The influenza A virus (IAV) is well known for its antigenic drift and shift, which enables this virus to be a continuous endemic and pandemic threat. Since its first detection in 1995, the highly pathogenic avian influenza A/H5N1 has spread throughout Asia and globally and is considered as high risk for future pandemics. Current antibody-inducing vaccines that target viral surface glycoproteins (i.e. hemagglutinin (HA) and neuraminidase (NA)) can combat infection, but they often fail to protect against antigenically distinct strains of the virus. In contrast, CD8+ T cells that recognise viral peptides presented on the cell surface by class I Human Leukocyte Antigen (HLA-I) molecules mediate direct killing of virus-infected cells. Viral peptides derived from internal proteins are often highly conserved across strains, and can provide broadly cross-reactive or “universal” immunity against different strains. In this study we examined a frequent HLA allotype associated with low morbidity and mortality rates following exposure to avian A/H5N1 influenza in South Asian populations, despite high exposure to A/H5N1-contaminated poultry. Here, we examined the HLA-A*33:03 immunopeptidome to identify A/H5N1 viral peptides recognised by CD8+ T cells that could be used to formulate a universal influenza vaccine.
Cells expressing HLA-A*33:03 (C1R.A*33:03) were used as antigen-presenting cells for transfection of targeted avian influenza H5N1 proteins. Using immunoaffinity purification coupled with mass spectrometry, we identified 33 potential CD8+ T cell epitopes from A/H5N1 proteins; including HA, polymerase basic protein 2 (PB2), matrix protein 1 (M) and nucleoprotein (NP). These candidate peptides were screened by in vitro stimulation of peripheral blood mononuclear cells from HLA-A*33:03-positive healthy donors to determine their immunogenicity. Strikingly, two immunogenic H5N1 epitopes (PB2128 & NP413) elicited strong immune reactivity and are >90% conserved among currently circulating avian and human influenza viruses. As these healthy donors have not been exposed to A/H5N1, T cell reactivity towards these two epitopes confers cross-strain protection. Moreover, single-cell profiling of NP413-induced T cells revealed a single dominant T cell receptor clonotype. These findings can inform the development of rationally designed influenza vaccines capable of inducing cross-strain protection from severe influenza disease in HLA-A*33:03 expressing individuals.