The study in phase I clinical trial demonstrated that DNA vaccine using full-length wild type spike was safe and could induce both antibodies and T cells responses [59]

The study in phase I clinical trial demonstrated that DNA vaccine using full-length wild type spike was safe and could induce both antibodies and T cells responses [59]. rigid accordance with the recommendations of the Honest Principles and Recommendations for the Use of Animals for Scientific Purposes. Mouse experimental methods and animal management with this study was authorized by the Committee of Animal Care and Use, Faculty of Medicine, Chulalongkorn University or college Foretinib (GSK1363089, XL880) (approval quantity 007/2563). Immunization and blood selections were performed under isoflurane anesthesia. All attempts to minimize the suffering of the animals were made throughout the study. Cell tradition and viruses The African Green Monkey Kidney (Vero, ATCC CCL81), HEK293T (ATCC? CRL-3216TM) were from ATCC (Old Town Manassas, VA, USA). Vero and HEK293T cell lines were managed in MEM and DMEM, respectively, and supplemented with 10% heat-inactivated fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin (Gibco, USA). Cells were incubated at 37 OC, 5% CO2 atmosphere. The highly pathogenic SARS-CoV-2 virus was initially isolated from a clinical specimen of Thai patient-infected with SAR-CoV-2 virus, strain hCoV-19/Thailand/47/2020 by the National Institute of Health, Department of Medical Sciences, Thailand and was further propagated on Vero cells twice to obtain a large amount of viruses. Construction and preparation of recombinant plasmid DNA To construct the recombinant plasmids, the protein sequence of SARS-CoV-2 S protein published in GenBank and GISAID during December 2019 to February 2020 were analyzed. Synthetic genes with humanized codon optimization encoding S, S1 or S2 Foretinib (GSK1363089, XL880) were synthesized by GeneScript (Piscataway, NJ, USA) then subcloned into pCMVkan expression vector which contain the cytomegalovirus promoter, the bovine growth hormone polyadenylation site, and the kanamycin-resistant gene [24], designated as pCMVkan-S, pCMVkan-S1 and pCMVkan-S2, respectively, (nucleotide sequences are shown in S1 Data). In the S and S2 constructs, cytoplasmic region which contained ER-retention signal was deleted to enhance S protein trafficking to the cell surface. Signal peptide (SP), the first 15 amino acids at N-terminus of S protein, were Foretinib (GSK1363089, XL880) included in all constructs (Fig 1A). The recombinant plasmids were propagated in E. coli, CXADR DH5-alpha (Invitrogen, Carlsbad, CA, USA) and purified by Qiagen endotoxin-free giga plasmid kit (Hilden, Germany) following the manufacturers protocol. Characterization of the plasmids were performed by nucleotide sequencing and gel electrophoresis. Open in a separate window Fig 1 (A) Schematic diagram of three DNA vaccine constructs; SP: signal peptide, RBD: receptor binding domain name, HR: heptad repeat, TM: transmembrane domain name. (B) Mice immunization and sample collection schedule. Plasmids transfection and protein expression analysis At 24 hr before transfection, 1×106 cells of HEK293T were seeded in 6-well plate (Thermo Fisher Scientific, MA, Foretinib (GSK1363089, XL880) USA). The cells with approximately 80C90% confluency were separately transfected with individual recombinant plasmid constructs (pCMVkan-S, -S1 and -S2) using lipofectamine 3000? (Invitrogen, Carlsbad, CA, USA) according to the manufactures protocol. Briefly, 2.5 g of plasmid and 3.75 L lipofectamine 3000? were separately diluted in 125 L and 250 L serum-free Opti-MEM? medium (Gibco), respectively. P3000?, a transfection enhancer reagent provided in lipofectamine 3000? kit, was also added into diluted plasmid tube to a final concentration of 2 L/g DNA. The diluted DNA was mixed with the diluted lipofectamine 3000? at 1:1 ratio (v/v) and incubated for 15 min at room temperature. The plasmid-lipofectamine 3000? complex was then added onto cells. At 24 hr post-transfection, cells were fixed, permeabilized with ice-cold acetone and stained with 1:200 dilution of anti-S1 or anti-S2 polyclonal antibodies (Sino Biological, Foretinib (GSK1363089, XL880) Beijing China). Donkey-anti-rabbit IgG-FITC, 1:5000 (BioLegend, USA), was used as a secondary antibody following anti-S1, or anti-S2 staining. Cell nuclei were counter stained with 4, 6-diamino-2-phenylindole hydrochloride (DAPI) (Sigma-Aldrich, USA). Stained cells were visualized under fluorescence microscope (Olympus, Japan). Immunization of mice Six-week old of female ICR mice (20C25 grams) were procured from the National Laboratory Animal Center, Mahidol University. Mice were randomly allocated into 4 groups.