Homozygous HLA-DQB1*06:02 combined with T-cell receptor alpha polymorphism results in narcolepsy onset - A familial case report.

Narcolepsy is a life-long neurological disorder with well-established genetic risk factors. Human leukocyte antigen-DQB1*06:02 remains the strongest genetic predeterminant; however, polymorphisms in genes encoding the T-cell receptor alpha chain are also strongly linked. This case report shows the inheritance pathway of these genetic markers contributing to narcolepsy onset in a 17-year-old female.

Characterization of T cell receptors reactive to HCRTNH2, pHA273-287, and NP17-31 in control and narcolepsy patients.

Narcolepsy type 1 (NT1), a disorder caused by hypocretin/orexin (HCRT) cell loss, is associated with human leukocyte antigen (HLA)-DQ0602 (98%) and T cell receptor (TCR) polymorphisms. Increased CD4+ T cell reactivity to HCRT, especially DQ0602-presented amidated C-terminal HCRT (HCRTNH2), has been reported, and homology with pHA273-287 flu antigens from pandemic 2009 H1N1, an established trigger of the disease, suggests molecular mimicry. In this work, we extended DQ0602 tetramer and dextramer data to 77 cases and 44 controls, replicating our prior finding and testing 709 TCRs in Jurkat 76 T cells for functional activation. We found that fewer TCRs isolated with HCRTNH2 (∼11%) versus pHA273-287 or NP17-31 antigens (∼50%) were activated by their ligand. Single-cell characterization did not reveal phenotype differences in influenza versus HCRTNH2-reactive T cells, and analysis of TCR CDR3αß sequences showed TCR clustering by responses to antigens but no cross-peptide class reactivity. Our results do not support the existence of molecular mimicry between HCRT and pHA273-287 or NP17-31.

Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

T cell reactivity to regulatory factor X4 in type 1 narcolepsy.

Type 1 narcolepsy is strongly (98%) associated with human leukocyte antigen (HLA) class II DQA1*01:02/DQB1*06:02 (DQ0602) and highly associated with T cell receptor (TCR) alpha locus polymorphism as well as other immune regulatory loci. Increased incidence of narcolepsy was detected following the 2009 H1N1 pandemic and linked to Pandemrix vaccination, strongly supporting that narcolepsy is an autoimmune disorder. Although recent results suggest CD4+ T cell reactivity to neuropeptide hypocretin/orexin and cross-reactive flu peptide is involved, identification of other autoantigens has remained elusive. Here we study whether autoimmunity directed against Regulatory Factor X4 (RFX4), a protein co-localized with hypocretin, is involved in some cases of narcolepsy. Studying human serum, we found that autoantibodies against RFX4 were rare. Using RFX4 peptides bound to DQ0602 tetramers, antigen RFX4-86, -95, and -60 specific human CD4+ T cells were detected in 4/10 patients and 2 unaffected siblings, but not in others. Following culture with each cognate peptide, enriched autoreactive TCRαß clones were isolated by single-cell sorting and TCR sequenced. Homologous clones bearing TRBV4-2 and recognizing RFX4-86 in patients and one twin control of patient were identified. These results suggest the involvement of RFX4 CD4+ T cell autoreactivity in some cases of narcolepsy, but also in healthy donors.

A/H1N1 hemagglutinin antibodies show comparable affinity in vaccine-related Narcolepsy type 1 and control and are unlikely to contribute to pathogenesis.

An increased incidence of narcolepsy type 1 (NT1) was observed in Scandinavia following the 2009-2010 influenza Pandemrix vaccination. The association between NT1 and HLA-DQB1*06:02:01 supported the view of the vaccine as an etiological agent. A/H1N1 hemagglutinin (HA) is the main antigenic determinant of the host neutralization antibody response. Using two different immunoassays, the Luciferase Immunoprecipitation System (LIPS) and Radiobinding Assay (RBA), we investigated HA antibody levels and affinity in an exploratory and in a confirmatory cohort of Swedish NT1 patients and healthy controls vaccinated with Pandemrix. HA antibodies were increased in NT1 patients compared to controls in the exploratory (LIPS p = 0.0295, RBA p = 0.0369) but not in the confirmatory cohort (LIPS p = 0.55, RBA p = 0.625). HA antibody affinity, assessed by competition with Pandemrix vaccine, was comparable between patients and controls (LIPS: 48 vs. 39 ng/ml, p = 0.81; RBA: 472 vs. 491 ng/ml, p = 0.65). The LIPS assay also detected higher HA antibody titres as associated with HLA-DQB1*06:02:01 (p = 0.02). Our study shows that following Pandemrix vaccination, HA antibodies levels and affinity were comparable NT1 patients and controls and suggests that HA antibodies are unlikely to play a role in NT1 pathogenesis.

