Autoantibodies in Pandemrix^®-induced narcolepsy: Nine candidate autoantigens fail the conformational autoantibody test

Abstract

Study objectives: Narcolepsy type 1 (NT1) is a chronic sleep disorder characterized by loss of hypocretin-producing neurons. Increased NT1 incidence was observed in Sweden following mass-vaccination with Pandemrix^®. Genetic association to HLA DQB1*06:02 implies an autoimmune origin, but target autoantigen remains unknown. Candidate autoantigens for NT1 have previously been identified in solid-phase immunoassays, while autoantibodies against conformation-dependent epitopes are better detected in radiobinding assays. The aims are to determine autoantibody levels against nine candidate autoantigens representing (1) proteins of the hypocretin transmitter system; Preprohypocretin (ppHypocretin), Hypocretin peptides 1 and 2 (HCRT1 and HCRT2) and Hypocretin receptor 2 (HCRTR2); (2) proteins previously associated with NT1; Tribbles homologue 2 (TRIB2), Pro-opiomelanocortin/alpha-melanocyte-stimulating-hormone (POMC/α-MSH) and Prostaglandin D2 Receptor DP1 (DP1); (3) proteins suggested as autoantigens for multiple sclerosis (another HLA DQB1*06:02-associated neurological disease); ATP-dependent Inwardly Rectifying Potassium Channel Kir4.1 (KIR4.1) and Calcium-activated chloride channel Anoctamin 2 (ANO2).

Methods: Serum from post-Pandemrix^® NT1 patients (n = 31) and their healthy first-degree relatives (n = 66) were tested for autoantibody levels in radiobinding assays separating autoantibody bound from free labelled antigen with Protein A-Sepharose. ¹²⁵I-labelled HCRT1 and HCRT2 were commercially available while ³⁵S-methionine-labelled ppHypocretin, HCRTR2, TRIB2, α-MSH/POMC, DP1, KIR4.1 or ANO2 was prepared by in vitro transcription translation of respective cDNA. In-house standards were used to express data in arbitrary Units/ml (U/ml).

Results: All radiolabelled autoantigens were detected in a concentration-dependent manner by respective standard sera. Levels of autoantibodies in the NT1 patients did not differ from healthy first-degree relatives in any of the nine candidate autoantigens.

Conclusions: None of the nine labelled proteins proposed to be autoantigens were detected in the radiobinding assays for conformation-dependent autoantibodies. The results emphasise the need of further studies to identify autoantigen(s) and clarify the mechanisms in Pandemrix^®-induced NT1.

Keywords:

• Autoantibodies
• narcolepsy type 1
• Pandemrix®-vaccination
• hypocretin/orexin
• autoimmunity

Acknowledgement

The authors would like to acknowledge all NT1 patients and their family members that have donated their blood to make this study possible. The Swedish Narcolepsy Association (Narkolepsiföreningen Sverige) is thanked for inviting us to one of their meetings to present the study and to inform their members about the opportunity to participate in the study. Thanks to Ida Jönsson and Falastin Salami for expert technical assistance.

Disclosure statement

The authors report no conflict of interest.

Additional information

Funding

The study was supported by Alfred Österlunds stiftelse, Anna och Edvin Bergers Stiftelse, Crafoord Foundation, Filip Lundbergs stiftelse, Fredrik och Ingrid Thurings Stiftelse, The Royal Physiographic Society of Lund – The Fund of the Hedda and John Forssman Foundation, Gunvor och Josef Anérs stiftelse, Jerringfonden, Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Linnéa och Josef Carlssons Stiftelse, Magnus Bergvalls Stiftelse, Neuro Sweden, Rune Ljungdahls Stiftelse, Stiftelsen Samariten, Stiftelsen till minne av Personalföreningarna i Holmia Försäkring AB, Gun och Bertil Stohnes Stiftelse, the Segerfalk Foundation, Svenska Läkaresällskapet, Region Skane FoU, ALF grants, the Swedish Research Council, Strategic Research Area Exodiab, Dnr 2009-1039, and the Swedish Foundation for Strategic Research Dnr IRC15-0067.The funding sources had no involvement in study design, sample collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the article for publication.