Genetic effects between TLR adjuvants and alum or MF59 formulations. (A) Heatmap depicting the effect of adding the TLR4 or 7 agonists to the alum formulation on gene module regulation. (B) Plots of representative genes showing the change in expression of MMP9 (top panel) and EIF2AK2 (bottom panel) for each alum-containing vaccine group compared with prevaccination. (C) Plots depicting the effect of TLR4 or 7 on the alum formulation. Symbols depict modules (top plot) and individual genes (bottom plot) found to be differentially regulated in the alum-only group, graphed by their change in expression found in the context of the TLR4 (x-axis) and TLR7 (y-axis) containing alum formulations. (D) Heatmap depicting the effect of coformulating ANE with the TLR4 or 7 agonists on gene module regulation. (E) Plots of representative genes showing the change in expression of IFNgR2 (top panel) and GBP1 (bottom panel) for each MF59 and ANE-based vaccine group compared with prevaccination. (F) Plots depicting the effect of ANE/TLR4 or 7 compared with the MF59 formulation. Symbols depict modules (top plot) and individual genes (bottom plot) found to be differentially regulated in the MF59-only group, graphed by their change in expression found in the context of the TLR4 (x-axis) and 7 (y-axis) containing ANE formulations. (G-H) Plots depicting the fold change of genes upregulated by TLR4 (G) or TLR7 (H) in the alum and ANE-based formulations. Blue lines (x = y) depict the hypothetical position of genes that have equal effects in both formulations; red dashed lines depict the linear regression of the actual data. The deviation in slope between both lines was evaluated by regression analysis; the correlation of each data set is indicated by the r_s coefficient and P value. Bar graphs depict the mean fold change and standard error between the TLR-regulated genes in either the alum or ANE formulations; P values are derived from a pairwise comparisons of the means. MMP9, matrix metalloproteinase 9.