Peptide and epitope DNA vaccine immunized HHD mice generated specific tetramer binding CD8 T cells that secreted intracellular interferon gamma (IFNγ) after in vitro culture. The spleen cells harvested from CRPVE1/149-157, 42-50, 245-253 and HPV16E7/82-90 peptide (A) or epitope DNA vaccinated mice (B and C) were stimulated with corresponding peptide pulsed mouse dendritic cells weekly twice. These bulk CTLs were then tested for A) Intracellular IFNγ labeling for peptide vaccination, B) tetramer binding and C) Intracellular IFNγ labeling for DNA vaccination. A) A very low but significant population of specific CD8 T cells secreting IFNγ in CRPVE1/149-157 peptide immunization was found (P<0.05, unpaired student t test) and no detectable IFNγ secreting CD8 T cells were found from either CRPVE1/42-50 or CRPVE1/245-253 peptide immunized HHD mice; B) Significantly more specific CD8 T cell binding to CRPVE1/149-157 and CRPVE1/42-50 tetramer was found in epitope DNA vaccinated HHD mice respectively (P<0.05, unpaired student t test). C) Significantly more IFNγ secreting CD8 T cells were also found in CRPVE1/149-157 epitope DNA vaccinated mice to corresponding peptide stimulation when compared with a reference peptide (P<0.05, unpaired student t test) but not in the other two epitope DNA vaccinated groups (N=2/ group).