Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine

Cell culture

LSCs were constructed from whole lung samples of healthy human from the University of North Carolina at Chapel Hill Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center and expanded as previously described22,23,24. LSCs were plated on a fibronectin-coated (Corning) flask and maintained in Iscove’s Modified Dulbecco’s Media (IMDM; Thermo Fisher) containing 20% fetal bovine serum (FBS; Corning). Media changes were performed every other day. LSCs were allowed to reach 70–80% confluence before generating serum-free secretome (LSC-secretome) as previously described26. LSC-secretome was collected and filtered with a 0.22 μm filter to remove cellular debris. C57BL/6 dendritic cells (1129–4807AU20) were purchased from Cellero and cultured in IMDM medium (Thermo Fisher) with 10% FBS. They are of the myeloid type and derive from bone marrow using murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation. Primary small airway epithelial cells (PCS-301-010) and primary bronchial/tracheal epithelial cells (PCS-300-010) were obtained from ATCC and cultured with airway epithelial cell basal medium. Splenocytes and pneumocytes were isolated from vaccinated mice as previously described54. All procedures in this study were in accordance with the ethical standard of the institutional research committee and with the guidelines set by the Declaration of Helsinki.

Exosome isolation and characterization

Exosomes were collected and isolated from human LSC-secretome via ultrafiltration55. Filtered secretome was pipetted into a 100 kDa Amicon centrifugal filter unit (Millipore Sigma) and centrifuged at 400 RCF at 10 °C. Once all medium was filtered, the remaining exosomes were detached from the filter and resuspended using Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher) with 25 mM trehalose (Millipore Sigma) for further analysis56. LSC-Exo, RFP-Exo, RFP-Lipo and RBD-Exo were analysed by nanoparticle tracking analysis (NTA; NanoSight NS3000, Malvern Panalytical). All samples were fixed onto copper grids and stained with vanadium negative staining for TEM (JEOL JEM-2000FX) to analyse exosomal composition and morphology before and after RBD binding. To determine the presence of RBD through TEM, RBD-Exo were incubated with anti-RBD primary antibody (40592-T62; Sino Biological) overnight at 4 °C. Unbound antibodies were removed via ultracentrifugation at 100,000 g for 30 min. Gold nanoparticles (15 nm) labelled with goat anti-rabbit IgG secondary antibody were added and incubated at room temperature for 2 h.

RFP loading

RFP (ab268535; Abcam) was loaded into exosomes and commercial liposome particles (300205, Avanti Polar Lipids) via electroporation, yielding RFP-Exo and RFP-Lipo32. The liposome consisted of fully hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (DSPE-mPEG2000) and cholesterol. The mass ratio of DSPE-mPEG2000, HSPC and cholesterol was 18.8:61.6:19.6. One billion nanoparticles from each sample were diluted in Gene Pulser electroporation buffer (Bio-Rad) at a 1:9 ratio of nanoparticles to buffer. RFP (10 μg) was added to the nanoparticle-electroporation buffer solution, which was transferred into the ice-cold 0.4 cm Gene Pulser/MicroPulser electroporation cuvette (Bio-Rad). The electroporation cuvette was inserted into the Gene Pulser Xcell Total System (Bio-Rad) and electroporated under the following conditions: pulse type, square waveforms; voltage, 200 V; pulse length, 10 ms; number of pulses, 5; pulse interval, 1 s. Electroporation buffer and unloaded RFP were removed from the fluorescently labelled nanoparticles by ultrafiltration via an Amicon centrifugal filter (Millipore, UFC510096, 100 kDa molecular weight cut-off), followed by three washes with DPBS buffer (10 mM, pH 7.4, 13,000 g, 10 min per wash, 4 °C). RFP encapsulation efficiency and loading efficiency were determined by RFP fluorescence at 595 nm, with excitation at 547 nm. The encapsulation efficiency (EE) was calculated using the following formula: \({{{\mathrm{EE}}}} = \frac{{{{{\mathrm{entrapped}}}}\;{{{\mathrm{RFP}}}}}}{{{{{\mathrm{total}}}}\;{{{\mathrm{RFP}}}}\;{{{\mathrm{added}}}}}} \times 100\%\). The loading efficiency (LE) was calculated as: \({{{\mathrm{LE}}}} = \frac{{{{{\mathrm{entrapped}}}}\;{{{\mathrm{RFP}}}}\;{{{\mathrm{amount}}}}}}{{{{{\mathrm{nanoparticles}}}}\;{{{\mathrm{amount}}}}}} \times 100\%\) (ref. 57).