Immunocyte single cell analysis of vaccine-induced narcolepsy.

Increased incidence of narcolepsy type 1 (NT1) was observed following Pandemrix®-vaccination, initiated as a preventive measure against the 2009 Influenza pandemic. Here, single cell analysis was conducted to suggest a lower number of CD8+ CD27+ T cells among these patients. These findings provide understanding into the autoimmune pathogenesis of NT1.

Activating autoantibodies against G protein-coupled receptors in narcolepsy type 1.

STUDY OBJECTIVES: Narcolepsy type 1 is a rare hypersomnia of central origin, which is caused by loss of hypothalamic neurons that produce the neuropeptides hypocretin-1 and -2. Hypocretin-containing nerve terminals are found in areas known to play a central role in autonomic control and in pain signaling. Cholinergic M2 receptors are found in brain areas involved with the occurrence of hallucinations and cataplexy. In addition to classical symptoms of narcolepsy, the patients suffer frequently from autonomic dysfunction, chronic pain, and hypnagogic/hypnopompic hallucinations. We aimed to test whether narcolepsy type 1 patients have autoantibodies against autonomic ß2 adrenergic receptor, M2 muscarinic receptors, or nociception receptors. METHODS: We tested the serum of ten narcolepsy type 1 patients (five female) for activating ß2 adrenergic receptor autoantibodies, M2 muscarinic receptor autoantibodies, and nociception receptor autoantibodies. RESULTS: Ten of ten patients were positive for muscarinic M2 receptor autoantibodies (P < 0.001), 9/10 were positive for autoantibodies against nociception receptors (P < 0.001), and 5/10 were positive for ß2 adrenergic receptor autoantibodies (P < 0.001). CONCLUSIONS: Narcolepsy type 1 patients harbored activating autoantibodies against M2 muscarinic receptors, nociception receptors, and ß2 adrenergic receptors. M2 receptor autoantibodies may be related to the occurrence of cataplexy and, moreover, hallucinations in narcolepsy since they are found in the same brain areas that are involved with these symptoms. The occurrence of nociception receptor autoantibodies strengthens the association between narcolepsy type 1 and pain. The connection between narcolepsy type 1, autonomic complaints, and the presumed cardiovascular morbidity might be associated with the occurrence of ß2 adrenergic receptor autoantibodies. On the other hand, the presence of the autoantibodies may be secondary to the destruction of the hypocretin pathways.

Reassessment of the risk of narcolepsy in children in England 8 years after receipt of the AS03-adjuvanted H1N1 pandemic vaccine: A case-coverage study.

BACKGROUND: Early studies of narcolepsy after AS03-adjuvanted pandemic A/H1N12009 vaccine (Pandemrix) could not define the duration of elevated risk post-vaccination nor the risk in children aged under 5 years who may not present until much older. METHODS/FINDINGS: Clinical information and sleep test results, extracted from hospital notes at 3 large pediatric sleep centers in England between September 2017 and June 2018 for narcolepsy cases aged 4-19 years with symptom onset since January 2009, were reviewed by an expert panel to confirm the diagnosis. Vaccination histories were independently obtained from general practitioners (GPs). The odds of vaccination in narcolepsy cases compared with the age-matched English population was calculated after adjustment for clinical conditions that were indications for vaccination. GP questionnaires were returned for 242 of the 244 children with confirmed narcolepsy. Of these 5 were under 5 years, 118 were 5-11 years, and 119 were 12-19 years old at diagnosis; 39 were vaccinated with Pandemrix before onset. The odds ratio (OR) for onset at any time after vaccination was 1.94 (95% confidence interval [CI] 1.30-2.89), The elevated risk period was restricted to onsets within 12 months of vaccination (OR 6.65 [3.44-12.85]) and was highest within the first 6 months. After one year, ORs were not significantly different from 1 up to 8 years after vaccination. The ORs were similar in under five-year-olds and older ages. The estimated attributable risk was 1 in 34,500 doses. Our study is limited by including cases from only 3 sleep centers, who may differ from cases diagnosed in nonparticipating centers, and by imprecision in defining the centers' catchment population. The potential for biased recall of onset shortly after vaccination in cases aware of the association cannot be excluded. CONCLUSIONS: In this study, we found that vaccine-attributable cases have onset of narcolepsy within 12 months of Pandemrix vaccination. The attributable risk is higher than previously estimated in England because of identification of vaccine-attributable cases with late diagnoses. Absence of a compensatory drop in risk 1-8 years after vaccination suggests that Pandemrix does not trigger onsets in those in whom narcolepsy would have occurred later.

