1. INFLUENZA, PANDEMRIX VACCINATION AND NARCOLEPSY – A REVIEW OF SOME RECENTLY PUBLISHED LABORATORY FINDINGS Reviewed by Anders Widell, Lund University Note that the publication by De la Herrán-Arita AK, which is cited in several slides, contains data that have been withdrawn by the authors due to difficulties to reproduce the findings /commentary by Anders Widell November 2014

2. The Fear The Spanish Flu, H1N1 1918 killed 40-50 millions Lethal virus then, lung effusion, still lethal today when reconstructed and given as challenge to macaques (P4 lab) One sequela in survivors was encephalitis lethargica Pandemia fear in the world since then Asian Flu pandemic in 1957 (H2N2) Hong Hong pandemic in 1968 (H3N2) Fort Dix 1976 – more people killed by post-vaccination Guillain Barré Syndrome than by the abortive H1N1 swine outbreak itself

3. The 2009 pandemic Winter - Spring 2009 severe flu cases reported from Mexico First un-typable, then typed to H1N1 pdm09 swine like Rapid spread world wide, WHO declared pandemic Prepared, safety–declared vaccine manufacturing platforms were adapted to include H1N1 pdm09 antigens Several manufacturers world wide. Some added potent adjuvants, GSK used squalene AS03

4. However, the A(H1N1)pdm09 pandemic turned out to be milder than expecetd Although some got very severe lung infection with deaths in immunocompromized persons and pregnant women, this pandemic killed fewer than a normal seasonal flu The vaccination locomotive was set in motion and the vaccination campaign was highly successful and conducted in an orderly way > 38.6 million people in EU/EEA countries were vaccinated: >30.5 million with Pandemrix, >560,000 with Celvapan >6.5 million with Focetria. In Sweden, that used Pandemrix, the take up was about 60%, few adverse events And everybody was happy ………until narcolepsy was reported in GSK Pandemrix vaccinees in Finland and Sweden

5. What is narcolepsy? Narcolepsy is a life long sleep disorder, due to loss of about 60.000 hypocretin (HCRT) secreting cells in the lateral hypothalamus. Cataplexy, a sudden loss of muscle tonus following emotions Normally starts in previously healthy persons - adolescents/ younger grown ups. Overall incidence about 1/100.000 pyr, seasonal Objective narcolepsy diagnosis is made either in a sleep lab or by measuring lower HCRT levels in CSF. A certain MHCII molecule (DQA1*01:02/DQB1*06:02) is present in 98% of cases Narcolepsy is of unknown etiology, autoimmunity suggested and streptococci have been implied

6. Narcolepsy outbreak 2010 An peak of narcolepsy in children in Finland and Sweden following Pandemrix Public outcry A very thorough case-control investigation by ECDC released in 2012 (Vaccine Adverse Event Surveillance and Communication, VAESCO; Finland, Sweden, Norway, Denmark, UK, Holland, Italy) Statistical increase only proven in ages 5-19 y and only after Pandemrix South –North gradient in Sweden In Finland, an increase in the incidence rate of narcolepsy diagnoses after September 2009 was observed with relative risk of 6.4 (95%CI 4.2-9.7). In Sweden, an increase with a relative risk of 7.5 (95%CI 5.2–10.7) No significant increase in other VAESCO countries By Aug 2012 600 cases in all VAESCO countries

7. Was Pandemrix the only culprit?

8. Pilot study possibly indicating broader H1N1pdm09 exposure as initiator than Pandemrix alone (Dauvilliers et al, Sleep 33, 2010)

9. Increased narcolepsy incidence in Beijing China in 2010, where H1N1pdm09 vaccination was rarely used (Han el al, Ann Neurol /0; 2011)

10. Key study by De la Herrán-Arita el al; Science Translational Med Dec 2013 Same Stanford team headed by E Mignot As Antigen Presenting Cells these authors use MHC class I– and class II–deficient lymphoblastic T-B fusion cell line, transfected to express the mandatory DQ0602 heterodimer Sets of peptides are added to the grove PBMCs carrying their T Cell Receptor from patients and DQ0602 controls react with APC and peptide. Readout as  -IFN from patient CD4 cells by ELISpot Analyzed first 127 aa Prepro-hypocretin peptide HCRT

11. Locating HCRT hot spots that compete in with EBV 490-503 epitope (RALLARSHVERTTDE; EBNA-1) in the DQ0602 grove. (EBV 490-503 is a known binder to DQ0602)

12. 2-log scale Pilot study

13. Pattern repeated in 23 patients and in 24 DQB1*0602 concordant controls, obtained and stored at different time points

14. … and further strengthened in family settings

15. The HCRT peptides binding to the APC grove and the T-cell receptor TCR

16. The HCRT variants can outcompete the binding of the reference EBV peptide in the DQ0602 grove

17. Potential flu peptide homologs in H1N1pdm09 101 H1N1pdm09 peptides from HA1,NA1, PB1 potentially binding to DQ0602 were screened by software for the best 9-mers to bind to APC (P1,P3, P4, P6, P9) thereafter to TCR (P5, P7, P8), at positions matching the key sites found for the HCRT peptides pHA1 273-287 matched best, with three key sites P5, P7, P8 at the TCR side There were also 3 weaker binders The pHA1 273-287 region was aligned with other H1N1 strains

18. Linear scale Pre and post seasonal trivalent influenza vaccination containing H1N1pdm09 protein – in vivo findings. HCRT and pHA1 275-287

19. Stronger findings when In vitro stimulated by the vaccine in solution

20. Mimicry and alignment across critical positions in the APC grove

21. Also the HA1 epitopes compete with the EBV reference peptides in the APC grove, strongest with HA1 2009 and HA1 1998, the latter a swine strain

22. Summing up T cell studies The case for binding of the HCRT peptides into the DQ0602 grove and subsequent activation of CD4 cells is strong Groups: Cases vs matched controls, twins, siblings A potentially H1N1pdm09 homolog pHA1 275-287 sharing 3 amino key acids binding to the TCR was found Vaccination of Pandemrix exposed narcolepsy patients with trivalent non-adjuvated vaccine evoked a weak in vivo but a stronger in vitro response The virus peptide may be culprit, enhanced by Pandemrix Caveats. Relevant peptide concentrations?, special APCs, neurons do not express MHC-II

23. A recent antibody study Lind et al, Autoimmunity 2014 Swedish narcolepsy victims (N= 47), 80 controls Using 35 S-labelled transcription-translation products corresponding to HA1 and TRIB2 (narcolepsy related protein), radiobinding to sera was measured Higher anti-HA antibodies were seen in narcolepsy patients, in particular the youngest TRIB2 not generally elevated but a correlation to anti- HA, strongest in children below 13

24. Over