Human papillomavirus (HPV) oncoproteins E6 and E7 which are constitutively expressed in cervical cancer cells are ideal targets for developing immunotherapies for treatment of existing HPV-associated carcinoma. In this project, we developed a simple, safe, and efficient, peptide-based therapeutic cancer vaccine, DOTAP/E7 complex, which comprises only two molecules: a DOTAP cationic lipid and an MHC class I-restricted peptide antigen derived from HPV-16 E7 protein. TC-1 cell line which is HPV-16 E7+ was used as a tumor model in an H-2b murine system. Tumor-bearing mice showed significant tumor inhibition following a single injection of DOTAP/E7 at the optimal lipid dose, suggesting that DOTAP liposome alone can be a potent adjuvant. E7 peptide formulated with DOTAP was taken up by dendritic cells (DC) and induced DC activation and migration to the draining lymph node (DLN), eliciting antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses. The mechanism of DOTAP as a vaccine adjuvant was revealed by DOTAP-mediated reactive oxygen species (ROS) production in DC. At the optimal lipid dose, DOTAP/E7 generates an adequate level of ROS for the initiation of the vaccine mechanism. In addition, we have improved the vaccine formulation by incorporation of E7-lipopeptide instead of the water-soluble native E7 peptide. The improved DOTAP/E7-lipopeptide vaccine showed a significantly enhanced therapeutic effect, including CTL response and anti-tumor activity. The vaccine was also effective for suppression of tumor growth in later stages of tumor progression, suggesting applications for advanced cancer treatment. Furthermore, we extended the studies of the vaccine efficacy observed in the mouse model to human cells in vitro. Instead of H-2Db-restricted peptide, an HLA-A2-restricted E7 peptide epitope (hE7) was formulated into the liposome. In vitro stimulation of naïve HLA-A2+ human T lymphocytes by DOTAP/hE7-activated autologous DC elicited a stronger clonal T cell proliferation and higher HPV-specific CTL response compared to those stimulated with DC pulsed with hE7 peptide alone. The in vivo CTL and anti-tumor activity induced by DOTAP/hE7 vaccine were demonstrated in an HLA-A2 transgenic mouse model. Overall, our data suggest that DOTAP/hE7 is a potent therapeutic cancer vaccine formulation with potential for clinical applications for the treatment of HPV-related neoplasia.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-10312007-185955 |
| Date | 19 November 2007 |
| Creators | Chen, Weihsu Claire |
| Contributors | Raman Venkataramanan, Leaf Huang, Lisa Rohan, Wen Xie, Louis Falo |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-10312007-185955/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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