Vaccine-linked chemotherapy with a low dose of benznidazole plus a bivalent recombinant protein vaccine prevents the development of cardiac fibrosis caused by Trypanosoma cruzi in chronically-infected BALB/c mice

Figures

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  Fig 1. Protective effect of the vaccine-linked chemotherapy. (A) Timeline for experimental infection. A total of 70 BALB/c mice were infected with 500 trypomastigotes of T. cruzi (H1 strain) by intraperitoneal injection. Four mice were included as non-infected control group and received only saline solution. At day 200 p.i mice were euthanized, and tissues samples were collected. (B) Survival curve. Survival was monitored during 200 days post-infection. (C) Cardiac parasite burden was quantified by quantitative real-time PCR. The dotted line represents the cut-off for the limit of detectable quantification (LOQ) based on serially diluted T. cruzi-enriched cardiac tissue DNA (1 parasite equivalents per 50 ng of DNA). Each point represents an individual mouse; horizontal lines denote median ± interquartile ranges values; significance calculated by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (D) Percentage fibrosis area for all experimental groups. The cardiac fibrosis was quantified from representative images of Masson´s trichrome-stained tissue sections using Image J software. Each point represents an individual mouse, horizontal lines denote means ± SD;…

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  Fig 2. Representative images of Masson´s trichrome stained-cardiac tissue from (A) non-infected, (B) infected untreated, (C) E6020-SE, (D) low BNZ, (E) vaccine alone and (F) low BNZ plus vaccine experimental groups. Five-nine representative photographs were taken from the myocardial region and were acquired at 10X magnification using a microscope adapted with a digital camera. Cell cytoplasm appears in red and collagen fibbers in blue. Black arrows show fibbers of collagen. Scale bar corresponds to 10μm.

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  Fig 3. Representative images of H&E stained-cardiac tissue from (A) non-infected, (B) infected untreated, (C) E6020-SE, (D) low BNZ, (E) vaccine and (F) low BNZ plus vaccine experimental groups. Five-nine representative photographs were taken from the myocardial region and were acquired at 20X magnification using a microscope adapted with a digital camera. Cell cytoplasm appears in pink and the nuclei of infiltrating inflammatory cells appear in purple. Scale bar corresponds to 10μm.

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  Fig 4. Effect of vaccine-linked chemotherapy on antigen-specific CD4+ and CD8+ T cells functional profile. Mononuclear cells isolated from mice at 200 days p.i were co-cultivated with macrophages stimulated in vitro with TSA-1-C4+Tc24-C4 (25 μg/mL) for 20 h. Data were analyzed using FlowJo X software. Stimulation index of (A) antigen-specific IFNγ-producing CD4+ cells, (B) perforin-producing CD4+ cells, (C) IFNγ-producing CD8+ cells and (D) perforin-producing CD8+ cells are presented. Each point represents an individual mouse, horizontal lines denote means ± SD or median ± interquartile ranges values according to the normality of the data. Data were analyzed using Student´s t-test or Mann-Whitney U-test. Significance is indicated as follows *, P≤0.05; **, P≤0.01.

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  Fig 5. Effect of vaccine-linked chemotherapy on antigen-specific CD4+ and CD8+ T cells memory response. Mononuclear cells isolated from mice at 200 days p.i. were co-cultivated with macrophages stimulated in vitro with TSA-1-C4+Tc24-C4 (25 μg/mL) for 96 h. Data were analysed using FlowJo X software. Stimulation index of antigen-specific (A) CD4+ Central Memory, (B) CD8+ Central Memory, (C) CD4+ Effector Memory and (D) CD8+ Effector Memory T cells are presented. Each point represents an individual mouse, horizontal lines denote mean ± SD or median ± interquartile ranges values according to the normality of the data. Data were analysed using Student´s t-test or Mann-Whitney U-test. Significance is indicated as follows *, P≤0.05.