Fig. 4

Evaluation of the safety profile of recombinant H5N1 vaccine strains in in vitro and in vivo models. The growth kinetics of recombinant H5N1 viruses (22W_PR8, 22W_MVV, and 22W_KY) were evaluated in mammalian (MDCK) and human respiratory (Calu-3) cells. Recombinant H5N1 viruses were inoculated at a multiplicity of infection (MOI) of 0.001 TCID₅₀/cell into (a) MDCK cells and (b) Calu-3 cells. Viral supernatants were harvested at 0, 24, 48, 72, and 96 h postinoculation, and virus titers at each time point were determined and are presented as the mean ± SD of TCID₅₀/mL values from triplicate experiments for each group. Statistical significance was determined using two-way ANOVA, followed by Tukey’s multiple comparison test. The blue asterisks indicate statistical significance between 22W_PR8 and 22W_KY, whereas the red asterisks represent significance between 22W_PR8 and 22W_MVV. The blue hash symbols (#) denote significant differences between 22W_MVV and 22W_KY (**p < 0.01, ##p < 0.01, ****p < 0.0001, ####p < 0.0001). (c) Body weight changes in recombinant H5N1 virus-infected mice were monitored. Groups of five six-week-old female BALB/c mice were intranasally inoculated with 10⁶ EID₅₀/50 µL of each virus, and body weight changes were recorded for 13 days. (d) Lung virus titers (TCID₅₀/mL) were measured at 3 days postinfection (dpi) in mice infected with each virus (n = 3 per group). The lung virus titer data are presented as mean ± SD, and statistical significance was determined using one-way ANOVA, followed by Tukey’s multiple comparison test. Significant differences are indicated by asterisks (*p < 0.05, **p < 0.01)