RBD conjugation on LSC-Exosomes

Recombinant SARS-CoV-2 RBD protein (40592-V08B, Sino Biological) was purchased and reconstituted in DPBS. RBD was conjugated to LSC-Exo using a DSPE-PEG-NHS linker. Briefly, RBD (100 μg) was conjugated with DSPE-(PEG)5000-NHS (10 mg) in 2 ml PBS (10 mM, pH 7.4) overnight at 4 °C. Unconjugated DSPE-(PEG)5000-NHS was removed by centrifugation and washing using an Amicon centrifugal filter unit with 10 kDa molecular weight cut-off (Millipore, UFC801096, triple wash with 10 mM PBS, 4 °C, 4,500 g, 10 min per wash). To demonstrate the conjugation of RBD-PEG-DSPE, RBD and RBD-PEG-DSPE were analysed by blue native SDS–PAGE (4–20% acrylamide precast Tris-glycine gel) and stained with Coomassie blue. Then, the resultant RBD-PEG-DSPE was conjugated with LSC-Exo (2 × 1011 particles) for 24 h at 4 °C. Unbound RBD-DSPE-NHS and free RBD were concentrated and washed three times with PBS buffer (10 mM, pH 7.4, 4,500 g, 10 min per wash) in an Amicon centrifugal filter (Millipore, UFC810096, 100 kDa molecular weight cut-off) at 4 °C. To further purify the RBD-Exo, the resulting RBD-Exo solution was ultracentrifugated at 100,000 g at 4 °C for 2 h, followed by washing with PBS buffer once (10 mM, pH 7.4) and resuspension in 2 ml PBS buffer (10 mM, pH 7.4).

To quantify the RBD moiety on LSC-Exo, 106 RBD-Exo particles were taken from the 2 × 1011 particles stock solution and diluted into RIPA lysis buffer and lysed on ice for 30 min. The amount of released RBD was quantified via ELISA (EH492RB, Thermo Fisher).

$$\begin{array}{l}{{{\mathrm{The}}}}\;{{{\mathrm{inserting}}}}\;{{{\mathrm{efficacy}}}}\\ = \frac{{{{{\mathrm{the}}}}\;{{{\mathrm{released}}}}\;{{{\mathrm{RBD}}}}\;{{{\mathrm{antigen}}}}\;{{{\mathrm{from}}}}\;{{{\mathrm{RBD – Exo}}}}}}{{{{{\mathrm{the}}}}\;{{{\mathrm{RBD}}}}\;{{{\mathrm{used}}}}\;{{{\mathrm{for}}}}\;{{{\mathrm{synthesizing}}}}\;{{{10^{6}}}}\;{{{\mathrm{of}}}}\;{{{\mathrm{RBD – Exo}}}}\;{{{\mathrm{particles}}}}}} \times 100\%.\end{array}$$

The conjugation efficiency of RBD with LSC-Exo via DSPE-PEG-NHS linker was determined to be 10.5%.

Stability studies on RBD-Exo VLPs

RBD-Exo lyophilisates were stored at −80 °C, 4 °C and room temperature (21 °C) for 21 d (3 weeks) or 3 months. Then, RBD-Exo lyophilisates were dispersed in PBS and their size and concentration were detected using NTA. In addition, the concentration of RBD on Exo was quantified using an ELISA kit.

An accelerated stability testing study of RBD-Exo lyophilisates was performed under the ICH guidelines at 40 ± 2 °C and 75 ± 5% relative humidity to further verify the potential of RBD-Exo as a vaccine58. The samples were evaluated periodically at 0, 3 weeks, 3 months and 6 months for stability analysis. Western blot and ELISA analyses were used for evaluating the stability and conformation integrity of RBD.

