Fig. 6: Protection and T-cellular immune response after the heterologous challenge.

a Body weight dynamics and survival of the BALB/c mice after the heterologous challenge with 5 × LD₅₀ (1 × 10⁴ PFU/mouse) of A/bald eagle/Florida/W22-134-OP/2022 (H5N1 reassortant with A/Puerto Rico/8/1934 vaccine backbone) or 5 × LD₅₀ (7.5 × 10³ PFU/mouse) A/New Caledonia/20/1999 (H1N1) (n = 8). b Viral titers in lungs and nasal turbinates of the BALB/c mice on day 4 after the challenge (n = 4). c Flow cytometry plots showing the percentage of IL-2- and TNFα-producing CD4⁺ EM T-lymphocytes (gated on live CD3⁺CD19^‒CD4⁺CD62L^‒CD44⁺ cells) in C57BL/6 mouse lungs and spleens 5 days after the challenge with 0.1 × LD₅₀ (2 × 10² PFU/mouse) of A/bald eagle/Florida/W22-134-OP/2022 (H5N1). Cells were incubated with overlapping peptides, covering the whole sequence of N1 NA-protein for 6 h in the presence of Brefeldin A and co-stimulatory anti-CD28 antibodies. Plots were produced by combining data from each separate sample into one file for each group. d, e Percentage of different cytokine-producing cell populations within the total CD4⁺ EM T-cell subset after background subtraction in the lungs (d) and spleen (e). Average and standard error (SE) values are shown. Statistical analyses were performed using one-way ANOVA with Tukey posthoc test. P-values for the groups with statistically significant differences are shown on the plots.