Fig. 3: M2e-tetramer specific CD4 T cell protection against influenza virus infection following i.n immunizations with CTA1-M2e-CD103 fusion protein.

Assessment of the binding ability of the M2e-specific fusion proteins -CD103 and -DD at 1 μM to bind to the three DC-subsets freshly isolated from mLN of Balb/c mice; CD103⁺CD11b⁻ (cDC1), CD103⁺CD11b⁺ (cDC2DP), CD103^-CD11b⁺ (cDC2SP) in vitro using a labeled anti-Flag antibody to detect MFI of the bound fusion proteins to the different cDC subset populations as determined by flow cytometry (a). Balb/c mice were i.n immunized three times with 5μM of CTA1-M2e-CD103 (n = 10) or CTA1-M2e-DD (n = 10) and immune protection was determined 3 weeks after the final immunization by a live challenge infection with 4× LD₅₀ of a highly virulent mouse-adapted PR8 strain³⁷. The frequency of surviving animals and changes in body weight was monitored over time. Anti-M2e IgG2a (most protective antibodies) in serum and IgA in BAL after the challenge infection in one representative experiment of three giving similar results was assessed by ELISA and given as log₁₀ titers ± SD (b). Representative FACS plots of M2e-tetramer-specific lung CD4 T cells isolated from i.n immunized or naïve control mice, as indicated. Prior to the challenge infection, the frequency of M2e-specific CD4 T cells that were CD69⁺ or CD69^- and the representation of Th17 cells (rorγt⁺) is given as % ± SD of all M2e-specific CD4 T cells (n = 3). Results are from three independent experiments giving similar results (c). Statistical significance was calculated using ANOVA with Dunnett’s T3 post-test analysis: ns; not significant, p values **p < 0.01, ***p < 0.001 and ****p < 0.0001.