Ad-tTA-SIINFEKL-Luc elicits an immune response comparable to those of other rHuAd5 vectors that can be fully attenuated by treatment with DOX. (A) C57BL/6 mice (three per group) were immunized with 109 PFU of Ad-tTA-SIINFEKL-Luc, and the numbers of SIINFEKL-specific CD8+ T cells in the spleens of mice sacrificed at various times postimmunization were assessed. Each point represents the mean ± standard error of the mean. (B) C57BL/6 mice (three per group) received a single bolus intraperitoneal injection of DOX 5 days prior to immunization and were subsequently given water containing either 50, 100, or 200 μg/ml DOX. Five days following the initiation of DOX treatment, mice were immunized with 109 PFU of Ad-tTa-SIINFEKLuc. Mice were sacrificed 22 days postimmunization, and the total number of SIINFEKL-specific CD8+ T cells in each spleen was assessed. Each data point represents the mean ± standard error of the mean. (C) C57BL/6 mice received 109 PFU Ad-tTA-SIINFEKL-Luc intramuscularly in the presence or absence of DOX. One group of mice (DOX day −5) started DOX treatment 5 days prior to immunization. The second group (DOX day +2) started DOX treatment 2 days after immunization. The third group of mice (no DOX) did not receive any DOX during the period of the experiment. Thigh muscles were harvested at the time points indicated and assayed for luciferase activity. The results are means ± standard errors of the means for six muscle samples per group.