Clinical utility of next generation sequencing based HLA typing for disease association and pharmacogenetic testing.

HLA associations have been linked to many diseases and are important for risk assessment of drug hypersensitivity reactions. The increasing number of HLA alleles discovered generated a list of ambiguities that cannot be resolved with the current clinical assays, which commonly include sequence-specific oligonucleotide probe (SSOP) genotyping, and real-time PCR with melting curve analysis. HLA typing by next-generation sequencing (NGS) has recently been adopted by clinical laboratories for transplantation testing, as it provides unambiguous and cost-effective HLA typing. The goal of this study was to evaluate the feasibility of using NGS-based HLA-B and DQ genotyping for clinical HLA disease association testing, and provide direct comparison with the currently used clinical tests, including SSOP genotyping, and real-time PCR with melting curve analysis. While the real-time PCR method is easy and inexpensive to perform, ambiguities are rapidly increasing as more and more HLA alleles are discovered. SSOP genotyping identifies the alleles present but limitations include ambiguities and underreporting less common alleles. Our data show that HLA typing by NGS is superior to the existing clinical methods for identifying HLA alleles associated with disease or drug hypersensitivity, and offers a viable approach for high volume clinical diagnostic laboratories.

Screening for autoantibody targets in post-vaccination narcolepsy using proteome arrays.

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n = 39), their first-degree relatives (FDR) (n = 66), population controls (n = 188), and disease controls representing multiple sclerosis (n = 100) and FDR to type 1 diabetes patients (n = 41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1.

[Narcolepsy: From the discovery of a wake promoting peptide to autoimmune T cell biology and molecular mimicry with flu epitopes]. / Narcolepsie : une maladie auto-immune affectant un peptide de l'éveil liée à un mimétisme moléculaire avec des épitopes du virus de la grippe.

Narcolepsy-cataplexy was first described in the late 19th century in Germany and France. Prevalence was established to be 0.05 % and a canine model was discovered in the 1970s. In 1983, a Japanese study found that all patients carried HLA-DR2, suggesting autoimmunity as the cause of the disease. Studies in the canine model established that dopaminergic stimulation underlies anti-narcoleptic action of psychostimulants, while antidepressants were found to suppress cataplexy through adrenergic reuptake inhibition. No HLA association was found in canines. A linkage study initiated in 1988 revealed in hypocretin (orexin) receptor two mutations as the cause of canine narcolepsy in 1999. In 1992, studies on African Americans showed that DQ0602 was a better marker than DR2 across all ethnic groups. In 2000, hypocretin-1/orexin A levels were measured in the cerebrospinal fluid (CSF) and found to be undetectable in most patients, establishing hypocretin deficiency as the cause of narcolepsy. Decreased CSF hypocretin-1 was then found to be secondary to the loss of the 70,000 neurons producing hypocretin in the hypothalamus, suggesting immune destruction of these cells as the cause of the disease. Additional genetic studies, notably genome wide associations (GWAS), found multiple genetic predisposing factors for narcolepsy. These were almost all involved in other autoimmune diseases, although a strong and unique association with T cell receptor (TCR) alpha and beta loci were observed. Nonetheless, all attempts to demonstrate presence of autoantibodies against hypocretin cells in narcolepsy failed, and the presumed autoimmune cause remained unproven. In 2009, association with strep throat infections were found, and narcolepsy onsets were found to occur more frequently in spring and summer, suggesting upper away infections as triggers. Following reports that narcolepsy cases were triggered by vaccinations and infections against influenza A 2009 pH1N1, a new pandemic strain that erupted in 2009, molecular mimicry with influenza A virus was suggested in 2010. This hypothesis was later confirmed by peptide screening showing higher activity of CD4+ T cell reactivity to a specific post-translationally amidated segment of hypocretin (HCRT-NH2) and cross-reactivity of specific TCRs with a pH1N1-specific segment of hemagglutinin that shares homology with HCRT-NH2. Strikingly, the most frequent TCR recognizing these antigens was found to carry sequences containing TRAJ24 or TRVB4-2, segments modulated by narcolepsy-associated genetic polymorphisms. Cross-reactive CD4+ T cells with these cross-reactive TCRs likely subsequently recruit CD8+ T cells that are then involved in hypocretin cell destruction. Additional flu mimics are also likely to be discovered since narcolepsy existed prior to 2009. The work that has been conducted over the years on narcolepsy offers a unique perspective on the conduct of research on the etiopathogeny of a specific disease.

