P100

1:Faculty of Medicine, Universidade Católica Portuguesa, 2635-631 Rio de Mouro, Portugal; 2:OMICS and Analytical Development Group, and BIOSCOPE Research Group, LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal; 3:Department of Medicine, University of Perugia, 06129 Perugia PG, Italy; 4:PROTEOMASS Scientific Society, 2825-466 Caparica, Portugal; 5:Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 6:Center for Interdisciplinary Research in Health, Universidade Católica Portuguesa, 2635-631 Rio de Mouro, Portugal; 7:Host-Pathogen Interactions Unit, Research Institute for Medicines, iMed-ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Portugal

Mycobacterium avium (M.a) is a slow growing Nontuberculous Mycobacteria (NTM). It is an opportunistic intracellular pathogen, which may affect the lungs of patients with Acquired Immunodeficiency Syndrome (AIDS), Cystic Fibrosis (CF), Chronic Obstructive Pulmonary Diseases (COPD), and others. M.a infections can be dramatic in patients with co-morbidities. Treatments are available with modest therapeutic efficacy, and there is no vaccine available. In AIDS patients, low counts of CD4⁺ T cells can associate with the onset of M.a Complex infection, and CD8⁺ T cells may contribute to a protective role against this pathogen. With the purpose to discover M.a antigens for the development of a new vaccine candidate, we used immunopeptidomics to identify peptides presented by MHC-I and MHC-II in M.a-infected human macrophages.

We have adapted and optimized mycobacterial growth conditions for M.a, characterized the kinetics of M.a survival in macrophages, and assessed the innate immune response of macrophages upon M.a infection. Following, we applied a well-established protocol of immunopeptidomics, based on immunoprecipitation of MHC-complexes, peptide purification, mass spectrometry, and peptide-spectrum match data analysis using Peaks 12.0 software.

We have identified 32 MHC-I and 58 MHC-II M.a peptides, with a Peaks Score higher than 15 (equivalent to a p-value < 0.05). Using NetMHCpan, we characterized the peptide binding affinity relative to the macrophage’s HLA alleles. Finally, combining Peaks Score hierarchization with peptide binding affinity, we shortlisted a number of peptides currently being evaluated for its capacity to stimulate T-cell responses for vaccine design and development.