RBD-Exo internalization by APCs

RBD was labelled using NHS-Rhodamine (Thermo Fisher) according to the manufacturer’s protocol. RBD-RhB and RBD-RhB-Exo were co-cultured with C57BL/6 dendritic cells for 4 h with the same amounts of RBD (1 μg). The free RBD-RhB and RBD-RhB-Exo were removed, and cells were washed with three times with DPBS buffer. Cells were imaged with an Olympus FLUOVIEW CLSM (Olympus; FV3000).

Mouse studies using SARS-CoV-2 mimics

All studies complied with the requirements of the Institutional Animal Care and Use Committee (IACUC, 19-806-B). Male CD1 mice (7–8 weeks old) (Crl:CD1(ICR)) were purchased from Charles River Laboratory (Wilmington, MA, USA). RFP-Exo and RFP-Lipo were administered via nebulization (Pari Trek S Portable 459 Compressor Nebulizer Aerosol System; 047F45-LCS, PARI). PBS, Exo, RBD, RBD-Exo treatments (1010 per kg of mouse weight) and RBD-Lipo treatments (1.26 × 1010 per kg of mouse weight) were given in two doses once a week for 2 weeks via nebulization or IV injection. Spike protein (Sino Biological) was modified onto the surface of lentivirus (Cellomics Technology) and the SARS-CoV-2 mimics were synthesized according to our previous report59. Mice were challenged with SARS-CoV-2 mimics labelled with AF647 (106 particles per kg of body weight) by nebulization 1 week after the second treatment dose. Lungs were excised and imaged at days 2 and 6 post vaccination with a Xenogen live imager (PerkinElmer). Blood and all other major organs were collected for further analysis.

IgG antibody titre

Microtitre plates (Nunc Cell Culture, Thermo Fisher) were coated with 10 μg ml−1 RBD in 100 μl coating buffer (R&D Systems) and incubated overnight at 4 °C. Wells were then blocked with 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) in 200 μl PBS-T for 1 h at 37 °C. After washing three times with PBS-T, serial dilutions (1:100, 1:1,000, 1:5,000, 1:10,000, 1:50,000, 1:100,000, 1:200,000) of sera samples were added and control sera samples with 1:100 dilution were added into wells for incubation for 1.5 h at 37 °C. Then, samples were washed with PBS-T three times and then incubated with HRP-labelled anti-mouse IgG secondary antibody at a 1/2,000 dilution (100 μl per well) or HRP-labelled anti-hamster IgG secondary antibody at a 1/20,000 dilution (100 μl per well) for 1 h at 37 °C. After washing four times with PBS-T, 3,3’,5,5’-tetramethylbenzidine soluble substrate (TMB; Thermo Fisher) was added to each well (100 μl per well). After incubation of 30 min at room temperature, 50 µl of stop solution (2 M H2SO4, Sigma-Aldrich) was added and optical absorption at 450 nm was determined by a plate reader. The end-point titre of IgG was quantified by the reciprocal of maximal serum dilution that exceeded twice the s.d. above the mean readout of the control group. The individual antibody titres are shown as [log10[\(\mathop {{{{\mathrm{x}}}}}\limits^ -\) ± s.d.]], calculated as the reciprocal of maximal serum dilution.

For IgG2a and IgG1 ELISA analyses, a similar protocol with minor modifications was followed as described above; goat anti-mouse IgG2a-HRP (A-10685, Thermo Fisher) and goat anti-mouse IgG1-HRP (ab97240, Abcam) were respectively used as secondary antibodies.