TITLE: Narcolepsie : une maladie auto-immune affectant un peptide de l'éveil liée à un mimétisme moléculaire avec des épitopes du virus de la grippe. ABSTRACT: La narcolepsie et la cataplexie sont décrites pour la première fois à la fin du XIXe siècle en Allemagne et en France. La prévalence de la maladie est établie à 0,05 % et un modèle canin est découvert dans les années 1970. En 1983, une étude japonaise révèle que les patients narcoleptiques sont porteurs d'un marqueur génétique unique, l'antigène leucocytaire HLA-DR2, suggérant l'auto-immunité comme cause de la maladie. Il faudra attendre 1992 pour qu'il soit montré, grâce à une étude chez des patients afro-américains, que DQ0602, un autre gène HLA, est la véritable cause de cette association. Des études pharmacologiques conduites sur le modèle canin établissent que la stimulation dopaminergique est le mode d'action des stimulants sur l'éveil, tandis que les antidépresseurs suppriment la cataplexie en inhibant la recapture adrénergique. Aucune association HLA n'est cependant mise en évidence chez les chiens, suggérant une cause distincte de la maladie humaine. Une étude de liaison génétique chez les chiens, initiée en 1988, révèle en 1999 que la narcolepsie canine est causée par des mutations du récepteur 2 de l'hypocrétine (orexine). En 2000, l'hypocrétine-1/orexine A est mesurée dans le liquide céphalo-rachidien (LCR) et on découvre qu'elle est indétectable chez la plupart des patients narcoleptiques, établissant qu'un déficit hypocrétinergique est la cause de la narcolepsie humaine. La diminution de l'hypocrétine-1 dans le LCR, secondaire à la perte des 70 000 neurones hypothalamiques produisant l'hypocrétine, est démontrée, ce qui, avec l'association au locus HLA, suggère qu'une destruction immunitaire de ces cellules est la cause de la maladie. D'autres études génétiques, notamment d'association à l'échelle du génome (GWAS), révèlent l'existence de nombreux facteurs génétiques prédisposant à la narcolepsie, la plupart étant également impliqués dans d'autres maladies auto-immunes. Une association forte et unique avec les loci des récepteurs lymphocytaires T (TCR) alpha et bêta est aussi observée, suggérant un rôle prépondérant des lymphocytes T. En dépit de nombreux efforts, toutes les tentatives visant à démontrer la présence d'auto-anticorps contre les cellules à hypocrétine dans la narcolepsie échouent, et la cause auto-immune présumée de cette maladie reste à l'état d'hypothèse. À la suite de la grippe pandémique influenza A pH1N1 en 2009, de nombreux cas de narcolepsie apparaissent, suggérant un mimétisme moléculaire avec le virus de la grippe qui pourrait déclencher la maladie auto-immune. Cette hypothèse est confirmée par un criblage peptidique montrant une plus grande réactivité des lymphocytes T CD4+ à un segment spécifique de l'hypocrétine (HCRTNH2) et une réactivité croisée des TCR correspondants à un segment d'hémagglutinine de pH1N1 qui partage une homologie avec HCRTNH2. De façon remarquable, le TCR le plus fréquent dans la population et qui reconnaît ces antigènes contient des séquences TRAJ24 ou TRVB4-2, segments modulés par des polymorphismes génétiques associés à la narcolepsie dans les études GWAS. Il est probable que les lymphocytes T CD4+ autoréactifs avec HCRTNH2 recrutent par la suite des lymphocytes T CD8+ qui détruisent les cellules à hypocrétine. On peut s'attendre à ce que d'autres séquences mimiques grippales inconnues soient découvertes prochainement puisque la narcolepsie existait avant 2009. Ces découvertes démontrent enfin la cause auto-immune de la narcolepsie. Les travaux menés au cours des années sur la narcolepsie offrent une perspective unique sur la conduite de la recherche sur l'étiopathogénie d'une maladie bien identifiée.

