Comparable moderate T cell immune responses were induced in both high-dose rNP- and RVJ1175NP-immunized mice. BALB/c mice were immunized i.m. with 10, 30, or 90 μg of rNP or PBS alone, three times, two weeks apart. Mice immunized with RVJ1175NP (2×107 PFU) twice, four weeks apart, were used as a positive control. Ten days after the last immunization, mice were sacrificed for ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays, as described. All data are shown as the mean ± standard deviation for two independent experiments. (A) Specific T cell responses were tested by ex vivo IFN-γ ELISPOT assays against NP147-155 in mice (n = 4 per group). Splenocytes (500,000) were incubated ex vivo with or without 5 μg/ml NP147-155. The plates were then incubated for 20-24 h. The left panel shows the number of IFN-γ-producing cells as determined by ELISPOT. Each histogram is representative of data from one mouse; a portion of the results are presented here. (B) In vivo cytotoxicity assays to determine the specific lytic potential of effector CD8+ T cells in mice. Target cells were transferred to immunized or naïve mice (n = 5 per group). The left histograms show the number of CFSElow unpulsed (M1) or the CFSEhigh NP147-155-pulsed (M2) mouse spleen cells. Each histogram is representative of data from one mouse; a portion of the results are presented here. The right graph indicates the percentage of specific killing for NP147-155-pulsed targets from the indicated groups.