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

Guo Luo,Mélodie Bonvalet,Ling Lin,Christopher M Adams,Aditya Ambati,Giuseppe Plazzi,Louis Jacob,Ryan Leib,Emmanuel Jean-Marie Mignot,Fabio Pizza,Markku Partinen

doi:10.1371/journal.pone.0187305

Guo Luo, Mélodie Bonvalet + Show 9 more

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https://doi.org/10.1371/journal.pone.0187305

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Abstract

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.

Highlights

There is strong genetic evidence suggesting autoimmunity in type 1 narcolepsy [1,2,3], notably a 97% association with human leukocyte antigen (HLA) class II alleles DQB1Ã06:02 and DQA1Ã01:02 that together form the heterodimer DQ0602 [4], the nature of the autoimmune antigen leading to hypocretin cell death has been elusive
Frequencies of NP 116I and 116M, HA1 146N (HA 146N) and 146D for Pandemrix1, Arepanrix1 and Focetria1 were shown in Table 2, with the caveat that much lower NP coverage was obtained with Focetria1
We found that the occurrences of mutation vs wild type in Pandemrix1, Arepanrix1, and Focetria1 were 47 vs 2, 76 vs 3, and 5 vs 0, respectively, indicating that the NP 116M mutation was predominantly present in all the three vaccines (Fig 1, Table 2)

Summary

Introduction

There is strong genetic evidence suggesting autoimmunity in type 1 narcolepsy [1,2,3], notably a 97% association with human leukocyte antigen (HLA) class II alleles DQB1Ã06:02 and DQA1Ã01:02 that together form the heterodimer DQ0602 [4], the nature of the autoimmune antigen leading to hypocretin cell death has been elusive Renewed interest in this area came from the observation of increased incidence of type 1 narcolepsy following the 2009– 2010 winter vaccination campaign against the pandemic H1N1 (pH1N1) “swine” flu (pH1N12009) that emerged in 2009 [5]. Observation of seasonality of onset was more obvious in young children where narcolepsy onset is very abrupt and can more be timed to the exact month All together, these data strongly suggest that narcolepsy is an autoimmune disease triggered by upper airway infections, notably influenza and maybe Streptococcus Pyogenes, with stronger susceptibility to pH1N1-2009. 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.

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Results

Conclusion