A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein

Ethics statements

The Ethics Review Board of the Kunming Institute of Zoology, Chinese Academy of Sciences (CAS) approved this study. All animal experiments were approved by the Ethics Committee of Kunming Institute of Zoology, CAS (assurance No.: SMKX-2021-01-006), and were carried out in strict compliance with the guidelines and regulations of the Animal Care and Use Committee, Kunming Institute of Zoology, CAS. All SARS-CoV-2 infection experiments were approved (assurance No.: KIZP3-XMZR-2021-05) and carried out in the biosafety level-3 (BSL-3) laboratory of Kunming Institute of Zoology, CAS. The human serum samples in this study were collected in accordance with the Declaration of Helsinki. Informed consent was obtained from each participant.

Viruses and cells

The prototype SARS-CoV-2 strain (Accession No.: NMDCN0000HUI in the China National Microbiology Data Center (NMDC)) was provided by the Guangdong Provincial Center for Disease Control and Prevention (Guangzhou, China). The SARS-CoV-2 Omicron BA.2 variant was isolated from a patient in Yunnan Provincial Infectious Disease Hospital, China. Its whole genome was sequenced by researchers at BSL-3 laboratory of Kunming Institute of Zoology, CAS, Kunming, China, and submitted to NMDC (No. SUB1663744906574). The viruses were propagated in African green monkey kidney epithelial cells (Vero-E6) (ATCC, #1586) and titrated. Virions from the 4th passage were used in the current experiments. VSV-G pseudotyped virus (G*ΔG-VSV-Rluc) was kindly provided by Prof. Geng-Fu Xiao (Wuhan Institute of Virology, CAS). HEK293T cells were obtained from ATCC. 293T-ACE2 cells were provided by Prof. Yuxian He (Institute of Pathogen Biology, Chinese Academy of Medical Sciences). These cells were cultured in basal DMEM (Gibco, Beijing, China) supplemented with 10% FBS (Gibco, New Zealand). Human pulmonary alveolar epithelial cells (HPAEpiCs) were purchased from the ScienCell Research Laboratory (San Diego, CA, USA) and were cultured in basal DMEM supplemented with 10% FBS,88 and passages 6–8 were used in this study.

Human serum samples

Nine serum samples from SARS-CoV-2-vaccinated participants and one healthy unvaccinated serum sample were collected from our laboratory. The participants were injected with two doses of inactivated SARS-CoV-2 vaccine (Sinovac Biotech, Beijing, China) within 1–2 months. Eleven serum samples from COVID-19 convalescent individuals were collected at Yunnan Provincial Infectious Disease Hospital. All convalescent patients had recovered from COVID-19 for 1–2 months.

Animals

BALB/c mice and New Zealand white rabbits were purchased from the Experimental Animal Center of Kunming Medical University, China. The hACE2 transgenic mice were established as we previously reported.89,90 Syrian golden hamsters were purchased from Charles River Company (Beijing, China). Rhesus macaques were purchased from Kunming Primate Research Center, Kunming Institute of Zoology.

Plasmid construction

Recombinant protein HR121 from the S protein of SARS-CoV-2 was designed as HR1–linker1–HR2–Linker2–HR1. Genes encoding the full amino acid sequence of HR1 (residues 912–988) and HR2 (residues 1163–1206) were derived from the S protein of SARS-CoV-2 isolate Wuhan-Hu-1 (accession No.: NC_045512).91 The nucleotide sequence encoding linker 1 (GSSGG) was GGAGGAAGCGGAGGA and the nucleotide sequence encoding linker 2 (SGGRGG) was AGCGGAGGAAGAGGAGGA. The gene encoding HR121 was synthesized and cloned into the E. coli expression vector pMCSG7 with an N-terminal SUMO tag using the ligation-independent cloning method.92

To express the GST-HR121 fusion protein, the full HR121 sequence was inserted into the E. coli expression vector, pGEX-6P-1, at the restriction enzyme sites of EcoRI and XhoI.

To express HR1, HR2, and HR12 proteins, the genes encoding these proteins were separately cloned into the E. coli expression vector, pET-30a, at EcoRI and XhoI restriction enzyme sites. Therefore, each of these expressed proteins contains the redundant 52 amino acid residues from the vector.

