Abstract B055: A pan-HLA HPV16 T cell epitope repertoire – validated by immunopeptidomics and immunogenicity analysis – for therapeutic vaccine design
Authors: Wellach Kathrin , Bonsack Maria , Förster Jonas D , Vuckovic Nika , Becker Jonas P , Riemer Angelika B 
Published: October 2024

Abstract
Human papillomavirus (HPV) causes ∼700.000 new cancer cases annually, with HPV16 being the most important HPV type. The viral oncoproteins E6 and E7 drive the malignant transformation of persistent HPV infections. Thus, epitopes derived from these proteins are optimal candidates for therapeutic vaccination against HPV-mediated tumors/precursor lesions. However, the epitopes presented are specific to the individual human leukocyte antigen (HLA) molecules. Fortunately, the number of epitopes required for broad population coverage can be reduced by exploitation of HLA supertypes. A combination of epitopes binding to six major HLA supertypes (A1, A2, A3/A11, A24, B7 and B62) allows &gt;99% world population coverage. To identify peptides against which successful immune responses have been induced naturally, interferon-γ ELISpot assays and flow cytometry-based intracellular cytokine staining (ICS) were performed with peripheral blood mononuclear cells (PBMCs) of healthy female donors above 40 years of age. In the ELISpots, 87 donors were analyzed with at least ten donors for each HLA supertype and an overall response rate of 76%. So far, we have identified 40 peptides inducing specific CD8 T cell activation in the ICS with at least 2 reactive peptides per HLA supertype. The most promising epitopes were selected for functional characterization in a newly established highly sensitive image-based cytotoxicity assay. This assay applies time-course analysis to enable the detection of specific killing executed by rare T cell populations derived from PBMCs. Up to now, several HLA-A2, -A24-and -A11-binding epitopes were validated to mediate epitope-specific killing by T cells in at least one donor. The analysis of additional donors, epitopes and HLA-supertypes is ongoing. In parallel to the immunogenicity analysis of healthy donors, the presentation of epitopes on HPV16-transformed cancer cell lines of different HLA backgrounds and malignancies was analyzed by targeted liquid chromatography-mass spectrometry (LC-MS). For this, LC-MS characteristics of target peptides (e.g. exact retention time, fragmentation pattern, and preferred precursor charge state) were acquired in order to determine optimal parameters for each peptide, resulting in ultra-high detection sensitivity. Peptide eluates from immunoprecipitated HLA-complexes of 22 cell lines were analyzed, leading to the identification of more than 20 peptides and at least one peptide per investigated HLA supertype. Interestingly, there is only a partial overlap between the data obtained by immunogenicity analysis and those acquired by targeted LC-MS. This might indicate a shift in the presented epitope repertoire in the course of disease progression and highlights the importance of inducing an immune response tailored to the correct target epitopes. In conclusion, the presented study provides a validated map of the HPV-16 E6-and E7-derived epitope repertoire, which will serve as a rational basis for the development of therapeutic HPV vaccines as well as other epitope-centric HPV16 immunotherapies.
Citation Format: Kathrin Wellach, Maria Bonsack, Jonas D Förster, Nika Vuckovic, Jonas P Becker, Angelika B Riemer. A pan-HLA HPV16 T cell epitope repertoire – validated by immunopeptidomics and immunogenicity analysis – for therapeutic vaccine design [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B055.