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Identification of target T cell epitopes for a therapeutic HPV16 vaccine

Hoppe, Stephanie

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To rationally design therapeutic human papillomavirus (HPV) vaccines, it is important to know which T cell epitopes are present on HPV-transformed cells. HPV affects the cellular antigen processing machinery, thus not every epitope derived from viral proteins is necessarily presented by human leukocyte antigen (HLA) molecules on the cancer cell surface. HPV16 has been identified as the causative agent in 50% of all cervical cancer cases and in approximately 95% of all extra-cervical mucosal HPV-induced tumors. The transforming potential of high-risk HPVs is mediated by two consistently expressed viral oncoproteins, E6 and E7. As the induction and maintenance of the malignant phenotype depend on these two proteins, they are ideal targets for immunotherapy. A therapeutic vaccine that is applicable to everyone without prior HLA typing needs to contain epitopes for the major HLA types. To date, HPV T cell epitopes have mostly been determined for the most prevalent HLA type, HLA-A2. We now aim to identify HPV16 E6 and E7 T cell epitopes for the HLA-A3, HLA-A11 and HLA-A24 supertypes. Different in silico prediction algorithms were used to predict prospective epitopes from the E6 and E7 proteins derived from the HPV16 reference sequence for the mentioned HLA supertypes. In total, 74 epitopes, comprised of 8 to 11mer peptides, were predicted for HLA-A3, 96 epitopes for HLA-A11 and 95 epitopes for HLA-A24. In competition-based cellular binding assays, 22 previously known binding peptides were confirmed and 78 novel binding peptides were identified. Additionally, HPV16 variants harbored in our HPV16-positive cell line collection were determined and peptide binding to HLA-A24 was shown for seven out of 20 tested HPV16 variant peptides. Evaluation of prediction server performance based on the generated data suggested different optimal prediction servers depending on peptide length and on the HLA type. NetMHC and NetMHCcons were shown to be the best predictors overall. Immunogenicity of identified binding peptides was investigated by screening long-term memory responses in healthy donors. To this end, peptide-specific short-term T cell lines were generated from peripheral blood mononuclear cells, which were HLA typed, stimulated with HLA-matching peptides and cultured for twelve days. Several peptides were identified to be immunogenic. Immunogenicity of the four most promising candidate peptides for HLA-A24 could be further confirmed by generation of peptide-specific long-term T cell lines from healthy HLA-A24-positive donors and subsequent coculture with autologous B cells pulsed with the respective peptide. Functional assays, such as IFNγ ELISpot assays and cytotoxicity assays, determined the best vaccine candidates. In conclusion, several novel HPV16 E6 and E7 CD8+ T cell epitopes were identified.Verified epitopes are the basis of rational therapeutic vaccine design and are also important for immunomonitoring purposes. In addition, they can be employed as a tool for the development of other immunotherapies such as adoptive T cell transfer with transgenic T cell receptors.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Date of thesis defense: 18 June 2015
Date Deposited: 01 Jul 2015 09:55
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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