Protein expression and purification

SUMO-tagged HR121 in the pMCSG7 vector was expressed in E. coli BL21 (DE3), and bacteria harboring the expression vector were grown at 37 °C in LB media supplemented with 100 μg/mL ampicillin. Protein expression was induced with 0.5 mM IPTG when the cells reached an optical density of 0.6 at 600 nm. The cells were then cultured at 16 °C for another 16 h. Then, the cells were harvested by centrifugation at 5000× g for 10 min at 4 °C. The cells were resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM β-ME), and lysed using ultrasonication. Then, the supernatant containing the recombinant proteins was separated by centrifugation at 12,000× g for 30 min at 4 °C. The fusion proteins were isolated by Ni Sepharose 6 FF (GE Healthcare, Beijing, China), and the SUMO tag was removed by TEV enzyme (1:100 w/w) cleavage. The target protein was loaded to a Superdex 200 increase column (GE Healthcare) with buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl and 2 mM DTT. Peak fractions containing HR121 dimer were pooled, concentrated to 25 mg/mL and stored in a –80 °C freezer.

The recombinant proteins HR1, HR2, and HR12 were expressed in E. coli BL21 (DE3) cells. Bacteria were induced with 1 mM IPTG for 12 h at 20 °C before harvesting by centrifugation. The collections were lysed in PBS buffer by ultrasonication. The supernatants were separated by centrifugation at 12,000× g for 30 min at 4 °C, and the target proteins were purified using Ni Sepharose 6 FF affinity column. Briefly, the column was washed in gradients with 20 mM and 50 mM imidazole. Proteins were then eluted by 100 mM imidazole and concentrated to 10 mg/mL in PBS through a concentrating column with a 3 kDa cutoff (Millipore, Bedford, MA, USA).

The fusion protein GST-HR121 was expressed and induced using the same method as that for the recombinant proteins HR1, HR2, and HR12. Purification was carried out by glutathione–Sepharose 4B affinity column (GE Healthcare).

Crystallization and structure determination

Crystals were obtained using the sitting-drop vapor diffusion method by commercial crystallization kits and incubation at 16 °C for 10 days. The crystals appeared in a solution containing 0.2 M sodium fluoride and 20% (w/v) polyethylene glycol 3350. Then, the crystals were harvested using 20% ethylene glycol (v/v) as a cryoprotectant before flash freezing in liquid nitrogen. The crystals were analyzed with beamline BL02U1 at the Shanghai Synchrotron Radiation Facility.93 The structure was determined by the molecular replacement method using PHASER94 and refined using PHENIX.95 The structure of 6-HB of SARS-CoV-2 (Protein Data Bank: https://www.rcsb.org/, with the accession number 6LXT) was used as the initial search model. The model of HR121 was manually adjusted in COOT96 and refined to a resolution of 2.41 Å with Rwork and Rfree values of 24.9% and 29.1%, respectively. Details of data collection and refinement statistics are provided in Supplementary information, Table S1. All figures representing structures were prepared using PyMOL (http://pymol.org).

GST pull-down assay

Excess HR1 or HR2 in the bacterial supernatants was mixed with glutathione–Sepharose 4B affinity gel containing GST-HR121. Blank glutathione–Sepharose 4B affinity gel mixed with HR1 or HR2 and glutathione–Sepharose 4B affinity gel containing GST-HR121 were used as negative controls. The mixture was incubated for 30 min at room temperature with gentle agitation. Then, the supernatants were removed by centrifugation at 500× g for 5 min. The gels were washed three times with PBS by the same method of centrifugation, and collected for 10% SDS-PAGE analysis.

Surface plasmon resonance assay

HR121 binding to HR2 or HR1 was determined by surface plasmon resonance using a BIAcore 3000 instrument (GE Healthcare). Briefly, HR121 was immobilized on the flow cell of a CM5 sensor. HR2 or HR1 protein (12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM) was injected to run across the chip. A separate channel was set as a control. The binding assays were performed at 25 °C. HR2 or HR1 was dissolved in BIAcore running buffer and injected at a constant flow rate of 35 μL/min for 3 min. Dissociation data were collected for 10 min. The kinetic parameters were obtained using an automated program.

