Immunostimulatory and adjuvant activity of TLR9 agonists derived from <i>T. cruzi</i> genome.

<p>(<b>A</b>) PBMCs derived from healthy donors were stimulated with human B-class-like CpG ODNs derived from the <i>T. cruzi</i> genome with four different concentrations (3.0, 1.0, 0.3, and 0.1 µM) and the levels of IFN-α measured in the cell culture supernatants 24 h later. The CpG ODN 2007 was used as positive controls for human B-class ODNs. PBMC experiments were performed in three different donors, yielding similar results. (<b>B</b>) Proinflammatory activity of mouse B class-like CpG motifs was evaluated in inflammatory macrophages from WT (C57BL/6), <i>TLR4</i>^−/− and <i>TLR9</i>^−/− mice. ODNs were tested at different concentrations (1.5, 0.3 and 0.06 µM) and LPS, as well as CpG ODN 7909 were used as positive controls for TLR4 and TLR9 activation, respectively. IL-12 (p40) was measured in the macrophage culture supernatants 24 h after cellular stimulation. (<b>C</b>) C57BL/6 mice received three immunization doses with alum alone, OVA plus alum or OVA plus alum associated with either CpG ODNs B344, B287, B128 or 7909 (positive control). The levels of OVA-specific total IgG, IgG1 and IgG2c were assessed by ELISA. (<b>D</b>) Amount of IFN-γ secreted by splenocytes after stimulation with OVA derived CD4⁺ T or CD8⁺ T cell epitopes was evaluated in culture supernatants 72 hours post-stimulation. Asterisks indicate that differences were statistically significant, when comparing T cell response from mice receiving different vaccine formulations.