Humoral immune responses induced in immunized mice. (A) Immunization and challenge experiment timeline. Immunization groups include soluble peptide mixture of 4M2e and the NP peptide (4M+NPep), NP-peptide/4M2e–layered nanoparticles (NPep/4M2e nano), NP-protein/4M2e–layered nanoparticles (NP/4M2e nano), OVA nanoparticles (OVA nano) and DPBS. (B) M2e-specific IgG endpoint titers. IgG titers against 4M2e were determined with serum samples collected 3-wk postboosting. (C) Determination of IgG1 and IgG2a titers against 4M2e using ELISA. (D) Bar chart showing logarithm values of the ratio IgG1:IgG2a. (E) M2e binding spectrum. Serum IgG binding capacity to various M2e peptides, including p09M2e, VtnM2e, and SHM2e was evaluated. Data are presented as mean ± SD (n = 10). Statistical significance was analyzed by t test for B, C, and E. P values shown in bar charts and N.S. indicates no significance between two compared groups. The experiments were repeated twice with the same samples with similar results.