Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2

ECDC comment

​A possible biological mechanism for development of narcolepsy following vaccination with one of the AS03-adjuvanted pandemic vaccines, Pandemrix, has been proposed by Ahmed et al. Narcolepsy results from destruction of hypocretin (HCRT) secreting neurons in hypothalamus, however further details on the disease mechanisms are poorly understood.

Ahmed SS, Volkmuth W, Duca J, Corti L, Pallaoro M, Pezzicoli A, Karle A, Rigat F, Rappuoli R,  Narasimhan V, Julkunen I, Vuorela A, Vaarala O, Nohynek H, Laghi Pasini F, Montomoli E, Trombetta C,  Adams CM, Rothbard J, Steinman L Science Translational Medicine vol 7 issue 294 (published 1 July 2015)

A possible biological mechanism for development of narcolepsy following vaccination with one of the AS03-adjuvanted pandemic vaccines, Pandemrix, has been proposed by Ahmed et al. Narcolepsy results from destruction of hypocretin (HCRT) secreting neurons in hypothalamus, however further details on the disease mechanisms are poorly understood.

Using previously published  observations regarding that most narcolepsy cases spontaneously reported to regulatory agencies in the EU occurred following Pandemrix vaccination, they hypothesized that there may be a difference between this and  other pandemic vaccines. They therefore conducted a series of experiments focusing on the two 2009 pandemic vaccines most used in Europe: Pandemrix produced by GSK (more than 30 million doses were administered) and Focetria produced by Novartis (more than 6 million administered doses) (1).
In their study they first compared protein sequences for the four influenza proteins expected to be present in the vaccines; matrix protein (M1), nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) of  the vaccine reassortant strains with protein sequences of the naturally circulating influenza A (H1N1)pdm09 (A/California/07/2009).
The protein sequence of the vaccine reassortant X-179A used in Pandemrix were identical with the protein sequence of the naturally circulating 2009 pandemic strain while the vaccine reassortant X-181 used in Focetria differed in one residue in the HA and in two residues in the NP. These results lead the investigators to further investigate the areas where differences had been identified.
Based on a known hypothesis that autoimmune disease may be triggered by molecular mimicry, the investigators then compared the protein sequences of  the three microbial protein sequences of the HA and NA with  fragments of the human HCRT  ligand and the HCRT receptors present in the human brain.
In this experiment the researchers identified a sequence of the NP 111-121 peptide in Pandemrix that was found to be similar to a sequence of the extracellular domain (N-terminus) of the HCRT receptor no. 2 34-45 (e value= 0.026). This sequence is located on the surface-exposed region of the influenza NP crystal structure.
This NP peptide seems to have been conserved in influenza strains from 1902 to 2013.
Having shown theoretically that molecular mimicry between the influenza NP111-121 and the HCRT 2 receptor could occur, the researchers continued to assess presence of antibodies in different patient groups to HCRT 2 receptor  using a cell-line expressing human HCRT receptor no. 2:

  • HLA-DQB1*0602 haplotype-positive narcoleptic patients with a history of Pandemrix vaccination;
  • sera from non-narcoleptic individuals with a history of a laboratory-confirmed 2009 influenza A (H1N1) infection;
  • sera from healthy control children collected in 2004/2005 prior to 2009 influenza pandemic or vaccination;
  • MF-59 adjuvanted influenza A (H1N1)pdm09 (Focetria) vaccination.

In 17/20 tested sera from narcolepsy patients that were vaccinated with Pandemrix, HCRT 2- specific antibodies were identified, while none of the 6 tested sera from Focetria vaccinated individuals (p<0.001) and only 5/20 naturally infected with a laboratory-confirmed influenza A(H1N1)pdm09 infection exhibited reactivity (p<0.001). However, many paediatric control sera from 2004/2005 also showed HRCT 2-specific antibodies. The authors point out an important limitation here, namely that none of the control groups were matched for the HLA-DQB1*0602 known to be associated with development of narcolepsy.

The investigators took the antibody detection one step further and showed by double-labelling immunofluorescence microscopy that the antibodies in analysed sera co-localised with staining by a commercial antibody for the human CRT receptor 2.

Further, the HCRT antibody reactions could be inhibited by either adding a NP peptide or a HCRT 2 peptide suggesting the reactivity is specific.

