Copyright © 2008 American Medical Association. All rights reserved. From: Development of an Adenoviral Vaccine Against E6 and E7 Oncoproteins to Prevent Growth of Human Papillomavirus–Positive Cancer Arch Otolaryngol Head Neck Surg. 2008;134(12):1316-1323. doi:10.1001/archoto.2008.507 Figure Legend: Infection of A549 cells with adenovirus 5 E6 and E7 (Ad E6/E7) oncoproteins results in functional protein expression. The E6/E7 transgene protein expression was tested after overnight transduction of A549 cells with Ad5 E6/E7 or control cells at a multiplicity of infection of 100. Western blot analysis was used to assess E7 protein expression and function. The top row shows production of E7 protein; the second row of proteins shows the E7-induced loss of Rb (retinoblastoma) (Jurkat cell lysate is shown as a positive control for Rb protein expression); and the third row of proteins shows E6-induced loss of p53. Date of download: 10/25/2017 Copyright © 2008 American Medical Association. All rights reserved.

Copyright © 2008 American Medical Association. All rights reserved. From: Development of an Adenoviral Vaccine Against E6 and E7 Oncoproteins to Prevent Growth of Human Papillomavirus–Positive Cancer Arch Otolaryngol Head Neck Surg. 2008;134(12):1316-1323. doi:10.1001/archoto.2008.507 Figure Legend: Coculture of splenocytes with tumor cells demonstrates antigen-specific production of interferon-γ (IFN-γ). For each group, splenocytes were harvested from mice (n = 3 in each group) 70 days after implantation of E6/E7/H-ras tumors in tumor-clearing mice and when euthanized in tumor-bearing mice. Naive mice served as sex- and age-matched controls. To show a specific response to E6/E7, the splenocytes from tumor-clearing mice were cocultured with E6/E7/H-ras mouse tonsil epithelial cells (MTECs) or small hairpin PTPN13/H-ras MTECs for 36 hours, and supernatants were analyzed for IFN-γ production using an enzyme-linked immunosorbent assay. To compare responses between naive, tumor-bearing, and tumor-clearing (spontaneously or after vaccination) mice, the splenocytes from these groups were again incubated with E6/E7/H-ras. Statistical analysis (Mann-Whitney test) showed that tumor-clearing mice splenocytes specifically produced IFN-γ during coculture with human papillomavirus (HPV)-positive cells (P < .05). Similar statistically significant results show that tumor-bearing mice responded with more IFN-γ production than naive mice to the HPV-positive cells. Tumor-clearing mice responded more than tumor-bearing mice, and inoculated mice had the most vigorous response at all doses of adenovirus 5 E6 and E7 (Ad5 E6/E7) vaccine tested. Gray bars indicate HPV-positive (+) E6/E7/H-ras) cells; black bar, HPV-negative (−) (small hairpin PTPN13/H-ras) cells. *5 × 105 plaque-forming units (PFUs); †5 × 106 PFUs; ‡5 × 107 PFUs). All experiments were performed in duplicate. Date of download: 10/25/2017 Copyright © 2008 American Medical Association. All rights reserved.

Copyright © 2008 American Medical Association. All rights reserved. From: Development of an Adenoviral Vaccine Against E6 and E7 Oncoproteins to Prevent Growth of Human Papillomavirus–Positive Cancer Arch Otolaryngol Head Neck Surg. 2008;134(12):1316-1323. doi:10.1001/archoto.2008.507 Figure Legend: Time course of antigen-specific immune response after vaccination with adenovirus 5 E6 and E7 (Ad5 E6/E7) oncoproteins. Interferon (IFN)-γ production from the splenocyte–tumor cell cocultures from mice inoculated with 5 × 107 plaque-forming units of Ad5 E6/E7. Splenocytes from these mice were harvested just before and 3, 15, 28, and 70 days after vaccination (n = 3 in each group). The splenocytes were cocultured with E6/E7/H-ras mouse tonsil epithelial cells followed by assessment of IFN-γ production. All experiments were performed in duplicate. The black bar indicates IFN-γ production from the splenocyte–tumor cell cocultures from mice that spontaneously cleared tumors as a positive control (n = 3). Mann-Whitney analysis was used to compare response after vaccination. On days 15, 28, and 70, all responses were significantly increased compared with prevaccination values. *P < .05. Date of download: 10/25/2017 Copyright © 2008 American Medical Association. All rights reserved.

Copyright © 2008 American Medical Association. All rights reserved. From: Development of an Adenoviral Vaccine Against E6 and E7 Oncoproteins to Prevent Growth of Human Papillomavirus–Positive Cancer Arch Otolaryngol Head Neck Surg. 2008;134(12):1316-1323. doi:10.1001/archoto.2008.507 Figure Legend: Representative in vivo images (In Vivo Imaging System [IVIS]) in mice that cleared tumors either spontaneously or after vaccination compared with mice that allowed tumor growth. E6/E7/H-ras/Luc mouse tonsil epithelial cells (1 × 106) were implanted into the thighs of wild-type mice with or without inoculation with adenovirus 5 E6 and E7. IVIS images were obtained in live mice on the indicated days. Date of download: 10/25/2017 Copyright © 2008 American Medical Association. All rights reserved.

Copyright © 2008 American Medical Association. All rights reserved. From: Development of an Adenoviral Vaccine Against E6 and E7 Oncoproteins to Prevent Growth of Human Papillomavirus–Positive Cancer Arch Otolaryngol Head Neck Surg. 2008;134(12):1316-1323. doi:10.1001/archoto.2008.507 Figure Legend: Adenovirus 5 E6 and E7 (Ad5 E6/E7) oncoprotein vaccine prevents tumor growth and enables 100% survival when tumor is implanted orthotopically. Wild-type mice were inoculated (n = 5 per group) with either Ad5 E6/E7 (5 × 105, 5 × 106, or 5 × 107 plaque-forming units) or a control Ad5 virus (5 × 107 plaque-forming units). Two weeks after intratracheal vaccination, human papillomavirus (HPV)–positive mouse tonsil epithelial cells (MTECs) were implanted into the posterior part of the oropharynx in mice. The log-rank test was used to demonstrate that survival differences between the Ad5 control and Ad5 E6/E7 groups were statistically significant at all doses of vaccine tested. *P < .05. Date of download: 10/25/2017 Copyright © 2008 American Medical Association. All rights reserved.