Comparison of immunogenicity of CRT/E6E7L2 DNA vaccine administered by various methods. (A) Schematic illustration of the experiment. Briefly, 5-8 week old female C57BL/6 mice (5 mice/group) were vaccinated with either 2 μg/mouse or 20 μg/mouse of CRT/E6E7L2 DNA, or CRT/E6E7L2 DNA formulated with 45 μg of aluminum phosphate by intramuscular injection, or followed by electroporation. The mice were boosted with the same regimen twice with a 2-week interval. Two weeks after last vaccination, serum and splenocytes were collected. Summary of HPV16 E6- (B) or E7- (C) specific CD8+ T cell responses analyzed by IFN-³ intracellular staining. Splenocytes were stimulated with 1 μg/ml of HPV16 E6aa48-57 or HPV16 E7aa49-57 peptide at the presence of GolgiPlug (1 μl/ml) overnight at 37°C. The cells were then stained with anti-mouse CD8 followed by intracellular IFN-³. The data were acquired with FACSCalibur and analyzed with CellQuest. (D) Summary of the HPV16 L2-specific neutralizing antibody responses.