Fig 2: PAA:nanoalum promotes TH1 immunity without exogenous TLR4 agonistsl. C57Bl/6 mice (five per group) were immunized twice with 0.5 µg of ID93 alone or adjuvanted as indicated. One week after a homologous booster immunization the frequency of antigen-specific CD4 T cells in the spleen was determined by intracellular cytokine staining after stimulation with the immunizing antigen. a A representative FACS plot is shown for antigen stimulated cells from the Alhydrogel and PAA:nanoalum animal (left and right, respectively). b Distribution of CD154, IFN-ƴ and TNF producing CD4 T cells. Representative FACS gating is shown in Supplementary Fig. 3A. N = 5 animals per group. Data are representative of three experiments with similar results. Bars are drawn to the means with whiskers to the standard deviations

Fig 3: Augmentation of TH1 immunity is not a general property of nanoalum formulations or free PAA. a ID93 was mixed with Alhydrogel, PAA, or PAA:nanoalum for detected by silver stain before and after centrifugation to pellet the alum particles. Lane 1: ladder, 2: ID93, 3: ID93 + Alhydrogel, 4: ID93 + PAA:nanoalum 5: ID93 + PEG:nanoalum. The full gel is shown in Supplementary Fig. 2. b–d C57Bl/6 mice (five per group) were immunized twice with 0.5 µg of ID93 alone or adjuvanted with Alhydrogel, PAA, PAA:nanoalum, or PEG:nanoalum. b One week after the second immunization, splenocytes were isolated and either unstimulated or stimulated with the ID93 protein in the presence of brefeldin A for 8 h at 37 °C. Cells were then stained for surface expression of CD4 and CD44, as well as intracellular expression of IFN-ƴ, TNF and/or CD154. c Splenocytes from immunized mice were stimulated with the ID93 protein and assessed for secretion of IFN-ƴ. d Serum was collected from immunized animals 3 weeks after the first immunization and assessed for ID93 binding antibody titers by ELISA for IgG1 and IgG2 subclasses. ***(P < 0.001), ****P < 0.0001 relative to the antigen alone group as determined by one-way ANOVA with Dunnett’s correction for multiple comparisons. Representative FACS gating is shown in Supplementary Fig. 3A. Data are representative of three experiments with similar results. a and d Bars are drawn to the means with whiskers to the standard deviations. c The line is drawn at the mean, the box at the interquartile range and the whiskers at the min and max