%0 Generic
%A Förster, Jonas Dieter
%C Heidelberg
%D 2024
%F heidok:34240
%R 10.11588/heidok.00034240
%T Mass spectrometry-based identification of HPV16 target epitopes for therapeutic vaccine design
%U https://archiv.ub.uni-heidelberg.de/volltextserver/34240/
%X Human papillomavirus (HPV) is the most common sexually transmitted infectious agent in humans. Infections are often asymptomatic and resolve spontaneously, but a subset of persisting infections can develop into warts, precancers, and eventually cancers in women and men. The so-called high-risk HPV (hrHPV) types are a major cause of cancer responsible for ~4.5 % of all annual cancer cases worldwide. Among hrHPVs, HPV16 is the most prevalent type and causes ~60 % of invasive cervical cancer and ~85 % of all other HPV-associated cancers. The primary oncoproteins of HPV16, E6 and E7, represent ideal targets for immunotherapy as they drive the malignant transformation of infected cells, are constitutively expressed, and are immunologically foreign. However, the development of a therapeutic vaccine has proven to be challenging, likely due to the low immunogenicity of persistent HPV16 infections. A vaccine must be highly immunogenic and elicit a strong antigen-specific cytotoxic response. To identify the most potent epitopes for immunotherapies, the research group set the aim of establishing an epitome map for the HPV16 oncoproteins E6 and E7 that specifies truly human leucocyte antigen (HLA)-presented and immunogenic epitopes. A major part of this effort is the detection of HLA-presented peptides on the surface of naturally HPV16-transformed cell lines by immunopeptidomics.  In this thesis, I developed a targeted immunopeptidomics workflow for the sensitive and highly specific detection of HLA class I-presented peptides. To this end, I developed effective quality control measures for the wet-lab technique and implemented chemical modification steps for peptide oxidation and alkylation that allow for optimal detection of the chemically diverse peptides. I optimized the liquid chromatography-mass spectrometry (LC-MS) acquisition of the peptides so that each target could be acquired with high sensitivity. In part, this was achieved by optimizing the LC gradient and tuning the scheduling of the MS acquisition in a manner that minimizes sensitivity-limiting parallel acquisition. Additionally, I showed that tuning the MS acquisition parameters enhances the sensitivity of peptide detection in complex samples. I characterized all targets in detail to allow for effective fine-tuning on a per-peptide basis. This included a newly developed approach for optimization of collision energies on a per-precursor basis, which I found to be especially effective for immunopeptidomics. The resulting method is scalable for targeting of hundreds of peptides, while allowing for maximum sensitivity where extra validation is required.  I applied the established method for the immunopeptidomics part of epitome map project. This involved the analysis of 20 cell lines covering six selected HLA supertypes (A01, A02, A03_A11, A24, B07 and B15), which are groups of HLA allotypes that bind similar peptides. Altogether, 239 distinct peptides were targeted. The HLA-restrictions of all peptide detections were validated through the complementary use of a newly developed untargeted immunopeptidomics analysis. This enabled me to detect the presentation of 25 HPV16-derived peptides on naturally HPV-transformed cell lines, 23 of which had not been MS-detected before. The associated HLA-restrictions cover all six of the targeted HLA supertypes, which I confirmed to allow for a robust and extensive predicted vaccine population coverage of more than 99 %.  These data complete the immunopeptidomics part of the HPV16 E6 and E7 epitome map. Ongoing experiments of the complementary immunogenicity assessment functionally characterize the peptides regarding their immunogenicity, immunodominance and frequency of memory responses in peripheral blood mononuclear cells from healthy donors. The most promising epitopes, as informed by these combined efforts, will be tested for their capability to induce specific killing of HPV-transformed cells. Taken together, this will inform the selection of epitopes for inclusion in epitope-specific vaccine formulations for future clinical trials. Ultimately, the presented findings will contribute significantly to the rational design of a therapeutic HPV16 vaccine applicable for a large part of the population.