Figure 2.

Interactions between antigen and PEI and between immune cells and PEI-antigen complexes. (a) Direct biophysical interaction between PEI and BSA or PEI and gp140 analyzed using SPR with surface-immobilized protein or PEI. Data are representative of 3 independent experiments. (b) Scanning electron micrographs of PEI alone, gp140 alone or PEI complexed with gp140 or DNA; bar = 500 nm. (c) PEI-antigen complex formation. Quantification of PEI-gp140 or PEI-DNA particle sizes determined by counting of randomly-chosen fields imaged by SEM. (d) BMDC uptake of gp140₆₄₇-PEI complexes. gp140₆₄₇ (2 μg/mL) complexed or not with 0.1 μg/mL PEI. Cells were surface stained with Alexa Fluor® 555-labelled wheat germ agglutinin (WGA₅₅₅) and nuclear stained with Hoechst 34580. Laser scanning confocal micrographs of BMDC incubated with gp140₆₄₇ alone (top panel) or gp140₆₄₇ mixed with PEI (bottom panel), bar = 20 μm; (e) 3D reconstructions of a single BMDC exposed to gp140₆₄₇ complexed with PEI, bar = 10 μm. (f) Quantification of gp140₆₄₇ uptake by BMDC or (g) LA-4 mouse lung epithelial cells after O/N exposure to antigen with or without PEI. (h-k) Uptake of gp140₆₄₇ into NALT (h) or CLN (j) cells, or cell recruitment into NALT (i) or CLN (k), 4h (h, i) or 24h (j, k) after intranasal immunization of BALB/c mice with 10 μg gp140₆₄₇ ± 20 μg PEI (n=5, two-tailed t-test). Leukocyte populations were identified by flow cytometry with appropriate labeling and gates. Data are representative of 2 or more independent experiments and are presented as mean values of replicates from one experiment ± SEM; * p<0.05, ** p<0.01, *** p=0.001