Fig. 6. Liposome nanoparticle-based pulmonary vaccine. (a) Schematic illustration of interbilayer-crosslinked multilamellar vesicles (ICMVs) synthesis and cryoelectron microscope images: (i) anionic, maleimide-functionalized liposomes are prepared from dried lipid films, (ii) divalent cations are added to induce fusion of liposomes and the formation of multilamellar vesicles (MLVs), (iii) membrane-permeable dithiols are added, which crosslink maleimide-lipids on apposed lipid bilayers in the vesicle walls, and (iv) the resulting lipid particles are PEGylated with thiol-terminated PEG. Cryo-EM images from each step of the synthesis show (i) initial liposomes, (ii) MLVs, and (iii) ICMVs with thick lipid walls. Scale bars = 100 nm. Right-hand image of (iii) shows a zoomed image of an ICMV wall, where stacked bilayers are resolved as electron-dense striations; scale bar = 20 nm. Reproduced with permission.49 Copyright 2011, Nature Publishing Group. (b) OT-I-luc CD8+ T cells were adoptively transferred into C57BL/6 mice (n = 5) 1 day before ICMV or soluble OVA vaccine immunization via intratracheal or subcutaneous routes. Trafficking and proliferation of OT-I-luc T cells were monitored by flow cytometry and bioluminescence imaging on days 3 and 5 after immunization. Flow cytometry histograms show representative CFSE dilutions in transferred T cells on day 3 after immunization at draining lymph nodes (dLNs) (mdLNs for intratracheal vaccines and ingLNs for subcutaneous vaccinations). Lungs (L) and gastrointestinal tracts (G) were dissected on day 5 and imaged to identify T cell localization. V, vaginal tract; PP, Peyer's patches. (c) Flow cytometry analyses of integrin a4b7+ OT-I T cells in blood on day 5. (d) C57BL/6 mice (n = 5 to 6 per group) inoculated with 5 × 105 B16F10-OVA cells subcutaneously in the flank on day 0 were treated on days 3 and 10 with intratracheal administration of ICMVs or soluble OVA vaccines. Survival of tumor-bearing mice was tracked for 18 weeks. *P < 0.05, log-rank (Mantel-Cox) test. (e) C57BL/6 mice were immunized subcutaneously or intratracheally on days 0 and 28 with antigen AL11 (AAVKNWMTQTL) and the universal CD4+ T cell helper epitope PADRE peptide ICMV or soluble vaccines, and then challenged by intratracheal administration of simian immunodeficiency virus (SIV) gag-expressing vaccinia virus (1 × 106 PFU) on day 42. Body weight changes were recorded over time. Dots show titers from individual animals. Data are means ± SEM with n = 3 to 7 animals per group. *P < 0.5, **P < 0.01, ***P < 0.001, by one-way ANOVA (c) or two-way ANOVA (e). Reproduced with permission.19 Copyright 2013, American Association for the Advancement of Science.
