Fig. 2: Mice vaccination and immune responses analysis.

a Mice groups. The Balb/c mice were randomly divided into four groups, with 20 mice in each group. b Mice vaccination and sample collection. The vaccines were administered intramuscularly with a dose of 10 µg on days 0 and 14, respectively. c A35R-specific antibodies. The sera collected from mice on days 7, 13, and 35 were tested for A35R-specific antibodies using ELISA against recombinant A35R protein. n = 12 to 15 for day 7 and day 13 and n = 6 for day 35 biologically independent mice. d M1R-specific antibodies. The sera were tested for M1R-specific antibodies using ELISA against recombinant M1R protein. n = 12 to 15 for day 7 and day 13 and n = 6 for day 35 biologically independent mice. e Neutralizing antibodies. The sera were tested for neutralizing antibodies against VACV using plaque reduction neutralization test (PRNT). n = 6 biologically independent mice. Data are presented as mean values ± SD. f CD4⁺ T cell immune response against A35R. The splenocytes were analyzed for the percentage of CD4⁺ T cells producing IFN-γ in response to A33R (VACV homolog of A35R) stimulation. n = 4 for day 7 and n = 5 for day 30 biologically independent mice. Data are presented as mean values ± SD. g CD8⁺ T cell immune response against A35R. n = 4 for day 7 and n = 5 for day 30 biologically independent mice. Data are presented as mean values ± SD. h CD4⁺ T cell immune response against M1R. n = 4 for day 7 and n = 5 for day 30 biologically independent mice. Data are presented as mean values ± SD. i CD8⁺ T cell immune response against M1R. n = 4 for day 7 and n = 5 for day 30 biologically independent mice. Data are presented as mean values ± SD. All the data in (c–i) were analyzed by one-way ANOVA with Tukey’s multiple comparison tests. “ns” indicates “not significant”; “*” indicates p < 0.05; “**” indicates p < 0.01; “***” indicates p < 0.001; “****” indicates p < 0.0001.