Modular Vaccine Design Using Carrier-Free Capsules Assembled from Polyionic Immune Signals

New vaccine adjuvants that direct immune cells toward specific fates could support more potent and selective options for diseases spanning infection to cancer. However, the empirical nature of vaccines and the complexity of many formulations has hindered design of well-defined and easily characterized vaccines. We hypothesized that nanostructured capsules assembled entirely from polyionic immune signals might support a platform for simple, modular vaccines. These immune-polyelectrolyte (iPEM) capsules offer a high signal density, selectively expand T cells in mice, and drive functional responses during tumor challenge. iPEMs incorporating clinically relevant antigens could improve vaccine definition and support more programmable control over immunity.

S2 were washed three times as above and the sequence was repeated for up to 3 cycles to form CaCO3 particles coated with (SIIN*/polyIC)3 or (SIIN*/ODN)3. The sacrificial templates were removed by collecting the particles with centrifugation (1 min, 1000 g), followed by resuspension in 300 µL of 0.1 M EDTA at the indicated pH values for 30 min. Particles were then washed twice to remove EDTA and finally resuspended in PBS.
iPEM characterization. iPEM build up on silicon and quartz chips was measured by a LSE stokes ellipsometer (Gaertner Scientific Corporation) and Evolution 60 UV-visible spectrophotometer (Thermo Scientific) to assess iPEM thickness and relative cargo loading, respectively. UV-visible spectrophotometry was used to assess relative cargo loading on quartz chips by measuring absorbance values from 200 nm to 700 nm at 1 nm intervals using a solid state sample holder. Wavelengths of 260 nm and 508 nm indicated loading of nucleic acid and peptide, respectively. At least five regions throughout each chip were measured after every 2 bilayers. A Leica SP5X confocal microscope was used to visualize co-localization of both fluorescently-tagged SIIN* (FITC) and polyIC (Cy5) in iPEM capsules. Loading of antigen and adjuvant on sacrificial cores was assessed by measuring the absorbance of the nucleic acid (260 nm) and peptide (FITC, 495 nm) dipping solutions and wash buffer by UVvisible spectrophotometery. Loading of immune signals in iPEM capsules was assessed by incubating capsules in 300 µL of trypsin (0.05%) at 37°C for 1 hr, then measuring the peptide (FITC; Ex: 495, Em: 520) and polyIC (Cy5; Ex: 650, Em: 670) signals by fluorimetry using a Gemini XPS fluorescence microplate reader (Molecular Devices). Capsule sizes were measured using ImageJ to analyze diameters of at least 50 particles. For stability studies, capsules were incubated in PBS, incomplete media (RPMI), or complete DC media (with 10% FBS; detailed below) at 37 °C and the size was measured at the indicated times. For cytokine studies, capsules were serially diluted and added to DCs (1x10 5 cells/well) to reach final capsule concentrations of 42, 21, 10, 5, 3, or 1 µg/mL. Controls included untreated DCs, soluble peptide SIIN (5 µg/mL), LPS (1 µg/mL), polyIC (10 µg/mL), LPS (1 µg/mL) + SIIN (5 µg/mL), and polyIC (10 µg/mL) + SIIN (5 µg/mL). After 24 hrs, supernatants were collected and analyzed by ELISA.
In vitro CD8 + T cell expansion. OT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). [1] Three days after immunization, CD11c-enriched DCs from naïve, soluble vaccine-immunized, and iPEM capsule-immunized C57BL/6 mice were isolated. T cells were isolated from the spleens of OT-I mice using a negative selection CD8 isolation kit (Stemcell). Briefly, splenocytes were resuspended at 1x10 8 cells/mL (up to 8 mL total volume) followed by adding 50 µL of normal rat serum (Stemcell) per 1 mL of cells. After mixing, 50 µL of mouse CD8 + T Cell Isolation Cocktail (Stemcell) per 1 mL of cells was added and incubated at room temperature for 10 min.
Streptavidin RapidSpheres (Stemcell) were then added to the cell suspension at 125 µL/mL of cells and incubated at room temperature for 5 min. After incubation, the cell suspension volume was brought to a total volume of 5 mL (for <4x10 8 cells) with recommended medium (0.5% BSA and 0.2 mM EDTA in PBS). The cell suspension was placed in a 14 mL tube without a cap in the magnet for 2.5 min and carefully the desired CD8 + T cells were poured into a new tube. Resulting cells were washed twice to remove any serum and labeled with 5 µM of proliferation dye eFluor 670 (eBioscience) for 10 min at 37°C in the dark. After 10 min, the dye was neutralized with 5 times volume of T cell medium (RPMI1640, 10% FBS, 1x non-essential amino acid, 10 mM HEPES, 2 mM L-glutamine, 0.5% Penicillin S5 Streptomycin, 50 µM 2-ME) followed by washing three times with T cell medium. Resulting cells (3x10 5 cells/50 µL) were added into wells containing DCs/capsules and incubated for 48 hrs for the T cell co-culture assay. After 48 hrs, the resulting cell population was divided into two portions for proliferation analysis and intracellular cytokine staining (ICCS).
To assess T cell proliferation, cells were blocked with anti-CD16/32 as described previously ELISA assay. All ELISA assays were conducted using mouse IL-1β, IL-6, and IFN-γ OptEIA reagents according to the manufacturer's instructions (BD Biosciences). Supernatants were collected and analyzed without purification using 4-10x dilutions.
Mechanistic in vivo studies. Six to eight week old C57BL/6 female mice from The Jackson Laboratory were immunized by intradermal injection with capsules, soluble SIIN and polyIC, or left untreated as above. Three days after immunization, DCs from draining lymph nodes and spleens were isolated by positive CD11c selection as described above. Cells were then S7 stained with antibodies against classical DC activation markers and analyzed by flow cytometry, as above. To test if DCs isolated from immunized mice present peptides from iPEM capsules in a manner that can expand antigen-specific CD8 + T cells (OT-I), DCs isolated from iPEM-immunized mice on Day 3 were co-cultured with CD8 + T cell from OT-I mice for 48 hrs. Proliferation and cytokine secretion were then assessed by fluorescence dilution assays and ELISA as described above. For immunohistochemical analysis, lymph nodes were removed on day 3, frozen, sectioned at 10 µm intervals, then the tissue was fixed.
Fixed sections were blocked with 5% donkey serum (Sigma) and 5% goat serum (Sigma) in PBS for 30 min. After a PBS wash, samples were stained for T cells with a purified rabbit anti-mouse antibody (CD3e, Abcam) for 1 hr at room temperature, then washed twice and stained with a fluorescently-conjugated antibody for B cells (rat anti-mouse B220 APC, eBioscience) and a goat anti-rabbit antibody (Dylight 405, Jackson Immunoresearch). Stained sections were washed then fixed in 4% paraformaldehyde before quenching in 1% glycerol.
Sections were then mounted with Prolong Diamond Antifade Mountant (Life Sciences), and imaged.
Statistical Analysis. Statistical analysis was carried out using one way analysis of variance (ANOVA) with a Tukey post test in GraphPad Prism v.6.02. Survival analysis was carried out using a Logrank test. Statistical significance was defined at p values ≤0.05 (95% confidence interval) and indicated as * = p≤0.05, ** = p≤0.01, *** = p≤0.001. Figure S1. Fluorescently-labeled antigen (FITC, green) and adjuvant (Cy3, red; Cy5, magenta) can be independently visualized without signal overlap between filter sets. iPEMs were assembled on quartz microscope slides using the indicated components, then a needle was used to remove of portion of the film to provide contrast for imaging (white lines).