Fig. 1: Optimization of electrostimulation conditions using electroacupuncture machine.

BALB/c mice (n = 4 per group) were intramuscularly immunized twice at a 3-week interval with vector or pSARS2-S DNA, followed by electrostimulation by an electroacupuncture machine under different peak-to-peak voltages (Vpp), total stimulation times, or amounts of DNA. Serum samples were collected at week 6 after the first immunization. a–c IgG antibodies against spike protein were assessed by ELISA. d Vaccine‐induced neutralizing activity against wild-type SARS-CoV-2 was evaluated by neutralization assay. The dashed line indicates the initial fold dilution of serum samples. e, f BALB/c mice were electrostimulated with 0, 5, 21, and 40 Vpp for 5 s. At the indicated timepoints after electrostimulation, muscle tissues at the injection site (gastrocnemius muscle) were collected and examined by hematoxylin and eosin staining. e Representative images of inflammation in the muscle tissue. Enlarged views of the area in the black box are shown at the bottom. Scale bar for low-magnification: 200 μm, for high-magnification: 50 μm. f The inflammatory severity score was evaluated based on the percentage of the inflammatory area. Antibody titers and severity scores are presented as the mean ± SEM, and neutralization titers are expressed as the geometric mean with a 95% confidence interval. *p < 0.05 by the Mann–Whitney test.