Immunization schedule for the BALB/c mice. Mice were immunized i.m. three times, two weeks apart with 10, 30, or 90 μg of rNP or PBS alone, respectively. Age- and sex-matched mice were immunized twice, four weeks apart with 2×107 PFU per mouse of RVJ1175NP as a positive control. Serum samples from four mice were collected ten days after each immunization. Ten days after the last immunization, five mice in each group were sacrificed for ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays. Next, eleven mice in each group were challenged intranasally with 104 TCID50 (10×MLD50) of influenza A/PR/8/34(H1N1) ten days after the last immunization.