CD spectroscopy

CD spectra were recorded using a Jasco spectropolarimeter (model J-815). 1 μM each of HR121 and HR2 or HR1 was dissolved in PBS. Using a 0.1 cm pathlength cuvette, wavelength spectra were recorded with a 1-nM step size and 1-nM bandwidth from 195 nm to 260 nm at 20 °C. The spectra were corrected by subtracting the solvent blank, PBS.

Animal immunization

Rabbits, mice, and hamsters were subcutaneously immunized with HR121 or HR12 formulated with CFA (Sigma-Aldrich, Saint Louis, MI, USA) and IFA (Sigma-Aldrich). Macaques were subcutaneously immunized with HR121 formulated with IFA. Another group of hamsters were intramuscularly immunized with HR121 formulated with aluminum adjuvant (Alhydrogel® adjuvant 2%, Invivogen, San Diego, CA, USA). All immunizations were performed in a prime boost-reboost manner.

For rabbit immunization, 11 adult female New Zealand White Rabbits (average weight: 2.8 kg) were used. Four and three rabbits were injected with HR121 plus CFA/IFA and HR12 plus CFA/IFA in the same procedure, respectively. Each rabbit was immunized with 100 μg protein on day 0, and then 150 μg protein on days 21 and 42. The other 4 rabbits were injected with equal volumes of Freund’s adjuvant as mock controls. Serum samples were collected 14 days after the third immunization. IgGs were purified from the serum samples using Protein A (BBI, Shanghai, China).

For BALB/c mouse (male, 8 weeks old) immunization, three groups of mice (n = 6 per group) were injected with 2 μg, 10 μg, or 50 μg HR121 plus CFA/IFA at 14-day intervals. Two groups of mice (n = 6 per group) were injected with equal volumes of CFA/IFA or PBS (mock controls). Serum samples were collected at 7 days post each immunization, and 60 days post the third immunization. At 90 days post the third immunization, the mice were euthanized, and their splenocytes were isolated as previously reported.9 To optimize the HR121 immunization interval in mice, another group of mice (n = 8) was injected with 10 μg HR121 at 21-day intervals. Serum samples were collected 7 days after the third immunization.

For hACE2 mouse (male, 8 weeks old) vaccination, 8 hACE2 mice were vaccinated with 10 μg HR121 plus CFA/IFA at 14-day intervals. Equal numbers of mice injected with Freund’s adjuvant or PBS were used as controls. Serum samples were collected 7 days after the third immunization. SARS-CoV-2 challenge was carried out 14 days after the third immunization.

Syrian golden hamsters (male, 8 weeks old) were injected with 15 μg HR121 formulated with adjuvant, or adjuvant, or PBS only at 14-day intervals. For SARS-CoV-2 challenge studies, 39 hamsters were divided into two groups for short- and long-term protection studies. In STP group (n = 25), 10 and 9 hamsters were immunized with HR121 plus CFA/IFA and CFA/IFA only, respectively, while 6 hamsters were injected with PBS as mock control. Serum samples were collected 7 days after the third immunization. SARS-CoV-2 challenge was carried out 14 days after the third immunization. In LTP group (n = 14), 8 and 6 hamsters were injected with HR121 plus CFA/IFA and CFA/IFA only, respectively. Serum samples were collected 83 days after the third immunization. SARS-CoV-2 challenge was carried out 90 days after the third immunization.

For Omicron BA.2 challenge studies, 48 hamsters were divided into 4 groups (12/group). Hamsters in Groups 1 and 2 were injected with HR121 plus CFA/IFA and CFA/IFA only, respectively, while those in Groups 3 and 4 were immunized with HR121/aluminum adjuvant and aluminum adjuvant only, respectively. Serum samples were collected 7 days after the third immunization. Omicron BA.2 challenge was carried out 14 days after the third immunization.

For rhesus macaque (3 males and 5 females, 9–13 years old) vaccination, 4 macaques were vaccinated with 50 μg HR121/IFA at 30-day intervals. Equal numbers of macaques injected with adjuvant were used as controls. Serum samples were collected 7 days after each immunization. SARS-CoV-2 challenge was carried out 7 days after the third immunization.

