Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens

Abstract

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

Figure 1

Mucosal adjuvant activity of PEI and protection from disease by mucosal pathogens. (a,b) BALB/c mice (n=6) were primed and boosted once intranasally with 10 μg HIV-1 gp140 combined with 10 μg CTB or 10 μg of linear (L) or branched (B) PEI at different molecular weights (kDa) as indicated; (a) gp140-specific serum IgG and (b) vaginal IgA four weeks post-boost immunization (Anova/Bonferroni test). (c-f) BALB/c mice (n=5) were immunized intranasally with two doses of 10 μg HIV-1 gp140 combined with 20 μg CTB, CpG, PEI, 2 μg CT or 1 mg PLGA nanoparticles. (c) gp140-specific serum IgG on day 21 post-prime immunization (Anova/Bonferroni test). (d) gp140-specific serum IgG avidity index (2 weeks post-boost) measured by chaotropic ELISA using NH₄SCN (Anova/Dunnett); high avidity interactions can tolerate higher chaotropic agent concentrations than low avidity interactions. (e) ratio of serum IgG endpoint titers against native versus SDS/DTT-denatured gp140 (2 weeks post-boost; Anova/Bonferroni). (f) Serum IgG1 / IgG2a titer ratio (2 weeks post-boost; Kruskal-Wallis/Dunn), data representative of 2 experiments. (g) BALB/c mice (n=6) were immunized intranasally with three doses of gp140 alone or adjuvanted with CTB or PEI and the ratio of local (CLN, cervical lymph node) proliferation versus systemic (spleen) proliferation was determined by ³H-thymidine incorporation (unpaired t-test). (h,i) Protection of mice from highly pathogenic influenza virus challenge, data shown are representative of three independent experiments. C57Bl/6 mice (n=4-5) were immunized intranasally with a single dose of 10 μg influenza A (PR8) HA alone or combined with 2 μg αGalCer, 20 μg CTB or 20 μg PEI, followed by intranasal challenge three weeks later with a highly pathogenic dose of live influenza A PR8 virus. (h) Weight loss post-challenge. (i) Kaplan-Meier survival plots. (j,k) Protection of mice from highly pathogenic vaginal HSV-2 challenge. C57Bl/6 mice (n=7) were immunized intranasally with one or three doses of 5 μg HSV-2 gD combined with 20 μg CpG, PEI or 2 μg αGalCer per administration, followed by vaginal challenge (n=5) with previously titrated HSV-2. (j) Disease score post-HSV-2 challenge (k) Kaplan-Meier survival plots. All data are presented as mean of replicates from individual experiments ± SEM; n.s., not significant; *p<0.05, **p<0.01, ***p<0.001

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

Figure 3

PEI-induced host dsDNA release and cytoplasmic recognition via IRF3-dependent signaling. (a-b) B16-blue™ type-I interferon reporter cells were exposed to synthetic dsDNA (poly(dA:dT)) lacking TLR9-activating CpG motifs, in the presence or absence of the indicated concentrations of alhydrogel (alum, a) or PEI (b) (parallel experiment). (c) BALB/c mice (n=5) were intraperitoneally injected with PEI (65 μg) or alhydrogel (Alum, 1 mg) and dsDNA in peritoneal lavage was detected 24 h post injection. (d) BALB/c mice (n=5) were intranasally immunized with 10 μg gp140 alone or co-formulated with 20 μg PEI, 2 μg CT or 2 μg αGalCer and treated intranasally at 2.5 h and 18 h post immunization with 0.5 mg DNase I or vehicle control. Data are representative of 2 or more independent experiments. (e-f) C57BL/6 WT control or Irf-3^−/− mice were intranasally immunized with gp140 alone or co-formulated with 20 μg PEI or 2 μg CT at day 0, 22 and 36 (gp140 alone n=3, other groups n=6-7). (e) Time course of gp140-specific serum IgG responses. (f) Day 35 serum IgG responses of immunized mice. * p<0.05, ** p<0.01, *** p<0.001