ABSTRACT: Genetic studies indicate a strong (97%) association of narcolepsy with HLA-DQA1*01:02/DQB1*06:02 (DQ0602), weaker associations with HLA-DP and class I HLA- A*11:01, and effects in other immune related genes such as the TCR αβ loci. The disease is caused by the loss of hypocretin (HCRT)/orexin neurons, as loss of HCRT or HCRT receptors recapitulates symptoms in animal models. Results from our laboratory and from others indicate that a critical step in the disease process is a CD4+ T cell autoimmune response targeting HCRT itself, notably a DQ0602 restricted response to the amidated, C-terminal end of secreted HCRT peptides (HCRTNH2). Indeed, increased T cell response to HCRTNH2 presented by DQ0602 is observed in narcolepsy (our observation), followed by a broader CD4+ T cell responses to other HCRT fragments presented by other HLA class II molecules (other investigators), a result of epitope spreading. This is likely followed by a CD8+ T cell response to HLA class I presented antigens that results in hypocretin cell death and life-long narcolepsy. Unique to narcolepsy, a known environmental factor, the flu, has been associated with initiation of the autoimmune process. Indeed, an increased incidence of narcolepsy in children has been observed following the 2009-2010 pH1N1 Influenza pandemic in China and following vaccination with Pandemrix (GSK), an AS03 adjuvanted pandemic H1N1 2009 (pH1N1) vaccine, but not other vaccines. These observations indicate that epitopes within the pH1N1 virus and vaccine can precipitate narcolepsy in some individuals, findings substantiated by the fact narcolepsy patients have increased T cells binding NP17-31 and pHA273-287, a flu hemagglutinin fragment with significant HCRTNH2 sequence homology. This mechanism is also supported by our preliminary data suggesting TRAJ24 and TRBVB4-2 involvement in pHA273-287, NP17-31 and HCRTNH2 TCR responses, as these TCR segments are modulated by SNPs associated with narcolepsy. In this application, we will further characterize antigenic differences in Pandemrix, Arepanrix and Focetria, vaccines used in the 2009-2010 pH1N1 vaccination campaign, capitalizing on the observation that they have differential risk for precipitating narcolepsy. Our hypothesis, supported by preliminary data, is that Pandemrix contains more pHA273-287 peptide, the main mimic of HCRTNH2 autoimmune responses. In addition, we will continue isolation and characterization of Pandemrix/H1N1 reactive and HCRTNH2 DQ0602 restricted CD4+ T cells. Our hypothesis is that we will ultimately discover pHA273-287/HCRTNH2 and other flu/HCRT cross-reactive cells that use TRAJ24 and TRBVB4-2 segments. Finally, we aim to characterize the phenotype of the corresponding pathogenic cells through single cell sequencing. This high impact study is expected to eventually lead to a) demonstration of molecular mimicry in narcolepsy and b) improved safety of influenza vaccines.