Fig 6. m-cNA-M2e VLP vaccination induces humoral and cellular immune responses and confers cross protection against influenza A viruses in aged mice.

BALB/c mice (14-months old, n = 10 per group) were intramuscularly immunized with m-cNA-M2e VLP (10 μg) prime-boost at a 3-week interval. (A-C) Concentrations of IgG and isotype antibodies specific for M2e, N2 NA (A/Brisbane/10/2007 H3N2), and N1 NA (A/California/4/2009 H1N1) protein in boost immune sera from aged mice. (D-F) Body weight changes monitored daily for 14 days and survival rates after challenge with influenza A viruses. (D) A/Phil/1982 H3N2 (3xLD₅₀, 7x10 EID₅₀), (E) A/Cal/2009 H1N1 (3xLD₅₀, 2x10⁴ EID₅₀), (F) rgA/SH/2013 H7N9 (H7 HA, N9 NA from A/Shanghai/2013) (3xLD₅₀, 5.6x10³ EID₅₀). (G) NA inhibition activity in 40-fold diluted boost immune sera against A/Phil (H3N2) virus by ELLA. (H) Lung viral titers at day 6 post infection with A/Phil (H3N2). (I) Antigen-specific IgG levels in mLN cells collected at day 6 post challenge with A/Phil (H3N2) after in vitro culture with M2e (4 μg/ml) or N2 NA protein (A/Brisbane/10/2007 H3N2, 200 ng/mL) for 1 (D1) or 5 (D5) days. (J) IFN-γ cytokine secreting spots in lung cells after stimulation with 5 μg/mL of M2e or N2 NA (A/Brisbane/10/2007, H3N2) pooled peptide. (K) IFN-γ secreting CD4 T cells in lungs after in vitro stimulation with 5 μg/mL of M2e or N2 NA pooled peptide by intracellular cytokine staining and flow cytometry. The statistical significances were performed with one-way ANOVA with Tukey’s Multiple comparison test or two-way ANOVA with Bonferroni posttest and indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001.