Animal passive vaccination

To evaluate the in vivo neutralizing activity of nAbs from HR121-immunized rabbits, sera from the 4 HR121-immunized rabbits were pooled. The IgGs purified from them (~5 mg IgGs obtained from 1 mL sera) were transferred intraperitoneally to 6 hACE2 mice (8 weeks old) or 9 hamsters (8 weeks old) at a dose of 5 mg IgGs/20 g body weight, while isotypical IgGs purified from adjuvant-immunized rabbits were transferred to 4 mice or 8 hamsters as mock controls. 24 h after transfer, SARS-CoV-2 challenge was carried out.

SARS-CoV-2 challenge

To challenge hACE2 mice vaccinated with HR121 (adjuvant and PBS as controls), the mice were anaesthetized with isoflurane (RWD, Shenzhen, China) and inoculated intranasally with 107 TCID50 of SARS-CoV-2 in 30 μL. Lungs were collected 5 days post infection (dpi).

To challenge Syrian golden hamsters vaccinated with HR121, the hamsters were anesthetized and inoculated intranasally with 104 TCID50 of SARS-CoV-2 or 103 TCID50 of Omicron BA.2 in 100 μL. Lungs were collected at 3 dpi.

To challenge rhesus macaques, the macaques were anesthetized with Zoletil-50 (FeiBo, Beijing, China) and inoculated equally by intranasal and intratracheal routes with 3 × 107 TCID50 of SARS-CoV-2 in 2 mL. Lungs were collected at 7 dpi.97

To challenge the animals passively administered with rabbit sera, hACE2 mice were inoculated with 106 TCID50 of SARS-CoV-2 and Syria golden hamsters were inoculated with 104 TCID50 of SARS-CoV-2, respectively. Lungs were collected at 3 dpi.

bAb detection

A sandwich ELISA was used to detect the bAbs of HR1, HR2, HR12, and HR121 in serum samples. Briefly, HR1, HR2, HR12, or HR121 protein (1 μg/mL) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) was coated on 96-well polystyrene plate at 4 °C overnight. After removing the coating buffer, the plate was washed 3 times with PBS containing 0.05% Tween-20 (PBST) and blocked with blocking buffer (PBST containing 5% BSA) at 37 °C for 2 h. Then the plate was washed 3 times, and serially-diluted serum samples were added to the plate and incubated for 2 h at 37 °C. After washing again, OPD substrate was added to each well. The reaction was stopped with 2 M H2SO4 and the optical density (OD) values of the wells were read on an ELISA reader at 490 nm/630 nm. The endpoint titers of the serum samples were determined as the reciprocal of the last dilution exhibiting an OD value ≥ 2.1-fold that of the average background values.

HR2/HR121 binding inhibition

Competitive ELISA was used to detect antibodies blocking the binding of HR2 and HR121.56 Briefly, HR121 protein (1 μg/mL) was coated onto the ELISA plate at 4 °C overnight. After the coating buffer was removed, the plate was washed with PBST and incubated with blocking buffer at 37 °C for 2 h. Then, the plate was washed 3 times with PBST. Meanwhile, serum or IgG samples from rabbits or humans were serially diluted in PBST and preincubated with HR2-labeled HRP (KPL, Gaithersburg, MD, USA) in 100 ng/mL for 20 min at 20 °C. Then the mixtures were added to each well of the plate and incubated for 1 h at 37 °C. After washing the plate and adding the OPD substrate, the OD values of the plates were determined at 490 nm/630 nm. The percentage inhibition of HR121/HR2 binding was calculated using the following formula: % inhibition = [1 – (E – N)/(P – N)] × 100, where E represents the OD value in the presence of a serum or IgG sample, P represents the OD value in the absence of the serum or IgG sample, and N corresponds to the OD value in the absence of the sample and HR2-labeled HRP. The inhibition data were plotted and log-transformed in GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA, USA). The NT50s in the serum and IC50s in the IgG samples were calculated using the “nonlinear regression (curve fit) — log(inhibitor) vs response -- variable slope (four parameters)” model.

