Exosome isolation, therapeutic peptide surface engineering, and characterization
The proprietary fabrication of ExoRelease immunomagnetic nanographene particles produces flower-like nano pom-poms morphology with photo click chemistry, which has been licensed by Clara Biotech Inc. The MHC binding peptide M with sequence NAITNAKII (RSV M187–195) and NS1 peptide with sequence ICPNNNIVV (RSV NS161–75) were synthesized by GenScript, confirming > 95% purity by high performance liquid chromatography (HPLC). These peptides conjugated onto ExoRelease particles respectively via NHS chemistry for affinity capture of MHC-I positive exosomes. The exosome isolation and surface engineering procedures were performed by incubating 100 mL of MHC binding peptide ExoRelease particles with 6 mL of cell culture medium at 4 °C overnight and shielded from light for MHC-I positive exosome capture and isolation. The solution tubes were placed onto the magnet for 1 min, then supernatant was discarded. Afterward, the particles were resuspended into 200 mL of 1 × phosphate buffered saline (PBS) buffer. Next, 15 mL of a solution containing β2-microglobulin (20 μg/mL) and peptide (100 μg/mL, either M peptide or NS1 peptide) for surface binding to form pMHC-I complex, was placed in a glass vial to incubate with rotation for 1 h at room temperature. The ExoRelease particles were separated from the reaction mixture by placing close to a magnetic bar for 1 min and discarding the supernatant; then the ExoRelease particles with surface engineered exosomes were washed three times with 1 × cool PBS buffer. Subsequently, exosomes were released from particles through photo-cleavage using a LED UV head at 365 nm wavelength exposed for 15 min (~ 6 mW/cm2). The conjugated PEG photo-cleavable linker with NHS moiety on bead surface allows the bond cleavage under light exposure to release intact, engineered peptide exosomes on demand, which further ensures the specificity for harvesting MHC-peptide modified exosomes. Harvested exosomes were characterized using the nanoparticle tracking analysis (NTA, Nano-Sight LM10, Malvern Panalytical) to determine size distribution and particle concentration of engineered exosomes. NTA post-acquisition parameters were adjusted to a screen gain of 10.0 and a detection threshold to 5. Standard 100 nm nanoparticles were used for calibration. Samples were diluted in 1 × PBS before every measurement and repeated for five times. The zeta potential was measured using ZetaVeiw (Particle Metrix).
NanoView microarray for characterization of exosome MHC-I expression level was conducted per standard protocols provided by NanoView Biosciences (Brighton, MA). Processed JAWS cell culture media was diluted appropriately using the EV binding buffer (solution A, pH 7.4), then 35 µL of diluted sample was dropped on microarray chips for incubation overnight. Each three chip spots were pre-coated with capture antibodies CD81 (clone Eat-2, mouse, BioLegend) and CD9 (clone MZ3, mouse, BioLegend), and negative controls HIgG (clone HTK888, mouse, BioLegend). Microarray chips were washed four times with solution A at 150 rpm/min, 3 min for each time. After washing, 300 µL blocking solution was incubated with each chip for 1 h at room temperature and protected from light, which contains two detection antibodies including 0.6 µL Alexa Fluor 647-conjugated CD63 (clone NVG-2, mouse, BioLegend) and 2.5 µL PE-conjugated MHC class I (clone AF6-88.5.5.3, mouse, Invitrogen). Subsequently, microarray chips were washed again with solution A, solution B and distilled water, then chips were kept air dry for imaging by ExoView R100 (NanoView Biosciences) equipped with 40 × objective lens (Olympus, Japan). Data was analyzed and quantified using off-line ExoViewer3 EAP_v3 software.
The bead-based flow cytometry analysis (CytoFlex, Beckman) was performed to characterize our ExoRelease particle captured exosomes and their surface engineering with RSV peptides. The exosomes were labeled by PKH67 (Sigma-Aldrich) per vendor’s instruction. The RSV peptides (M and NS) were labeled by Alexa Fluor 555 (Thermo Fisher).