Finally, the investigators detected NP by gel electrophoresis and Western blot in different pandemic and seasonal influenza vaccines and subsequently quantified NP to show that the NP content containing the protein sequence with similarity to the HCRT no 2 receptor vary by product. The highest content was identified in the two AS03-adjuvanted pandemic vaccines Pandemrix and Arepanrix. 
In short, the investigators propose a mechanism by which Pandemrix vaccination, and possibly also influenza infection as suggested in studies from China but not elsewhere, in genetically susceptible individuals, could lead to narcolepsy. The presentation of NP antigen during immunisation with adjuvanted influenza vaccines containing higher amounts of NP or infection may generate high titres of NP antibodies that can persist in the systemic circulation for months. In some individuals these antibodies may cross-react with the HCRT 2 receptor and trigger narcolepsy.
The article ends with a number of limitations of the current work including:

  • lack of knowledge on how common HCRT receptor 2 antibodies are in healthy populations;
  • lack of a direct link between the presence of anti-HRCT 2 antibodies and the development of narcolepsy;
  • lack of studies in CD4 T cells purified from the individuals that provided sera for testing; 
  • lack of sera and peripheral blood mononuclear cells from age-matched HLA-DQB1*0602 positive subjects vaccinated with Focetria and subjects not receiving Pandemrix but who developed narcolepsy in this time period;
  • lack of an appropriate humanized animal model in which to test the hypothesised mechanism.

Earlier studies addressing a possible biological mechanism
There are six earlier studies, all published in the last 12 months that have explored factors that could contribute to a possible biological mechanism and therefore worthwhile mentioning in this context. Notably, two of those studies focused on the two AS03-adjuvanted vaccines - Pandemrix and Arepanrix (2-3), and considered the role of the influenza NP. 
The first study by Vaarala et al published in December 2014 already pointed to a role for influenza NP noting higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix  and antigenic changes induced by detergent recognized by antibodies in sera collected from children with narcolepsy (2).
A more recent study published by Jacob et al showed that Pandemrix contained more NP and NA protein than Arepanrix.  Further this study showed a 10-fold higher deamidation in Arepanrix versus Pandemrix in the HA protein (3).

Other studies focused on possible autoimmune responses including cellular immune response to β-haemolytic group A streptococci in individuals with narcolepsy (4), and TRIB2 autoantibodies in conjunction with influenza A(H1N1)pdm09 antibodies (5).

In another study the adjuvant component α-tocopherol of AS03 was shown to  activate the transcription factor Nrf2, that possibly can lead to increased formation of longer hypocretin-derived fragments that then can be presented by HLA-subtype DQB1*602 and, due to their increased sensitivity to apoptotic stimuli, can be destroyed and lead to hypocretin deficiency (6). 

Finally, in a rat model researchers showed that narcolepsy patients have antibodies that stain distinct cell populations in rats’ brains and influences their sleep pattern upon passive transfer experiments of serum from narcolepsy individuals (7).

ECDC comment

It is now almost five years since the first reports of narcolepsy following use of Pandemrix were received by European regulatory agencies, some time after pandemic influenza vaccination campaigns had ceased.  An epidemiological association between narcolepsy development and receipt of Pandemrix has subsequently been shown in six European countries (9-15) while essentially none has been identified in Canada that used a similar but not identical AS03-adjuvanted vaccine Arepanrix (16-17).
This new publication from one of the pandemic vaccine producers Novartis in collaboration with public health and academic researchers in Finland and the US adds to the evidence-base on how narcolepsy might have been triggered in genetically susceptible individuals.
However, the biological mechanism described and tested in this publication is still only presented as a hypothesis, backed up by a series of experiments, that needs further verification, as also the authors note.  The implications, particularly if further research suggests that the hypothesis is valid, will also need to be carefully assessed by regulators.

Although this study proposes one possible biological mechanism there may be other mechanisms, as well as many outstanding research questions not addressed in this study:

  • what role, if any, did the adjuvant have in development of narcolepsy;
  • whether co-infections, notably streptococcal infections played a role in development of narcolepsy; 
  • whether other  host susceptibility genetic factors, such as the presence of the  DR1 gene, that facilitates replication of influenza A viruses (8) play a role in  individuals that developed narcolepsy.

There is also a need for animal models to assess the blood-brain-barrier, to understand how antibodies can transfer across the blood-brain-barrier and have an impact on neurons in the hypothalamus.

An increased understanding of the reasons for the increased number of reported cases of narcolepsy following use of Pandemrix is essential to further the public health community’s understanding of the event to avoid anything similar happening in the future.

Means for an independent confirmation should be explored and facilitated by all relevant stakeholders.