VSV pseudotyped SARS-CoV-2 env variant production

To construct VSV-based SARS-CoV-2 pseudoviruses, pCMV3 plasmid containing the full sequence of the codon-optimized spike gene was purchased (Sino Biological, Beijing, China). The spike gene with a C-terminal 18-amino acid truncation of the SARS-CoV-2 Wuhan-Hu-1 strain was constructed by PCR and inserted into the eukaryotic expression plasmid pcDNA3.1(+) between the BamH1 and EcoR1 sites. A Kozak sequence (GCCACC) was introduced in front of the spike gene to generate the recombinant plasmid pcDNA3.1-SARS-CoV-2-SΔ18.98 Based on pcDNA3.1-SARS-CoV-2-SΔ18 sequence, a panel of plasmids containing the SARS-CoV-2 spike-related mutants of S477N, E484K, A222V, N439K, K417N, D614G, and D839Y were introduced using a Fast mutagenesis system kit (Transgen Biotech, Beijing, China). Another panel of plasmids containing the spike genes of 17 important SARS-CoV-2 variants, including B.1.617, B.1.617.1, B.1.617.2.V2, B.1.429, B.1.525, B.1.526, B.1.1.7, B.1.351, B.1.1.28, B.1.617.2, C.37, B.1.621, B.1.1.529 (Omicron BA.1), Omicron BA.2, Omicron BA.3, and Omicron BA.4/5, were synthesized in codon optimization with the corresponding gene fragments. The primers for the point mutants are listed in Supplementary information, Table S2, and the multiple mutations introduced in the spikes of 13 important SARS-CoV-2 variants are listed in Supplementary information, Table S3.

SARS-CoV-2 pseudovirus was prepared using a VSV pseudotyped SARS-CoV-2 S packaging system as described previously.99 Briefly, 1 × 106 293T cells were seeded on a T75 cell flask in DMEM low-sugar medium (Gibco) at 37 °C overnight. After reaching 80% confluence, the cells were transfected with 10 μg pcDNA3.1-SARS-CoV-2-variant-SΔ18 using jetPRIME transfection reagent (Polyplus-transfection, Illkirch, France). After 24 h, the cells were infected with G*ΔG-VSV-Rluc virus at an M.O.I. = 1. Six hours after infection, the cells were washed three times with PBS containing 1% FBS. Thirty-six hours after infection, the supernatant was collected, centrifuged at 1000× g for 10 min, aliquoted, and stored at −80 °C. The TCID50 of the SARS-CoV-2 pseudotyped virus was determined in 293T-ACE2 cells, as previously described.99

Pseudovirus-based neutralization assays

To measure the neutralizing activity of sera or IgGs against various SARS-CoV-2 variant pseudotyped viruses, 1 × 104 293T-ACE2 cells (100 μL) were seeded in 96-well plates in DMEM low-sugar medium (Gibco). The next day, the sera from HR121-immunized animals were three-fold serially diluted in another 96-well plate in a volume of 60 μL. Then, 60 μL SARS-CoV-2 pseudovirus (M.O.I. = 0.1) was added to the diluted sera and incubated for 1 h at 37 °C. Thereafter, 100 μL of the mixture was incubated with 293T-ACE2 cells for 24 h at 37 °C, and the supernatant was removed from the cells after incubation. Renilla luciferase activity was determined using a Renilla luciferase assay kit (Promega, Madison, WI, USA). The NT50s of the sera or IC50s of the IgGs were calculated in GraphPad Prism 8.0.1 software as mentioned above.

SARS-CoV-2-based neutralization assays

8 × 105 HPAEpiC cells (200 μL) were seeded into each well of a 48-well plate and incubated at 37 °C overnight. The next day, sera or IgGs from HR121-immunized animals were two-fold serially diluted in another 48-well plate at a volume of 100 μL. Then, 100 μL SARS-CoV-2 (M.O.I. = 1) were added into the diluted sera or IgG and incubated for 1 h at 37 °C. Thereafter, the medium was removed and replaced with the virus-serum or virus-IgG mixture. After incubating at 37 °C for another 1 h, the mixture was removed and washed 3 times with PBS. Subsequently, fresh medium (200 μL) containing the same diluted sera or IgGs was added. The cells were cultured at 37 °C for 48 h, and then the supernatants were collected for viral RNA extraction by kit (Roche Diagnostics, Mannheim, Germany), followed by analysis of viral load (viral genome RNA). The NT50 and IC50 values were calculated using GraphPad Prism 8.0.1 as mentioned above.

