(A) HLA-A*11:01 transgenic mice immunized with LO protein plus GLA-SE were protected compared with control mice inoculated with PBS when they were challenged with 20,000 T. gondii prugneaud strain (Fluc) luciferase expressing parasites after 4 and 6 days. (n = 4 mice per group, 2 replicate experiments.) (B) Assay demonstrating that GLA-SE is a TLR4 ligand that leads to production of IL-6, IL-12, and TNF-α by PBMC. Stimulation of human whole blood with GLA-SE. Heparinized whole blood was collected from 6 healthy donors, and 200 μl was stimulated with 5 μg GLA-SE in 96-well plates at 37^oC CO₂. After 24 hours, plasma was removed and assayed for IL-6, IL-12(p40), and TNF-α by a custom Luminex-based multiplex immunoassay kit (Affymetrix eBioscience). Data were analyzed using the Masterplex QT software (Miraibio). The cytokine production stimulated by adjuvant was statistically significant for IL-6, IL-12, and TNF-α (P < 0.05) compared with the unstimulated groups as assessed by the Mann-Whitney U test (GraphPad Prism software). The plots show median, with box extending from the 25th to 75th percentile and the whiskers extending from minimum and maximum values of the data set. (C) Multi-epitope proteins with GLA-SE are captured by the Antigen-presenting cells (APCs), and the peptides contained are presented by MHC molecules on the APCs to T lymphocytes in both a class I and a class II pathway. This demonstrates that cross presentation into a class I pathway must occur by virtue of effector function. APCs are also activated through recognition of GLA-SE by TLR4 receptors molecules. This activation leads to the production of proinflammatory cytokines (IL-12, IL-6, TNF-α) and the expression of costimulatory molecules on the cell surface.