期刊
PROTEOMICS
卷 18, 期 11, 页码 -出版社
WILEY
DOI: 10.1002/pmic.201700390
关键词
human papillomavirus (HPV); immunopeptidomics; immunotherapy; neoepitopes; targeted mass spectrometry (MS)
资金
- Hector Foundation II
- Manfred Lautenschlager foundation
- Marie Curie Career Integration Grant of the European Commission [MC-CIG 294098]
- German Center for Infection Research (DZIF) [TTU 07.706]
- Eduard and Melanie zur Hausen Foundation
- Helmholtz International Graduate School of the German Cancer Research Center (DKFZ)
- ZMBH
- DKFZ Proteomics Core Facilities
For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation-derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC-MS3-based targeted detection of low-abundant human leukocyte antigen (HLA) class-I-presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen-derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA-peptide complexes, while keeping high isolation yields of low-abundant target peptides. The subsequent targeted LC-MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA-A2-restricted epitope E7(11-19) and ten additional E7-derived peptides on the surface of HPV16-transformed cells. T-cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low-abundant candidate epitopes to be true immunotherapy targets.
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