(A) Pre-existing IgG antibody responses to virus by earlier vaccine immunization. Split-p: Prime, Split-b: Boost. (B) IgG antibody responses post M2eVLP additional immunization. Split-b(7wk): 7wk after Split-b. Split-b(11wk): 11wk after Split-b. (C) IgG isotype antibody responses to vaccine antigen A/Cali virus. (D) Ratios of IgG2a/IgG1 isotype antibodies to homologous A/Cali virus. Split-b: 7wk after Split-b. BALB/c mice were immunized with split vaccine alone (Split, N=12) or received subsequent additional M2e5x VLP immunization after split vaccine (S-M2e5x, N=12). Sera were collected 3 weeks after each vaccination. The IgG level was detected using homologous influenza A virus, A/California/04/09 (A/Cali, H1N1), as an ELISA coating antigen (2ug/ml). Error bars indicate mean ± SEM.