Yu et al. analyzed by IFN- ELISpot assay following activation of indicated peptides and recombinant proteins for 48 h.(TIF) pntd.0009374.s002.tif (766K) GUID:?B17BEADE-A52C-4DA7-B4DA-812F53571B79 Data Availability StatementAll relevant data are within the manuscript and its Supporting Info files. Abstract The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is definitely a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is definitely a novel option, beyond the traditional inactivated computer virus vaccine or recombinant protein vaccine. Here, we statement a DNA vaccine comprising the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell manifestation and then cloned into mammalian cell manifestation vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient manifestation in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced related levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding website of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell reactions and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protecting immunity against SARS-CoV-2 challenge for plasmid amplification. Plasmids were extracted and purified using an endotoxin-free Qiagen column system (EndoFree Plasmid Mega Kit). Transient manifestation and Western blot HEK293T cells were transfected with the indicated Kobe2602 DNA plasmids using PolyJet? reagent (SignaGen Laboratories) following a manufacturers protocol. At 24 hours post transfection, cell lysates were harvested and subjected to electrophoresis on 8% SDS-PAGE. The proteins were then transferred to PVDF membranes and blotted with rabbit anti-Spike polyclonal antibody (40592-T62, Sino Biological). Horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were used as the secondary antibody. Specific proteins within the membrane were visualized by ECL reagent (Thermo Scientific). Animal immunization BALB/c, C57BL/6 mice and Syrian hamsters were from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). Mice or hamsters were used between Kobe2602 6 and 12 weeks of age. Anesthetized mice or hamsters were vaccinated with 100?L of answer containing indicated DNA inside a 3-week interval, followed by electroporation having a BTX electroporator (ECM830) using Kobe2602 two-needle array electrodes (5-mm diameter (BTX 45C0121)). Intramuscular electroporation was performed at 75 V constant voltage with 10 pulses at 50 msec/pulse and 100-msec intervals between pulses. Blood samples of mice and hamsters were collected by submandibular or retroorbital blood samplings, respectively. All animals were housed in the Laboratory Animal Center of the National Health Study Institutes (NHRI) and managed in accordance with institutional animal care protocols. Immunoassay The presence of S-specific antibodies in sera was determined by ELISA. Briefly, 50 L of 4 g/mL recombinant protein (Sino Biological) in 0.1 M carbonate buffer (pH 9.5) was coated onto 96-well microplates by overnight incubation at 4C. Coated plates were washed twice with 0.05% Tween 20 in PBS and then blocked with 3% BSA in PBS at room temperature for 2 hours. Diluted sera from immunized animals were applied to wells at space heat for 2 hours. Following a addition of HRP-conjugated goat anti-mouse IgG (Thermo Scientific) or HRP-conjugated goat anti-hamster IgG (Arigo Biolaboratorie), the assay was developed by using SureBlue TMB 1-Component Peroxidase Substrate (KPL). The absorbance was measured using an ELISA reader at 450 nm. Neutralization of SARS-CoV-2 computer virus illness Vero cells were seeded (2.4104 cells/well) in 96-well plates for 24 h to form a monolayer. Preimmune sera and antisera against SARS-CoV-2 S protein were pretreated at 56C for 30 minutes to ruin heat-labile nonspecific viral inhibitory substances. The sera were diluted to an initial dilution of Kobe2602 1/20 with M199 medium, added into a well comprising 200 TCID50 of SARS-CoV-2 computer virus in a volume of 0.2 mL, Rabbit Polyclonal to CCRL1 and then incubated at 37C for 2 h. Subsequently,.