PMC

Partially overlapping B-cell, MHC-I and MHC-II epitopes on MASP-XP_820771.1-derived peptide, as predicted by immunoinformatic analyses.

Effect of in vivo depletion of CD4+ or CD8+ T cells in mice immunized with KLH or MASPpep-KLH. Kaplan-Meier curves for survival and parasitemia of mice immunized with MASPpep-KLH or KLH, and then treated with anti-CD4 (A and B) or anti-CD8 mAb (C and D). N=2 animals per group.

Cytokine profile of immunized mice following parasite challenge. Anti-inflammatory (IL-4 and IL-10) and proinflammatory (IFN-γ, IL-12, and IL-17) cytokines were assayed in triplicate by ELISA four weeks after the last immunization and approx. two weeks after the challenge. Each bar represents the mean value±SEM (n=4 animals per group) for each treatment. Statistical analysis: Student's t-test. *, p≤ 0.05; ns, not significant.

Parasitemia (A), Kaplan-Meier curves for survival (B), and parasite load (C) in mice immunized with MASPpep-KLH or MASPpep-KLH/Al, and then infected with T. cruzi trypomastigotes (1×10⁶ cells). (A) Parasitemia levels are shown as trypomastigotes/ml. Each point corresponds to the mean parasitemia level. Blood parasitemia was followed in all four groups (n=4 animals per group). From the 3^rd to the 20^th day post-infection, five microliters of blood was taken from the tail and the number of trypomastigotes was determined. (B) Survival was monitored daily in all groups (n=4 animals per group). Three independent studies were performed with similar results. (C) Parasite load as measured by qRT-PCR of whole heart, liver, and spleen of mice immunized with MASPpep-KLH or MASPpep-KLH/AlOH, and infected with T. cruzi trypomastigotes (Y strain). Parasite load represents the equivalent of parasites in 50 ng gDNA obtained from each specific organ. Each determination was done in duplicate. Statistical analysis: two-way Anova with Bonferroni multiple comparisons.*, p<0.05; **, p≤ 0.01; ****, p≤ 0.0001.

Specific antibody responses against MASPpep in mice and humans. (A) Anti-MASPpep-KLH-specific antibodies were detected by CL-ELISA in sera of MASPpep-KLH-immunized mice and patients with chronic Chagas disease. MASPpep-KLH (10 μg/well) was incubated with a serum pool (at 1:100 dilution) from the specific immunized mouse group (PBS, KLH, Al, MASPpep-KLH, or MASPpep-KLH/Al) or with a serum pool (at 1:100 dilution) from patients with chronic Chagas disease (ChSP) or healthy individuals (NHSP). RLU, Relative luminescence units. Statistical analysis: Student's t-test. *, p<0.05; ***, p<0.001. (B) Western blotting analysis with pooled sera from MASPpep-KLH-immunized animals (MASPpep-KLH antiserum). TCT, Host cell-derived trypomastigote lysate immunoprecipitated with MASPpep-KLH antiserum; TCT_EV, TCT-derived extracellular vesicles purified by differential ultracentrifugation from conditioned culture medium. Epi and Ama, whole-cell lysates from epimastigotes and intracellular amastigotes, respectively. (C) Trypomastigote lysis assay. TCTs (1 × 10⁷/ml) were incubated with pooled sera from non-immunized (Placebo, PBS) or MASPpep-KLH-immunized mice. The assay was performed in the presence of inactive (iC) or active (aC) complement. Live, untreated TCTs were used as negative control (-C), whereas TCTs treated with 200 μM H₂O₂ were used as positive controls (+C). All experiments were performed with T. cruzi Y strain. The data are representative of three independent experiments.