Children with Narcolepsy type 1 have increased T-cell responses to orexins.

Narcolepsy type 1 (NT1) is caused by severe loss of the orexin neurons, and is highly associated with HLA DQB1*06:02. Using intracellular cytokine staining, we observed a higher frequency of IFN-γ- and TNF-α-producing CD4+ and CD8+ T-cells in response to orexins in 27 children with NT1 compared to 15 healthy control children. Conversely, no such difference was observed between 14 NT1 and 16 HC adults. In addition, priming with flu peptides amplified the T-cell response to orexins in children with NT1. Our data suggests that NT1 may be caused by an autoimmune T-cell response to orexins, possibly triggered by flu antigens.

In vivo clonal expansion and phenotypes of hypocretin-specific CD4+ T cells in narcolepsy patients and controls.

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαß clonotypes encoded by identical α/ß gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87-97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development.

The olfactory bulb: A link between environmental agents and narcolepsy, from the standpoint of autoimmune etiology.

Narcolepsy type 1 is a lifelong sleep disorder characterized by the loss of hypocretin-producing neurons in the brain. Environmental agents, including influenza, neurotoxic metals, and combustion smoke, have been implicated in the pathogenesis, especially in carriers of the human leukocyte antigen class II DQB1*06:02 allele. Sensitive experimental approaches have recently revealed hypocretin-autoreactive CD4+ and CD8+ T cells in the blood of narcoleptic patients. However, such potentially harmful cells are also detectable, to a lesser degree, in control DQB1*06:02 carriers, suggesting that the integrity of the blood-brain barrier (BBB) provides a neuroprotective effect. Here, we present the hypothesis that external toxic agents induce neuroinflammation in the olfactory bulb and concomitant overproduction of proinflammatory cytokines (e.g., tumor necrosis factor-α and interferon-γ); this, in turn, compromises the BBB, allowing autoimmune cells to access and kill hypocretinergic neurons. Such sequential pathological alterations could occur insidiously, passing unnoticed and consequently being underestimated. The elevated number of autoreactive T cells in narcoleptics relative to controls might reflect externally induced immunomodulation rather than a direct disease trigger.

[Autoimmune mechanisms and new opportunities for treatment narcolepsy]. / Autoimmunnye mekhanizmy i novye vozmozhnosti lecheniia narkolepsii.

Narcolepsy is a disease related to hypersomnia type-sleep disorders and characterized by the following manifestations: excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, sleep paralysis and disturbed night sleep. There are several models of the disease pathogenesis each of them assumes the autoimmune nature of the disease. In this regard, it is necessary to develop etiological approaches to the treatment of the disease.

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

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.

Meeting report narcolepsy and pandemic influenza vaccination: What we know and what we need to know before the next pandemic? A report from the 2nd IABS meeting.

A group of scientific and public health experts and key stakeholders convened to discuss the state of knowledge on the relationship between adjuvanted monovalent inactivated 2009 influenza A H1N1 vaccines used during the 2009 influenza pandemic and narcolepsy. There was consensus that an increased risk of narcolepsy was consistently observed after Pandemrix (AS03-adjuvanted) vaccine, but similar associations following Arepanrix (AS03-adjuvanted) or Focetria (MF59-adjuvanted) vaccines were not observed. Whether the differences are due to vaccine composition or other factors such as the timing of large-scale vaccination programs relative to H1N1pdm09 wild-type virus circulation in different geographic regions is not clear. The limitations of retrospective observational methodologies could also be contributing to some of the differences across studies. More basic and epidemiologic research is needed to further elucidate the association between adjuvanted influenza vaccine and narcolepsy and its mechanism and to inform planning and preparation for vaccination programs in advance of the next influenza pandemic.

H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls.

Following the 2009 H1N1 influenza pandemic, an increased risk of narcolepsy type 1 was observed. Homology between an H1N1 hemagglutinin and two hypocretin sequences has been reported. T cell reactivity to these peptides was assessed in 81 narcolepsy type 1 patients and 19 HLA-DQ6-matched healthy controls. HLA-DQ6-restricted H1N1 hemagglutinin-specific T cell responses were detected in 28.4% of patients and 15.8% of controls. Despite structural homology between HLA-DQ6-hypocretin and -H1N1 peptide complexes, T cell cross-reactivity was not detected. These results indicate that it is unlikely that cross-reactivity between H1N1 hemagglutinin and hypocretin peptides presented by HLA-DQ6 is involved in the development of narcolepsy.