Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi

Abstract

To stimulate both local and systemic immune responses against Trypanosoma cruzi, Salmonella enterica serovar Typhimurium aroA was exploited as a DNA delivery system for cruzipain (SCz). In a murine model we compared SCz alone (GI) or coadministered with Salmonella carrying a plasmid encoding granulocyte-macrophage colony-stimulating factor (GII), as well as protocols in which SCz priming was followed by boosting with recombinant cruzipain (rCz) admixed with either CpG-ODN (GIII) or MALP-2, a synthetic derivative of a macrophage-activating lipopeptide of 2 kDa from Mycoplasma fermentans (GIV). The results showed that protocols that included four oral doses of SCz (GI) elicited mainly a mucosal response characterized by immunoglobulin A (IgA) secretion and proliferation of gut-associated lymphoid tissue cells, with weak systemic responses. In contrast, the protocol that included a boost with rCz plus CpG (GIII) triggered stronger systemic responses in terms of Cz-specific serum IgG titers, splenocyte proliferation, gamma interferon (IFN-gamma) secretion, and delayed-type hypersensitivity response. Trypomastigote challenge of vaccinated mice resulted in significantly lower levels of parasitemia compared to controls. Protection was abolished by depletion of either CD4+ or CD8+ T cells. Parasite control was also evident from the reduction of tissue damage, as revealed by histopathologic studies and serum levels of enzymes that are markers of muscle injury in chronic Chagas' disease (i.e., creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Enhanced release of IFN-gamma and interleukin-2 was observed in GI and GII upon restimulation of splenocytes in the nonparasitic phase of infection. Our results indicate that Salmonella-mediated delivery of Cz-DNA by itself promotes the elicitation of an immune response that controls T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.

FIG. 1.

Humoral immune responses in mice immunized using Salmonella as a delivery system for a Cz-based DNA vaccine. Two weeks after the last immunization, sera and intestinal lavage fluids were collected and assayed by ELISA for the presence of rCz-specific antibodies. (A) Antigen-specific serum IgG titers. (B) Antigen-specific sIgA titers in intestinal lavage fluids. Results are expressed as Cz-specific sIgA titers per μg of total IgA. (C) Antigen-specific serum IgG isotype titers. Results are expressed as endpoint titers. The SEMs are indicated by vertical lines. The results are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 2.

Cellular responses stimulated in vaccinated mice. (A) Proliferative responses of mesenteric lymph node (MLN), Peyer's patch (PP), and spleen cells from immunized mice. Fifteen days after the last immunization, cells were collected and stimulated with 10 μg/ml of rCz. Four days later, 1 μCi of [³H]thymidine per well was added and the counts per minute were measured 18 h later. Results are expressed as the ratio of values from stimulated and nonstimulated samples (proliferation index). Each bar represents the group mean ± SEM. *, P < 0.05; **, P < 0.01. (B) Detection of IFN-γ produced by spleen cells of vaccinated mice. Cells were harvested 2 weeks after the last immunization and cultured by duplicate in the presence of rCz (10 μg/ml). Supernatant fluids were collected 48 h later and assayed in duplicate by capture ELISA for the presence of IFN-γ. **, P < 0.01. (C) Detection of IFN-γ-secreting CD8⁺ T cells. Spleen CD8⁺ T-cell-enriched preparations recovered from immunized mice were incubated for 16 h in the presence of rCz, and secretion of IFN-γ was determined. Then, the number of IFN-γ-producing cells was determined by ELISPOT. Results are presented as spot-forming units per 10⁵ cells (mean ± SEM of triplicate wells), which were subtracted from the values obtained from nonstimulated cells. **, P < 0.01; ***, P < 0.001. The results are representative of three independent experiments.

FIG. 3.

Delayed-type hypersensitivity test in vaccinated mice. Mice were immunized as indicated previously and intradermally challenged on day 13 after the last immunization with 5 μg of either nCz in the right footpad (A) or rCz in the left footpad (B). The results are expressed as the difference between the thickness of the footpad 48 h after and before the inoculation. Each bar represents the group mean (n = 6) ± SEM. The results are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 4.

Parasitemia levels during the acute phase of T. cruzi infection. Mice were challenged 15 days after the last immunization with 50 trypomastigotes of the RA strain by the intraperitoneal route. Parasitemia was determined by counting in a Neubauer chamber the number of trypomastigotes in 5 μl of fresh blood collected from the tail vein every 2 to 3 days. The results are representative of three independent experiments. Each bar represents the group mean ± SEM.

FIG. 5.

Cytokine secretion by spleen cells from vaccinated mice after challenge with T. cruzi. One hundred days after challenge with trypomastigotes (chronic stage of the infection), spleen cells from mice were aseptically removed. Cultures were performed in duplicate in the presence of whole soluble antigens of T. cruzi (F105; 10 μg/ml). Supernatants were collected 48 h later and assayed by capture ELISA for IFN-γ (A) and IL-2 (B). Each bar represents the group mean ± SEM. The results presented are representative of three independent experiments. *, P < 0.05; **, P < 0.01. Spleen cells from all groups exhibited demonstrable concanavalin A responsiveness (not shown).

FIG. 6.

T. cruzi challenge in vaccinated mice depleted of CD4⁺ or CD8⁺ T cells. Intraperitoneal challenge with 50 trypomastigotes of the RA strain was performed 15 days after the last immunizing dose. SCz-vaccinated mice were treated with rat IgG, anti-CD4, or anti-CD8 MAb. Parasitemia was determined by counting in a Neubauer chamber every 2 to 3 days (A). Mortality was recorded daily (B). Results are representative of three independent experiments.

FIG. 7.

(A to C) Serum levels of myopathy-linked enzyme markers and histopathological studies on skeletal muscle from immunized mice after challenge. Blood was collected on day 100 postinfection with trypomastigotes, and assays were performed to determine the levels of CK (A), LDH (B), and AST (C) by spectrophotometry. The bars represent the average of six determinations; the SEMs are indicated by vertical lines. The results are representative of three independent experiments. *, P < 0.05; **, P < 0.01. (D) Tissue sections were collected at the same time and stained with hematoxylin and eosin. Histological sections of skeletal muscle from mice immunized with Salmonella as a Cz-DNA delivery system, showing limited focal cellular infiltrates (panel 1), and reactions in control groups (panels 2, 3, and 4), showing severe inflammatory foci and fiber muscle infarction. Magnification: ×40 (panels 1, 2, and 4) or ×5 (panel 3).