PMC

RAW264.7 macrophages were seeded in 8 well chamber slides at a density of 2×10⁴ cells per well and stimulated with 5 µg/ml of either RS01 or RS09. After 30 min incubation, cells were fixed (4% para-formaldehyde), permeabilized (0.2% Triton X-100), blocked (10% goat serum/1% BSA), and incubated with anti-NF-κB followed by DAPI staining of nuclei. The stained cells were visualized using the Axiovert 200 M microscope with a 40× magnification. NF-κB (green) was more diffusely spread throughout the cytoplasm in control cells but appeared more circular in RS01, RS09, and LPS treated cells. Upon merging green NF-κB with DAPI nuclei, nuclear localization of NF-κB was observed by the teal color in treated but not in control cells.

Nuclear translocation studies of NF-κB were performed by Western blot analysis on both RAW264.7 (), HEK-BLUE™-4 (), and HEK293 () cells. Cells were stimulated with peptide, either RS01or RS09, at a concentration of 5 µg/ml per 5×10⁴ cells at various time points (15, 30, 60, and 120 min). Nuclear protein fractions were assayed for NF-κB while cytoplasmic proteins fractions were assayed for both NF-κB and IκB-α. HDAC was used as a nuclear protein loading control and actin was used as a cytoplasmic protein loading control. For HEK293 cells, no differences in the levels of cytoplasmic and nuclear NF-kB were observed suggesting that LPS, RS01, and RS09 do not activate the non-TLR-4 expressing HEK293 cells ().

LPS antibody was immobilized at a concentration of 1 µg per well of a 96-well plate. Phages (2×10¹¹) expressing 7-mer peptides were initially added to the well of a 96-well plate containing immobilized LPS antibody after which non-specific phages were removed. Twelve random phage clones were selected from three rounds of panning and specificity to LPS antibody (black bars) was confirmed by ELISA using HSP70 antibody (white bars) as a negative control. All 12 (RS01-RS12) clones selected displayed specific reactivity to LPS antibody, as determined using the HRP labeled anti-M13 phage antibody which was detected by the HRP substrate SIGMAFAST™ OPD and absorbance measured at 490 nm. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.

LPS peptide mimics were dissolved in endotoxin free water and added to 96-well plates at three concentrations; 1 µg/ml (black bar), 5 µg/ml (gray bar), and 10 µg/ml (white bar). HEK-BLUE™-4 were then added to each well at a concentration of 5×10⁴ cells per well in complete media. The supernatant was then harvested after 24 h and assayed for alkaline phosphatase secretion indicating activation of NF-κB. NF-κB activation for peptides RS01-RS07 is represented in and peptides RS08-RS12 in . LPS was used as a positive control in assaying all peptides for NF-κB activation. Absorbance was taken at 630 nm. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.

Four groups of mice (3 mice/group) were vaccinated with X-15 (100 µg) conjugated to KLH. The mice were bled on days 0, 14, and 28. The serum obtained from each animal was analyzed for presence of X-15 specific antibodies by direct ELISA with immobilized X-15 peptide. The difference between groups was the adjuvant used, which was Alum, RS01 or RS09. OVA peptide (8-mer peptide) was used as a negative peptide control. As shown in , the highest X-15 specific antibody concentration was observed on Day 28 post vaccination (White bars) for all groups. RS09 was the only peptide capable of enhancing the X-15 specific antibody response on day 28. The standard deviation represents triplicate values for each group of animals.

RAW264.7 cells were seeded at a density of 1×10⁶ cells per well of a six well plate followed by addition of either RS01 () or RS09 () and incubated for 24 h after which the culture media was collected. LPS was used as a positive control for both RS01 and RS09 and therefore represented on both . Culture media was then analyzed for specific cytokine using an antibody array kit. Both RS01 (triangle) and LPS (square) were capable of inducing inflammatory cytokines and chemokines from RAW264.7. Each cytokine is represented as an IDV value which was calculated by comparing the density of each spot with respect to the density of the internal positive controls. The antibody array represents a qualitative comparison of each cytokine and is not qualitative. Each experiment was repeated three times with similar results observed and the standard deviation shown above each sample represents three replicates in one experiment.