Human papillomaviruses (HPVs) are implicated in the etiology of cervical cancer and HPV type 16 (HPV 16) is commonly identified in malignant cervical cancer tissues. Genetic analysis has revealed that the E6 and E? genes of HPV16 are sufficient for the transformation of both mouse fibroblasts and primary human keratinocytes. This will allow the performance of fundamental tumorvirus-immunological experiments in mice and the extrapolation of the results to humans. The objectives of this application are 1) to determine the role of cellular immunity in the eradication of HPV induced tumors in mice and in humans. 2) to determine if there is a relation between cervical cancer, HLA type and HPV type. 3) to develop a peptide based vaccine against HPV and to explore its effectiveness in mice. 4) to develop a protocol for immunotherapy of HPV induced tumors by adoptive transfer of cultured cells in mice and 5) to discover possible immune escape mechanism of HPV induced tumors and to explore the neutralization of these mechanisms in mice. To achieve these goals the following methodology will be used: 1) mouse and human HPV16 specific CTL will be generated from immunized mice and cervical carcinoma patients. These CTL clones will be analyzed for their peptide specificity by using a set of overlapping peptides spanning the HPV16 protein sequences. Mouse CTL clones will be adoptively transferred to explore their capacity to eradicate these tumors. 2) MHC binding peptides of HPV16 will be identified with the same set of peptides. For this purpose cellines of mouse (RMA-S) or human (T2) origin both with """"""""empty"""""""" MHC molecules will be used. Peptides that bind into the groove of these empty MHC molecules induce an increase of MHC expression which can be analyzed by immunofluorescence. 3) The identified peptides from 1 and 2 will be used to vaccinate mice. Three weeks after vaccination these mice will be challenged with live tumor cells to assess the degree of protection against tumor growth. HPV peptides identified for the mouse and human models will be used to induce primary CTL responses. The peptides will be presented by the RMA-S and T2 celllines. Mouse CTL clones obtained in this manner will also be used for adoptive transfer experiments. 4) Cervical carcinoma patients will be HLA typed and their surgically removed tumor material will be analyzed for the presence of HPV subtypes with the use of RNA-PCR. 5) HPV 16 induced tumors of mouse and man will be analyzed by immunofluorescence, histochemistry and RNA- PCR techniques for the levels of MHC expression, HPV16 gene expression and TGF beta gene expression to detect possible mechanisms that these tumors use to escape from T cell destruction. Eventually neutralization of these mechanisms will be explored. The combined analysis will shed light on a possible role of cellular immunity in the defense against HPV16 induced tumors and on ways of applying cellular immunity for therapeutic or preventive purposes in this type of cancer.
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