Immunogenicity of recombinant MVA expressing ANYNFTLV. C57BL/6 mice were administered intramuscularly (A, B) or intraperitoneally (C, D) with two different doses of either MVA-MANY or MVA-p3. The mice were sacrificed 11 days after the immunization, and their spleens were removed. A half of splenocytes from individual mice were cultured with irradiated EL-4 cells pulsed with ANYNFTLV peptide for 1 week. The freshly isolated splenocytes (A, C) or the one-week cultured splenocytes (B, D) were subjected to the ELISPOT assay for IFN-γ-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-γ-secreting cells/106 cells was counted 24 h later. The number of IFN-γ-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-γ-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± standard deviation of three mice in each group. The data are representative one of three independent experiments.