IgA antibody titre

RBD-specific IgA from NPLF and BALF was measured using ELISA. To collect NPLF, the trachea was cut in the middle and the nasopharynx was rinsed upwards from the incision with 200 μl DPBS. The fluid was collected and rinsing was repeated three times for a total of 600 μl wash fluid. To collect BALF, the trachea was exposed by thoracotomy and a transverse incision was made at the top of the bronchial bifurcation. A needle was inserted into the trachea to wash the lungs with 200 μl DPBS. The wash fluid was collected and rinsing was repeated three times for a total of 600 μl wash fluid. Microtitre plates (Nunc Cell Culture, Thermo Fisher) were coated with 10 μg ml−1 RBD in 100 μl coating buffer (R&D Systems) and incubated overnight at 4 °C. Then, RBD solution was removed and wells were blocked with 1% (w/v) BSA (Sigma-Aldrich) dissolved in 200 μl PBS-T for 1 h at 37 °C. After washing with PBS-T three times, serial dilutions (1:50, 1:100, 1:1,000, 1:5,000, 1:10,000, 1:50,000) of NPLF and BALF, and control samples (1:50 dilution) were added and incubated for 1.5 h at 37 °C. After washing three times with PBS-T, HRP-labelled anti-mouse IgA secondary antibody at a 1/2,000 dilution (100 μl per well) was added and incubated for 1 h at 37 °C. Samples were removed and washed with PBS-T four times and TMB soluble substrate (Thermo Fisher) was added to each well (100 μl per well) for 30 min incubation at room temperature. Stop solution (50 µl) (2 M H2SO4, Sigma-Aldrich) was added and optical absorption at 450 nm was measured on a plate reader. Calculation for the end-point titre of IgA is the same as that for IgG as described above.

Cytokine measurement in splenocytes

Splenocytes from each vaccinated mouse were challenged with 1 μg ml−1 RBD and plated into ELISpot wells (106 per well) (R&D Systems) that were coated with anti-mouse IFN-γ capture antibody. RBD-specific cells secreting IFN-γ were measured using an ELISpot assay according to the manufacturers’ protocol. Spot-forming units (s.f.u) were analysed using an anatomical microscope (Nikon) and the spots were counted using ImageJ software (NIH; https://imagej.nih.gov/ij/). Splenocytes from each vaccinated mouse were cultured in 6-well plates (5 × 106 cells per well) and restimulated with 5 μg ml−1 RBD. After a 48 h incubation, antigen-specific cytokine amounts of IL-6 and TNF-α from culture medium were detected by ELISA using a mouse IL-6 ELISA kit (RAB0308, Sigma-Aldrich) and mouse tumour necrosis factor α ELISA kit (RAB0477, Sigma-Aldrich) following the manufacturer’s protocols.

T-cell immunity in pneumocytes and splenocytes

Mouse pneumocytes and splenocytes were obtained at day 7 post vaccinations and plated at a 1 × 106 per well concentration. A peptide pool composed of 15-mers (overlapping by 15 amino acids) spanning the SARS-CoV-2-S RBD (PP002-A, Sino Biological) was employed to stimulate pneumocytes and splenocytes for 12 h at a cell incubator. Brefeldin A (1 μg ml−1) was then added into pneumocytes and splenocytes for a 4 h incubation, followed by collection and staining of cells with anti-CD4-FITC (100406, Biolegend) or anti-CD8-FITC (ab22504, Abcam). Then, cells were washed, fixed using 4% paraformaldehyde and permeabilized with saponin for intracellular staining of anti-IFN-γ-PE (507806, Biolegend), anti-IL-14-PE (504103, Biolegend) or anti-IL-17a-APC (17-7177-81, Invitrogen). After a 1 h incubation, cells were collected, washed and tested with a CytoFLEX flow cytometer (Beckman Coulter) and analysed using FCS Express V6 (De Novo software; https://denovosoftware.com).

Hamster studies with live SARS-CoV-2

Fifteen male and female Syrian golden hamsters (6–8 weeks old; Envigo) were randomly divided into three treatment groups. All hamsters were housed at Bioqual. Hamsters were administered with two doses of PBS (placebo), RBD or RBD-Exo 1 week apart by nebulization (n = 5 per group, 3 females/2 males). At 1 week after the vaccinations, the hamsters were challenged with 1.99 × 104 TCID50 of SARS-CoV-2 using the intranasal and intratracheal routes (50 μl in each nare). BAL, OS and blood were collected at the indicated time. Hamsters were necropsied at day 7 post challenge. All immunologic and virologic analyses were conducted blindly. All hamster studies were performed in compliance with all relevant local, state and federal regulations and were approved by the Bioqual Institutional Animal Care and Use Committee (20–091P).

