Fig. 6
Intranasally administered 22W_KY inactivated with binary ethylenimine provides robust protection efficacy against heterologous and heterosubtypic virus challenges in mice. (a) Schematic representation of the experimental procedure for immunization and challenge studies. Groups of eight six-week-old BALB/c mice were intranasally immunized with 5 µg/50 µL 22W_KY inactivated with formaldehyde (F/A), binary ethylenimine (BEI), beta-propiolactone (BPL) or PBS (negative control). Two weeks after priming immunization, the mice received a booster immunization via the same route. Three weeks after the booster immunization, the mice were challenged with 10 LD50 of either heterologous A/Wild duck/Korea/SNU50-5/2009 (SNU50-5, H5N1) or heterosubtypic A/Puerto Rico/8/1934 (PR8, H1N1) virus. (b) Schematic representation of the genome composition of the 22W_KY vaccine strain, the SNU50-5 virus, and the PR8 virus. Each bar represents a genomic segment in descending order of length, corresponding to PB2, PB1, PA, HA, NP, NA, M, and NS. The color coding indicates the origin of each segment: green: 310-MVV PB2 segment; blue: HA and NA segments of 22W_KY; pink: PR8 virus segments; light blue: SNU50-5 virus segments. The SNU50-5 virus shares the same subtype as the vaccine strain but exhibits antigenic differences, whereas the PR8 virus, despite being a different subtype, shares identical internal genes with the vaccine strain, except for the PB2 segment. (c-f) Body weight changes and survival rates were monitored for 14 days postchallange (n = 5). (g-j) Viral loads in lung and nasal turbinate (NT) tissues were assessed five days postchallange to determine the effectiveness of each vaccine in reducing virus replication (n = 3). The data are presented as the mean ± SD, and statistical significance was determined by one-way ANOVA, followed by Tukey’s multiple comparison test. Asterisks directly above the bars indicate statistical significance compared with the negative control (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001)