Characterization of engineered exosomes using SEM and TEM imaging
For scanning electron microscope observation, exosome bound particles were resuspended in 200 μL PBS solution and washed 2 times with pure water. 5 μL exosome bound particle solution was added to clean silicon chips and immobilized by drying under a ventilation hood. Samples on silicon chips were mounted on a SEM stage by carbon paste with 30 s sputtering coating. SEM imaging was performed under low beam energies (7 kV) with 10 nA on Hitachi SU8230 field emission scanning electron microscope.
For TEM imaging, ~ 5 μL of harvested exosomes were dropped onto formvar carbon coated copper Grid 200 mesh (Electron Microscopy Sciences) for 5 min followed by 3 min of negative staining with 2% aqueous uranyl acetate. Excess liquid was blotted by a filter paper. Total grid preparation was performed at room temperature until completely air-dried under a ventilation hood for 25 min. The imaging was performed immediately after fixation using the Tecnai G2 Spirit TWIN Transmission Electron Microscope at 120 kV.
RSV isolation
Human epithelial type 2 (HEp-2) cells (ATCC, CCL-23) were maintained in complete minimal essential media (cMEM), composed of MEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mM l-glutamine (Gibco), 1% antibiotic antimycotic (Gibco), and 1 mM sodium pyruvate (Gibco) until they are approximately 80 to 90% confluent. RSV strain A2 (ATCC, VR-1540) was inoculated into the HEp-2 cells with serum-free MEM. After 2 h of virus inoculation, the supernatant was removed, and cMEM was added. For storage, RSV infected HEp-2 cells were frozen at − 80 °C from 48 to 72 h after infection. Virus-infected HEp-2 cells were thawed, and cultures were centrifuged at 3200×g for 15 min at 4 °C to remove cell debris. Virus supernatants were collected and added to the polyethylene glycol 8000 in NT buffer containing 150 mM NaCl and 50 mM Tris–HCl (10% w/v). The supernatants were incubated at 4 °C for an hour with stirring and then centrifuged to precipitate the RSV at 3200×g for 40 min at 4 °C. The virus was suspended in 3% sucrose Dulbecco’s Modified Eagle Medium (DMEM, Gibco) and stored at − 80 °C. HEp-2 cells were inoculated with tenfold serial dilutions of RSV in a 96 well flat bottom plate. The titration was measured by cytopathic effects. The Reed and Muench calculation was used to get the virus titer.
Animal study and ex vivo experiments
All mice procedures were in accordance with the relevant guidelines and regulations, and experimental protocols were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC) 18-074. Additionally, all animal experiments were conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Six to seven week old C57BL/6J female mice were purchased from Jackson Laboratories. Mice were challenged intranasally with RSV. Animals were anesthetized with isoflurane prior to intranasal inoculation with 107 PFU RSV in 60 μL 3% sucrose in DMEM and were sacrificed on day 8 post-infection by carbon dioxide (CO2) asphyxiation, and then the lung tissues were harvested. Five RSV infected C57BL/6 mice were euthanized and spleens and lungs were collected for CD8+ T cells. Another five non-infected C57BL/6 (naïve) mice were euthanized and spleens were collected for CD11c + cells. Magnetic activated cell sorting (MACS) was used to sort CD8+ T cells and CD11c+ DCs according to the manufacturer’s instructions (Miltenyi Biotec). The exosome:T cell:DC co-culture model was adapted from previously described methods16. CD8+ T cells and CD11c+ DC cells (5:3 ratio) were incubated for 72 h with escalating concentration (5 μL, 10 μL, 25 μL) of exosomes engineered with M187–195 or NS161–75 peptides with or without LPS (100 ng/mL, InvivoGen) . Cells were cultured in complete RPMI (cRPMI), composed of RPMI 1640 medium (Gibco) containing 10% FBS, 2 mM l-glutamine, 1% antibiotic antimycotic, and 1 mM sodium pyruvate. cRPMI was used as a negative control, and M peptide (5 μg/mL, GenScript), NS1 peptide (5 μg/mL, GenScript), and ConA (5 μg/mL, MP Biomedicals) were used as positive controls.