Histopathology and immunohistochemistry in infected hamsters

Tissues were fixed with 4% paraformaldehyde for 24 h and transferred to 70% ethanol. The samples were paraffin embedded within 7 d of fixation and blocks were sectioned at 5 µm. Slides were then baked for 1 h at 65 °C, deparaffinized in xylene and rehydrated by a series of graded ethanol to distilled water. Subsequently, the slides were stained with hematoxylin (HSS16, Sigma-Aldrich) and eosin Y (318906, Sigma-Aldrich). Trichrome (HT10516, Sigma-Aldrich) assay was conducted according to the manufacturer’s instruction. Optical microscopy was performed to analyse these slides. For SARS-N, CD3, MPO and MX1 for IHC staining, dewaxing and rehydration were first performed, followed by retrieval in citrate buffer (AP9003125, Thermo Fisher) and treatment with 3% H2O2 in methanol for 10 min. Slides were permeabilized and blocked with Dako Protein blocking solution (X0909, DAKO) containing 0.1% saponin (47036, Sigma-Aldrich). Then, slides were incubated with primary rabbit anti-SARS-N antibody (Novus, NB100-56576, 1:200), rabbit anti-CD3 (Abcam, ab16669, 1:200), rabbit anti-MPO (Thermo Fisher, PA5-16672, 1:200) and anti-MX1 (Millipore Sigma, MABF938, 1:200) overnight at 4 °C, followed by goat anti-rabbit HRP secondary antibody (Abcam, ab6721, 1:1,000) or goat anti-mouse HRP secondary antibody (Abcam, ab6789, 1:1,000) for 1 h at r.t., counterstaining with hematoxylin and then bluing with 0.25% ammonia water.

SARS-CoV-2 genomic RT-qPCR assay

A QIAsymphony SP (Qiagen) automated sample preparation platform along with a virus/pathogen DSP midi kit and the complex800 protocol were used to extract viral RNA from 800 µl of oral swabs or bronchoalveolar lavage fluid. A reverse primer specific to the orf1a sequence of SARS-CoV-2 (5′-CGTGCCTACAGTACTCAGAATC-3′) was annealed to the extracted RNA and then reverse transcribed into complementary DNA using SuperScript III Reverse Transcriptase (Thermo Fisher) along with RNase Out (Thermo Fisher). The resulting cDNA was then treated with RNase H (Thermo Fisher) and added to a custom 4x TaqMan gene expression master mix (Thermo Fisher) containing primers and a fluorescently labelled hydrolysis probe specific for the orf1a sequence of SARS-CoV-2 (forward primer 5′-GTGCTCATGGATGGCTCTATTA-3′, reverse primer 5′-CGTGCCTACAGTACTCAGAATC-3′, probe 5′-/56-FAM/ ACCTACCTT/ZEN/GAAGGTTCTGTTAGAGTG GT/3IABkFQ/-3). All PCR setup steps were performed using QIAgility instruments (Qiagen). The qPCR was then carried out on a QuantStudio 3 real-time PCR system (Thermo Fisher). SARS-CoV-2 genomic (orf1a) RNA copies per reaction were interpolated using quantification cycle data and a serial dilution of a highly characterized custom RNA transcript containing the SARS-CoV-2 orf1a sequence. Mean RNA copies per ml were then calculated by applying the assay dilution factor (11.7). The limit of quantification for this assay is approximately 31 RNA copies per ml (1.49 log10) with 800 μl of sample.

RNAscope in situ hybridization in hamsters

SARS-CoV-2 antisense specific probe v-nCoV2019-S (ACD, 848561) and SARS-CoV-2 v-nCoV2019-S-sense (ACD, 845701) were used to respectively target the positive-sense and the negative-antisense of the Spike sequence. Before performing RNAscope assay, slides were first deparaffinized in xylene, rehydrated and incubated with RNAscope H2O2 (ACD, 322335) for 10 min at room temperature, followed by treatment with retrieval in ACD P2 retrieval buffer (ACD, 322000) for 15 min at 98 °C. Then, slides were incubated with protease plus (ACD, 322331) for 30 min at 40 °C. Probe hybridization and detection were performed through the RNAscope 2.5 HD Detection Reagents-RED (ACD, 322360) according to the manufacturer’s instructions.

