Identifikation von natürlich prozessierten HLA- Liganden des Mycobacterium tuberculosis mittels massenspektrometrischer Analyse

Abstract

Mycobacterium tuberculosis (Mtb) is the causative pathogen for tuberculosis (TB) in humans. Most of the individuals infected with Mtb do harbour the pathogen without sign of infection, and the reactivation risk remains lifelong. The current vaccine against TB is Bacillus Calmette–Guérin (BCG), which fails to protect against pulmonary TB in adolescent and adults. For the development of optimal vaccination based on antigens or for immune monitoring strategies, it is essential to characterize the human histocompatibility leucocyte antigen (HLA)-ligandome of cells infected with Mtb. The presented mycobacterial ligands on infected cells can lead to identification of novel ligands and resultant antigens. Further, the identified ligandome could give a glance at the life cycle of intracellular persisting Mtb, in active replicating or dormant state, and help to understand the biology of the pathogen. In the past various epitopes from mycobacterial antigens could be identified by epitope prediction and screening of T cells. We showed an approach to isolate naturally processed mycobacterial peptides presented by HLA molecules and analysed them by HPLC-coupled mass spectrometry. For the first time, we were able to identify novel mycobacterial HLA ligands with mass spectrometric analysis of B-lymphoblastoid cell line (B-LCL) infected with modified vaccinia virus Ankara (MVA) encoding mycobacterial antigens and macrophages infected with mycobacterial laboratory strain H37Rv. Several additional viral peptides from MVA vectors could be identified. The identified Mtb peptides were screened in highly sensitive enzyme-linked immunospot (ELISPOT) assays for single cell IFNγ release to monitor the presence of reactive memory T cells in BCG-vaccinated or purified protein derivative (PPD) -positive donors. Three of nine identified ligands were immunogenic. This demonstrates the suitability of the mass spectrometry-based technique to identify bacterial and viral ligands from infected cells.