  1. EMA Twenty-second pandemic pharmacovigilance update published August 19, 2010
  2. 2. Vaarala O, Vuorela A, Partinen M, Baumann M, Freitag TL, Meri S, Saavalainen P, Jauhiainen M, Soliymani R, Kirjavainen T, Olsen P, Saarenpaa-Heikkila O, Rouvinen J, Roivainen M, Nohynek H, Jokinen J, Julkunen I, Kilpi T Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for Pandemrix-associated narcolepsy risk PLOS ONE 2014, December 15, 9(12): e114361. Doi:10.1371/journalpone.0114361
  3. Jacob L, Leib R, Ollila HM, Bonvalet M, Adams CM, Mignot E Comparison of Pandemrix and Arpanrix, two pH1N1 AS03-adjuvanted vaccines differentially associated with narcolepsy development Brain, Behaviour and Immunity 2015;47 44-57
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  5. Lind A, Ramelius A, Olsson T, Arnehim-Dahlstrom L, Lamb F, Khademi M, Ambatti A, Maeurer M, Nilsson AL, Bomfim IL, Fink K, Lernmark A A/H1N1 antibodies and TRIB2 autoantibodies in narcolepsy patients diagnosed in conjunction with the Pandemrix vaccination campaign in Sweden 2009-2010. J Autoimmun 2014 May;50:99-106 doi: 10.1016/j.aut.2014.01.031
  6. Masoudi S, Ploen D, Kunz K, Hildt E The adjuvant component α-tocopherol triggers via modulation of Nrf2 the expression and turnover of hypocretin in vitro and its implication to the development of narcolepsy Vaccine 2014; 32 2980-2988
  7. Bergman P, Adori C, Vas S, Kai-Larsen Y, Sarkanen T, Cederlund A, Agerberth B, Julkunen I, Horvath B, Kostyalik D, Kalmar L, Bagdy G, Huutoniemi A, Partinen M, Hokfelt T Nacrolepsy patients have antibodies that stain distinct populations in rat brain and influence sleep patterns PNAS 2014 18 August
  8. Hsu SF, Su W-C, Jeng K-S, Lai MM A host susceptibility gene, DR1, facilitiates influenza A virus replication by suppressing host innate immunity and enhancing viral RNA replication J Virol April 2015 doi: 10.1128/JVI.03610-14
  9. Dauvilliers Y, Arnulf I, Lecendreux M, Monaca Charley C, Franco P, Drouot X, d'Ortho MP, Launois S, Lignot S, Bourgin P, Nogues B, Rey M, Bayard S, Scholz S, Lavault S, Tubert-Bitter P, Saussier C, Pariente A; Narcoflu-VF study group. Increased risk of narcolepsy in children and adults after pandemic H1N1 vaccination in France. Brain. 2013 Aug;136(Pt 8):2486-96.
  10. Heier MS, Gautvik KM, Wannag E, Bronder KH, Midtlyng E, Kamaleri Y, Storsaeter J. Incidence of narcolepsy in Norwegian children and adolescents after vaccination against H1N1 influenza A. Sleep Med. 2013 Sep;14(9):867-71.
  11. Miller E, Andrews N, Stellitano L, Stowe J, Winstone AM, Shneerson J, Verity C. Risk of narcolepsy in children and young people receiving AS03 adjuvanted pandemic A/H1N1 2009 influenza vaccine: retrospective analysis. BMJ. 2013 Feb 26;346
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  13. Nohynek H, Jokinen J, Partinen M, Vaarala O, Kirjavainen T, Sundman J, Himanen SL, Hublin C, Julkunen I, Olsén P, Saarenpää-Heikkilä O, Kilpi T. AS03 adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland. PLoS One. 2012;7(3):
  14. O'Flanagan D1, Barret AS, Foley M, Cotter S, Bonner C, Crowe C, Lynch B, Sweeney B, Johnson H, McCoy B, Purcell E. Investigation of an association between onset of narcolepsy and vaccination with pandemic influenza vaccine, Ireland April 2009-December 2010. Euro Surveill. 2014 May 1;19(17):15-25.
  15. Persson I, Granath F, Askling J, Ludvigsson JF, Olsson T, Feltelius N. Risks of neurological and immune-related diseases, including narcolepsy, after vaccination with Pandemrix: a population- and registry-based cohort study with over 2 years of follow-up. J Intern Med. 2014 Feb;275(2):172-90.
  16. T Harris, K Wong, L Stanford, J Fediurek, N Crowcroft, S L Deeks Did narcolepsy occur following administration of AS03-adjuvanted A(H1N1) pandemic vaccine in Ontario, Canada A review of post-marketing safety surveillance data Eurosurveillance, 2014;  Volume 19, Issue 36, 11 September
  17. Montplaisir J, Petit D, Quinn MJ, Ouakki M, Deceuninck G, Desautels A, Mignot E, De Wals P.
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