Influenza A virus is a contagious respiratory pathogen responsible for regular epidemics and occasional pandemics, causing substantial damage to life and the economy. The first case of highly pathogenic avian influenza H5N1 was reported in 1997 in Hong Kong. Since then, the virus has spread throughout 68 countries, displaying greater than 50% mortality and representing a high pandemic threat. In virally infected cells, antigens are processed by proteasome complex and transported to ER where it further trims to short peptides (8-11 aa) and presents through Human Leukocyte Antigen (HLA) onto the cell surface for the T cells scanning and response. Immune cross-protection against different influenza strains has been demonstrated by some influenza-specific CD8+ T cells, foreshadowing the development of universal influenza vaccines. Recent progress in immunopeptidomics has facilitated the identification of many influenza peptides displayed on the surface of infected cells using mass spectrometry, providing a comprehensive insight into potential T cell epitopes. Some populations in South-Asia exhibit low avian influenza H5N1 incidence despite high levels of exposure to the virus, suggesting they are less susceptible than other populations to severe disease. We explored the immunopeptidome presented by the prevalent HLA-A*33:03 allotype within these populations, in order to map peptides that stimulate CD8+ T cells and confer protection, which in turn may be used as a potential vaccine target.
Peptides were isolated from HLA-A*33:03+ cell lines transfected with avian influenza H5N1 genes HA, PB2, M and NP by immunoaffinity purification and sequenced by LC-MS/MS (Orbitrap Q-Exactive). Raw data were analysed using PEAKS software version X+. A total of 30 H5N1 peptides were tested for their ability to stimulate T cells from HLA-A*33:03+ healthy donors. A number of T cell epitopes were identified using this approach, including two epitopes (PB2128-136 & NP413-422) that are conserved among circulating influenza strains. These findings have implications for incorporating immunogenic H5N1 influenza epitopes in T cell-mediated vaccines to protect people from influenza viruses globally.