Figure 1.
Synthesis of trimeric imidazoquinoline dendrimer 3.
Figure 2.
Synthesis of Click reaction derived imidazoquinoline dendrimer 8.
Figure 3.
TLR7- and TLR8-agonism and IFN-α and IFN-γ induction by Compounds 3, 4, and 8.
Top: TLR7- and TLR8-agonistic activity. Reporter gene assays specific for human TLR7 and TLR8 were used. Data points represent means and SD of quadruplicates. Bottom: IFN-α and IFN-γ induction by Compounds 4 and 8 in human PBMCs, assayed by analyte-specific ELISAs. Data points represent means of duplicates of three representative experiments.
Figure 4.
Cytokine induction by Compounds 4 and 8 in human PBMCs.
Data points of dose-response profiles represent means of duplicates of three representative experiments. Cytokines were quantified using cytometric bead array assays.
Figure 5.
Anti-bovine α-lactalbumin-specific IgG titers.
Top: Anti-bovine α-lactalbumin-specific IgG titers in rabbits adjuvanted with 4, 8, a TLR7-specific imidazoquinoline (1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine; reported as Compound 31 in Ref. 9), and unadjuvanted controls (n = 3 per cohort). Ratios of immune/pre-immune titers yielding absorbance values of 1.0 are shown for the individual samples. Each symbol corresponds to the titer of a single animal. Bottom: Chaotropic ELISA showing apparent titers as a function of chaotrope (NaSCN) concentration. Means and SD are shown.
Figure 6.
Linear peptide epitope mapping results of immune sera.
Top: Foreground fluoresence intensities of secondary goat anti-rabbit IgG (H+L) conjugated with DyLight680 antibody to each overlapping 13-mer peptide in the microarray are shown (means and SD). Bottom: Raw fluorescence images of representative peptide microarrays are shown. Each array is framed by a fusion tag (Flag) peptide (DYKDDDDKGG, 72 red spots) and influenza virus hemagglutinin (HA) epitope tag peptide (YPYDVPDYAG, 72 green spots) which were used as internal controls.
Figure 7.
Mapping of immunoreactive linear epitopes of bovine α-lactalbumin to its primary sequence and crystal structure.
Top: Sequence homology between bovine and rabbit α-lactalbumin. Conservative and non-conservative changes are shown in blue and red, respectively. Bottom: Mapping of immunoreactive linear epitopes to the crystal structure of bovine α-lactalbumin (PDB code: 1F6R). Regions colored green show contiguous immunodominant epitopes elicited by both 4 and 8, while regions colored red show additional epitopes specifically adjuvanted by 8.
Figure 8.
Correlation of observed and predicted epitopes.
Overlay of normalized mean background-corrected fluorescence intensity (13-mer peptide-specific immunoreactivity) of the dendrimer 8-adjuvanted rabbit sera and normalized Parker Hydrophilicity parameter computed with a moving window of 13 residues.