Fig 1: Development of modified DNA vaccine construct. (a) Expression of each or all mutated spike glycoprotein in HEK293 cells evaluated through western blotting. Spike was detected using polyclonal SARS-CoV-2 spike antibody (GTX135356, GeneTex). GAPDH was used as a loading control. Lower graph shows the ratio between spike protein and GAPDH, analyzed by densitometric measurement. (b) Localization of the spike glycoprotein expressed by the parental DNA vaccine construct, or the GP?-DNA vaccine construct, assessed through immunostaining in HEK293 cells. Nuclei were stained by DAPI. Scale bars: upper panel (control IgG) = 20 µm, lower 2 panels (anti-Spike Ab) = 10 µm. (c) Spike expression on the cell membrane, assessed using cell surface protein isolation. Spike was detected using monoclonal SARS-CoV-2 spike antibody (#42172, Cell Signaling Technology). a1-Na+/K+-ATPase expression was used as a loading control for isolated surface proteins. (d) GP?-DNA vaccine-induced antibody titer determined using ELISA. Parental DNA vaccine (666.6 µg); Parental, GP?-DNA vaccine (666.6 µg); GP?, or half amount of GP?-DNA vaccine (333.3 µg); GP? (Half), with alum adjuvant was intramuscularly injected into male SD rats three times at 2-week intervals. Control rats were not treated. The antibody titer was evaluated for 10 weeks after the first vaccination using ELISA. *p < 0.05 vs. control group (Two-way ANOVA with Tukey’s multiple comparison test). Data are shown as mean ± SEM. See also Supplementary Figs. 1–3.