-γ knockout mice died during GFP-DDDHA strain infection. Eight-week-old IFN-γ knockout mice were infected with 5 × 103 GFP-DDDHA Tulahuen strain parasites and received TMP-lactate treatment on day 7 postinfection. All mice died within 35 days postinfection, indicating that IFN-γ is essential for the development of immunity against T. cruzi (n = 10).
FIG 6
FIG 6
Immunization with the GFP-DDDHA strain induces early and strong recall responses in immune cells. Immunized C57BL/6 mice at 42 days postimmunization were reinfected with half a million WT Tulahuen parasites at 12 and 24 h postinfection. Splenocytes were analyzed by FACS and gated on dendritic cells, monocytes, neutrophils, and CD4+ and CD8+ T cells, and the percentage of cells expressing activation or effector molecules was determined. Note that dendritic cells and monocytes from immunized mice have early strong responses, by expressing CD86, CD40, MHCII, iNOS, TNF-α, and IL-12, compared to those of nonimmunized mice. A subset of effector memory T cells (CD44+ CD62L− CD4+ and CD44+ CD62L− CD8+) from immunized mice quickly and strongly expressed granzyme B compared to nonimmunized mice, and neutrophils expressed a higher level of TNF-α than that of nonimmunized mice. Data were analyzed with an unpaired two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data presented are one representative example of three separate experiments. All three experiments produced similar results.
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