Immunofluorescence staining of hamster lung sections

In brief, the pretreatment of slides was the same as for IHC, including dewaxing, rehydration, retrieval and 3% H2O2 treatment. Then, slides were blocked with 5 % BSA for 30 min, followed by 3 rinses with DPBS. Subsequently, slides were incubated with primary rabbit anti-SARS-N antibody (1:200) overnight at 4 °C and goat anti-rabbit Alexa Fluor 647 (Abcam, ab150079, 1:500), AF-488-CD206 (Santa Cruz Biotechnologies,sc-376108 AF488, 1:150) and Alexa Fluor 568-Iba-1 (Abcam, ab221003, 1:200) were incubated at r.t. for 1 h, or goat anti-rabbit Alexa Fluor 647 (Abcam, ab150079, 1:500) and FITC-pan-CK (Abcam, ab78478, 1:200) were incubated at r.t. for 1 h. Finally, slides were mounted with ProLong Gold antifade mountant with 4′,6-diamidino-2-phenylindole (Invitrogen) and imaged on an Olympus FLUOVIEW CLSM (Olympus, FV3000).

SARS-CoV-2 D614G pseudovirus neutralization assay

Female CD1 mice (7 weeks old) were immunized with PBS, RBD, RBD-Exo or RBD-Exo LTS in two doses once a week via nebulization. At 7 d after vaccinations, each mouse was challenged with 8 × 108 genomic copies (GC) of SARS-CoV2 D614G pseudovirus carrying the GFP reporter (C1120G, Montana Molecular). Lungs were collected and imaged at 24 h post vaccination with a Xenogen live imager (PerkinElmer) and an Olympus FLUOVIEW CLSM (Olympus, FV3000).

IgA and IgG purification

IgA antibodies in BALF and NPLF of immunized mice were collected and purified. IgG antibodies were collected from serum of mice and purified according to previous reports44. Briefly, serum, BALF or NPLF (500 μl) were diluted with DPBS, heat-inactivated at 56 °C for 1 h and mixed with protein L/agarose beads (gel-protl-2; InvivoGen) overnight at 4 °C. The above solution was transferred to chromatography columns and washed with PBS (10 mM, pH 7.4) at a volume of 10 column. IgA and IgG were then collected by eluting with pH 3.0 glycine buffer (0.1 M). Then, pH was immediately adjusted to 7.5 using 1 M tris (pH 8.0). The collected IgA and IgG were transferred to PBS (1×) buffer using Amicon ultra centrifugal filters with a 10 kDa membrane. IgA and IgG concentrations were determined by measuring the absorbance at 280 nm with a NanoDrop (Thermo Fisher) instrument.

Blockage activity of IgA antibodies

Primary small airway epithelial cells were co-cultured with primary bronchial/tracheal epithelial cells (lower chamber) using transwells for 24 h. Purified IgA antibodies from mice vaccinated with PBS, free RBD, RBD-Exo or RBD-Exo LTS were incubated with SARS-CoV-2 D614G pseudovirus for 1 h at 37 °C. The concentration of SARS-CoV-2 D614G pseudovirus used was 6.6 × 108 GC ml−1. After incubation, the mixture was added to the transwell and incubated for another 24 h. The primary small airway epithelial cells in the upper chamber were collected for flow cytometry assay, while the primary bronchial/tracheal cells in the lower chamber were imaged by microscopy.

IgA and IgG neutralization assay

To compare their protection activity against SARS-CoV-2 variant, purified IgA and IgG antibodies from PBS or RBD-Exo treatments were incubated with SARS-CoV-2 D614G pseudotyped virus for 1 h at 37 °C. Subsequently, they were incubated with ACE2-expressing A549 cells (a549-hace2tpsa, Invivogen) for 24 h. After incubation, cells were washed with PBS and the GFP positive cells were counted by microscopy.

Statistical analysis

All quantitative experiments were conducted in triplicate independently. Data are shown as mean ± s.d. Two-tailed unpaired Student’s t-test was used to analyse differences between any two groups. Comparisons of more than two groups were conducted using one-way analysis of variance (ANOVA) followed by post hoc Bonferroni test. Grouped data were analysed by two-way ANOVA followed by Tukey post hoc test for multiple comparisons. P < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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