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

BackgroundA 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.MethodsMS 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 [35S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [35S]-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).ResultsNP 116M mutations were predominantly present in all batches of Pandemrix®, Arepanrix® and Focetria®. The wild-type NP109-123 (ILYDKEEIRRIWRQA), a mimic to HCRTR234-45 (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.ConclusionAnti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix® autoimmune responses.背景 近期一项研究提出，A/波多黎各/8/1934（A/Puerto Rico/8/1934, PR8）毒株的核蛋白（nucleoprotein, NP）序列存在分子模拟现象——该毒株是构建Pandemrix®疫苗所用重配株X-179A的骨架毒株；同时该研究报道了发作性睡病（narcolepsy）患者体内存在抗促泌素（hypocretin, HCRT）受体2（anti-HCRTR2）自身抗体，此类抗体主要见于Pandemrix®疫苗接种后发作性睡病病例（20份血清中17份呈阳性）。本研究通过质谱（mass spectrometry, MS）对Pandemrix®以及另外两款2009年甲型H1N1流感大流行疫苗（Arepanrix®与Focetria®）进行表征，并采用三种独立检测策略分析发作性睡病患者与对照人群体内的抗HCRTR2自身抗体，以此重新验证前述假说。 方法 本研究对2个批次的Pandemrix®、4个批次的Arepanrix®以及1个批次的Focetria®进行质谱表征，完成NP 116I或116M的谱图映射。本研究纳入两组发作性睡病病例与对照人群：第一组为40例Pandemrix®疫苗接种后发作性睡病（post Pandemrix® narcolepsy, PP-N）病例与18例年龄匹配的Pandemrix®疫苗接种后对照（post Pandemrix® controls, PP-C）；第二组为48例近期（≤6个月）早发性发作性睡病（early onset narcolepsy, EO-N）病例与70例年龄匹配的其他对照（other controls, O-C）。本研究采用三种策略检测抗HCRTR2自身抗体：（1）将人血清与瞬时表达HCRTR2的人胚肾（human embryonic kidney, HEK）293T细胞共孵育染色，随后通过流式细胞仪进行分析；（2）将经[³⁵S]放射标记的HCRTR2体外翻译产物与人血清共孵育，采用放射配体结合试验定量自身抗体与[³⁵S]放射标记HCRTR2形成的免疫复合物；（3）采用细胞内酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测人血清免疫球蛋白G（immunoglobulin G, IgG）与稳定表达HCRTR2的中国仓鼠卵巢（Chinese hamster ovary, CHO）-K1细胞结合后的450nm光密度（OD450）值。 结果 所有批次的Pandemrix®、Arepanrix®与Focetria®疫苗中均主要存在NP 116M突变。作为HCRTR234-45（YDDEEFLRYLWR）模拟表位的野生型NP109-123序列（ILYDKEEIRRIWRQA）未被发现可与DQ0602结合。分别通过两种检测策略或第三种检测策略，仅3或4名受试者的抗HCRTR2自身抗体呈阳性。在全部三种检测策略下，40份Pandemrix®疫苗接种后发作性睡病病例血清均未呈阳性（0/40）。 结论 抗HCRTR2自身抗体并非发作性睡病或Pandemrix®疫苗接种后自身免疫应答的显著生物学特征。