(A to D) Human PBMCs collected from young adults (A, C) and older adults (B, D) were cultured in vitro for 24 hours with CpG alone, aluminum hydroxide (AH) alone, or a combination of both. Supernatants were collected for multiplexing bead array. n=6 per age group. (A and B) Radar plot analysis presented as a fold-change over the CpG alone group for the 20 μg/mL CpG and 40 μg/mL AH conditions. (C and D) Cytokine and chemokine concentrations are shown at the indicate doses of CpG and AH. Results are presented as mean ± SEM. Unpaired Mann-Whitney tests were applied at each concentration. Blue and yellow colored asterisks indicate comparisons of AH:CpG formulation to AH and CpG alone groups, respectively. Degree of synergy was calculated using an adapted Loewe definition of additivity (D <1: synergy, D=1: additivity, D >1: antagonism). (E) Human monocyte-derived dendritic cells from young adults were stimulated with spike protein and indicated adjuvants and co-cultured with CD4+ T cells for 5 days. The percent of CD25+CD134+ CD4+ T cells of total CD4+ T cells was assessed. n=5. Results are presented as mean ± SEM. Statistical significance was determined by two-way repeated measures ANOVA corrected for multiple comparisons. * indicates comparisons among AH, CpG, and AH:CpG at the same concentration. # indicates comparisons among same adjuvant groups to the baseline control (antigen only). Comparisons among experimental groups are indicated by color code. * P <0.05, **/ ##
P <0.01, ####
P <0.0001.