Absence of anti-hypocretin receptor 2 autoantibodies in post pandemrix narcolepsy cases

BACKGROUND: A recent publication suggested molecular mimicry of a nucleoprotein (NP) sequence from A/Puerto Rico/8/1934 (PR8) strain, the backbone used in the construction of the reassortant strain X-179A that was used in Pandemrix(®) vaccine, and reported on anti-hypocretin (HCRT) receptor 2 (anti-HCRTR2) autoantibodies in narcolepsy, mostly in post Pandemrix(®) narcolepsy cases (17 of 20 sera). In this study, we re-examined this hypothesis through mass spectrometry (MS) characterization of Pandemrix(®), and two other pandemic H1N1 (pH1N1)-2009 vaccines, Arepanrix(®) and Focetria(®), and analyzed anti-HCRTR2 autoantibodies in narcolepsy patients and controls using three independent strategies. METHODS: MS characterization of Pandemrix(®) (2 batches), Arepanrix(®) (4 batches) and Focetria(®) (1 batch) was conducted with mapping of NP 116I or 116M spectrogram. Two sets of narcolepsy cases and controls were used: 40 post Pandemrix(®) narcolepsy (PP-N) cases and 18 age-matched post Pandemrix(®) controls (PP-C), and 48 recent (≤6 months) early onset narcolepsy (EO-N) cases and 70 age-matched other controls (O-C). Anti-HCRTR2 autoantibodies were detected using three strategies: (1) Human embryonic kidney (HEK) 293T cells with transient expression of HCRTR2 were stained with human sera and then analyzed by flow cytometer; (2) In vitro translation of [(35)S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [(35)S]-radiolabelled HCRTR2 were quantified using a radioligand-binding assay; (3) Optical density (OD) at 450 nm (OD450) of human serum immunoglobulin G (IgG) binding to HCRTR2 stably expressed in Chinese hamster ovary (CHO)-K1 cell line was measured using an in-cell enzyme-linked immunosorbent assay (ELISA). RESULTS: NP 116M mutations were predominantly present in all batches of Pandemrix(®), Arepanrix(®) and Focetria(®). The wild-type NP(109-123) (ILYDKEEIRRIWRQA), a mimic to HCRTR2(34-45) (YDDEEFLRYLWR), was not found to bind to DQ0602. Three or four subjects were found positive for anti-HCRTR2 autoantibodies using two strategies or the third one, respectively. None of the post Pandemrix(®) narcolepsy cases (0 of 40 sera) was found positive with all three strategies. CONCLUSION: Anti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix(®) autoimmune responses.