Viral load measurement

RNA was extracted from lung tissues using Trizol (Life Technologies, Carlsbad, CA, USA). The RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific, USA). Viral gRNA and sgRNA were measured by real-time qPCR using a one-step qRT-PCR kit (QRZ-101, Toyobo, Osaka, Japan) on a ViiA7 Real-Time PCR System (Life Technologies). The primers for gRNA detection were derived from the nucleocapsid (N) gene of SARS-CoV-2, as previously described,88 including forward, 5′-GGGGAACTTCTCCTGCTAGAAT-3′; reverse, 5′-CAGACATTTTGCTCTCAAGCTG-3′; and probe, 5′-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′. The primers for sgRNA detection were derived from the envelope (E) gene of SARS-CoV-2, as previously described,7 including forward, 5′-CGATCTCTTGTAGATCTGTTCTC-3′; reverse, 5′-ATATTGCAGCAGTACGCACACA-3′; and probe, 5′-FAM-CGAAGCGCAGTAAGGATGGCTAGTGT-TAMRA-3′.

Real-time qPCR

Real-time qPCR was performed to evaluate the mRNA expression of inflammation-related cytokines in the lung tissues of hACE2 mice after SARS-CoV-2 challenge. Primers (for genes including IFNG, IL-2, IL-4, IL-6, IL-10, IP-10, MX2, and TNFA) were designed according to mouse mRNA sequences (Supplementary information, Table S4). cDNA was generated using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Beijing, China). Real-time qPCR was performed using a ViiA7 Real-Time PCR System and SYBR Premix Ex Taq II (Takara). Expression levels of the genes of interest were analyzed using the comparative cycle threshold (Ct) method, where Ct is the cycle threshold number normalized to that of ACTB mRNA. The fold change was calculated using the 2−ΔΔCt method by dividing the normalized quantity of post-infection samples by that of healthy hACE2 mouse samples.

Histopathology and immunohistochemistry

Lung tissues of SARS-CoV-2-infected animals were collected and fixed in 4% paraformaldehyde for 3 days, followed by embedding in paraffin and cutting into 3 μm sections. Some sections were stained with H&E for evaluation of lung injury, and other sections were stained with the SARS-CoV-2 nucleocapsid protein (Sino Biological) to evaluate SARS-CoV-2 replication levels in the lung tissues.

ELISpot assays

SARS-CoV-2 HR1-specific cytotoxic T lymphocytes (CTLs) were evaluated using a murine IFN gamma set (Diaclone Research, Besancon, France) according to the manufacturer’s instructions. Briefly, a MultiScreenHTS IP filter plate (Millipore) was pre-coated with anti-murine IFNγ. Then, 1 × 106 splenocytes isolated from BALB/c mice were co-cultured with a pool of 15-amino-acid overlapped peptides covering the full HR1 sequence in 1 μg/mL (Supplementary information, Table S5, synthesized by Generay Biotech) for 24 h. After incubation, the wells were washed and colored, and images were collected using an ImmunoSpot S6 universal analyzer (Cellular Technology Limited, Cleveland, OH, USA). Using an automated program, the spots were counted with parameters such as size, intensity and gradient.

To evaluate SARS-CoV-2 HR121-specific humoral responses, a MultiScreenHTS IP filter plate was pre-coated with HR121 (1 μg/mL), and washed 3 times. Then, 1 × 106 splenocytes isolated from BALB/c mice were added into each well and cultured for 24 h. Using the same method as that for IFNγ, the plate was imaged.

Statistics

All statistical analyses were performed using GraphPad Prism 8.0.1. P values were labeled in the figures. The titers of the bAbs or nAbs were presented as geometric mean ± geometric SD, and the copies of viral gRNAs or sgRNAs were presented as median ± interquartile range. Two-tailed Mann-Whitney test or Wilcoxon test was used to compare the difference between HR121 and control groups.

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