Exosome immunization for challenging RSV infected mouse model
Five mice per group were immunized (prime-boost) subcutaneously with 100 μL (1.7 × 109 particles/mL) of engineered exosomes harboring either M or NS1 peptides on their surface with or without Quil A adjuvants (10 μg/mouse). There were 2 weeks gap between prime and boost vaccination. Mice were challenged intranasally with RSV, 2 weeks after boost. The same protocols as ex vivo experiments were used to inoculate the virus and sacrifice the mice. They were anesthetized with isoflurane prior to intranasal inoculation with 107 PFU of RSV in 60 μL 3% sucrose in DMEM. Mice were sacrificed on day 3 or 6 post-infection by carbon dioxide (CO2) asphyxiation and lung tissues were harvested (same as ex vivo). Lung cells were stimulated and incubated with cRPMI media, M peptide (5 μg/mL), and NS1 peptide (5 μg/mL). Positive controls used were PMA (50 ng/mL, Sigma-Aldrich) and Ionomycin (1 μg/mL, MP Biomedicals) for flow cytometry and ConA (5 μg/mL) for ELISA.
Lung cell isolation
The lungs were diced and digested by collagenase type I (3 mg/mL, Gibco) in cRPMI for 30 min at 37 °C. After incubation, the tissues were processed through a 70 μm strainers to prepare a single cell suspension. The cell suspension was washed twice with cRPMI at 1500 rpm for 5 min at 4 °C.
Tetramer and intracellular cytokine staining (ICS)
After 12-h incubation with stimuli followed by 6-h incubation with Brefeldin A (Becton Dickinson Biosciences) for all wells and PMA/Ionomycin only for positive controls, virus-specific T cells were detected with tetramers of H-2Db RSV M187–195 peptide. Cells were stained with fluorochrome-conjugated antibodies against FITC anti-mouse CD45 (clone 30-F11, mouse, eBioscience,), PerCP/Cyanine5.5 anti-mouse CD8a (clone 53–6.7, mouse, BioLegend,), APC H-2Db RSV M187–195 tetramer (MBL), and aqua live/dead fixable dead cell stain (Invitrogen) for 30 min at 4 °C. Cells were fixed and permeabilized according to the manufacturer’s instructions (Becton Dickinson Biosciences) and stained with APC/Cyanine7 fluorochrome-conjugated anti-mouse IFN-γ antibodies (clone XMG1.2, mouse, BioLegend) for 30 min at room temperature. After staining, cells were washed and analyzed by flow cytometry. Cells were analyzed on a Becton Dickinson FACSCanto. Data were analyzed by using FlowJo software version 10.7.1 (FlowJo, LLC). A representative gating strategy for analysis of tetramer and intracellular cytokine staining is included in Supplementary Fig. 1.
ELISAs
Mouse IFN-γ DuoSet ELISA Development kit was purchased from R&D systems and ELISAs were performed according to manufacturer’s instructions. Sandwich ELISAs were used to quantify mouse IFN-γ in cell culture supernatants. As previously described, lung cells were stimulated and incubated with cRPMI media, M peptide (5 μg/mL), NS1 peptide (5 μg/mL), and ConA (5 μg/mL). The supernatants were collected after 72-h or 5-day stimulation with those stimuli. The supernatant samples were diluted 1:4 with reagent diluent buffer. All samples and standards were plated in duplicates. The results of ELISA are representative of three independent ex vivo experiments and that of one in vivo experiment.
Statistics
Results were shown as averages ± standard errors of the mean (SEM). Statistical analysis was determined by one-way and two-way Analysis of Variance (ANOVA) using Prism software version 9.0.0 (GraphPad Software, LLC).
