Fig. 1: TLR9 agonists increase CAF®-mediated Th1/Th17 responses in mice.

A CB6F1 mice immunized 2x SC with H56 protein antigen adsorbed to DDA/MMG liposomes alone (Ø) or combined with various TLR agonists (TLR4, monophosphoryl lipid; TLR7, 3M-052; TLR9, CpG1826) in independent experiments and assessed after 2 weeks by antigen stimulation of splenocytes for IFNγ and IL-17 A secretion by ELISA. Bars, mean ± SD fold change compared to DDA/MMG alone (Ø). Symbols, individual mice, n = 5/grp (Exp1) n = 67grp (Exp2). B HEK-Blue^TM reporter cells expressing human TLR9 (left) or murine TLR9 (right) were stimulated in vitro with decreasing concentrations (100, 10, 1 µg/ml) of CpG2006 (grey bars) or CpG1826 (black bars) for 17 hrs and supernatants assayed for SEAP activity by HEK-Blue detection QUANTI-Blue™ (shown as OD₆₅₅). The experiment was performed twice. C Splenocytes from CB6F1 mice immunized 2x SC with H56 protein combined with 10 µg CpG2006 alone or DDA/MMG liposomes ± 10 µg CpG2006 assessed 2 weeks after immunization for IFNγ and IL-17A secretion by ELISA. Bars, mean ± SD; symbols, individual mice n = 10/grp and n = 2 naïve, ANOVA with Tukey’s posttest adjusted for multiple comparisons, p values < 0.1 shown D CB6F1 mice immunized 2x SC with DDA/MMG liposomes combined with various amounts of CpG2006 (left) or CpG1826 (right) adsorbed with H56 protein antigen, and two weeks later, PBMCs stimulated ex vivo with protein antigen to assess secreted IFNγ (blue) and IL-17A (red) by ELISA. Symbols indicate mean ± SD, n = 8/grp and n = 2 naïve, ANOVA with Dunnett’s posttest adjusted for multiple comparisons, p values < 0.1 shown. E The depicted CAF®10b adjuvant was assessed for zeta potential (ZP) (left), particle size (top right) and polydispersity index (bottom right) over 25 days. Source data are provided as a Source Data file.