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Abstract

Persistent infections with high-risk human papillomaviruses (HPVs) cause >730,000 new cancer cases per year, with HPV16 being most prevalent. The viral oncoproteins E6 and E7 drive malignant transformation of infected cells and are consistently expressed during disease progression. The presence of these non-self proteins makes HPV-driven tumors the ideal model systems for therapeutic cancer vaccination and epitopes derived from E6 and E7 optimal vaccine candidates. However, due to human leukocyte antigen (HLA) type-specific presentation of epitopes, short epitope-based vaccines need to cover various HLA types to provide a broad population coverage. Fortunately, the number of required epitopes can be reduced by exploitation of HLA supertypes, and a combination of epitopes binding to six prevalent HLA supertypes (A1, A2, A3/A11, A24, B7 and B15) was computed to provide >99% world population coverage. Within this thesis, I completed a long-term project within the Division of Immunotherapy and Immunoprevention that aimed at providing an HPV16 E6-/E7-derived epitome map for the indicated HLA supertypes. To this end, I completed an immunogenicity screening for HPV16 E6-/E7-derived HLA ligands in healthy female donors, by applying interferon-γ ELISpot assays and intracellular cytokine stainings. The whole screening resulted in the identification of 168 immunogenic epitopes. In addition, I established a highly sensitive live-cell imaging-based cytotoxicity assay that allowed the analysis of CD8+ T cell cytotoxicity against epitopes naturally presented by HPV16-transformed tumor cell lines. By applying this cytotoxicity assay, I was able to functionally validate at least one epitope for each of the analyzed HLA supertypes. Finally, I combined all the immunogenicity and cytotoxicity data with pre-existing immunopeptidomics data from our group about the surface presentation of the analyzed epitopes to obtain an overview on the epitope repertoire that can be derived from HPV16 E6 and E7. This overview enabled the selection of the most promising candidates for therapeutic vaccination and informs on suitable epitopes for different intervention time points. For selected HLA-A24 epitopes, I further analyzed their capacity to induce de novo immune responses in vivo using HLA-A24-humanized mice. The obtained data validated the results of the in vitro screenings, as two of the most promising epitopes also mediated in vivo immune responses upon vaccination. In a side project, I generated a syngeneic tumor cell line for A24 mice that models HPV16-driven malignancies. This cell line provides the basis for the establishment of an HPV16 tumor model in A24 mice, which can then serve as target to assess anti-tumor efficacy of multi-epitope vaccine formulations. Overall, this thesis provides valuable data for the rational design of a multi-epitope therapeutic HPV16 vaccine and other epitope-centric therapies, as well as tools that can be used for epitope validation.