Autoimmunogenetic Aspects of Vaccine-induced Narcolepsy. As part of the AMINA - Autoimmune Multiple Sclerosis and Narcolepsy Study

Sammanfattning: Narcolepsy type 1 (NT1) is a neurological sleep disorder. The biological cause is the degeneration of hypocretin neurons located in the hypothalamus. It has been suggested that NT1 has an autoimmune pathogenesis, as in most patients, the Human Leukocyte Antigen (HLA)-DQB1'06:02:01 is present. An increased incidence of NT1 was observed in Sweden following the 2009 influenza Pandemrix®-vaccination. The research underlying this thesis represented a focused attempt to identify immunological mechanisms to explain vaccine-induced NT1. The specific aims were to: 1. Determine antibody levels and affinity to influenza nucleoproteins (NPs), NP1934 in the Pandemrix®-vaccine and NP2009 in the 2009 pandemic. 2. Determine HLA Class II -DRB3, DRB4, DRB5, -DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genetic predispositions for vaccine-induced NT1. 3. Determine immunocyte signatures in whole blood and in peripheral blood mononuclear cells (PBMC) for association with NT1 patients as compared to their first-degree relatives as controls. 4. Determine autoantibodies against previously suggested and newly identified NT1 candidate autoantigens. Research subjects were included from two study groups; a) The Karolinska Narcolepsy Cohort with 43 Pandemrix®-vaccinated NT1 patients ascertained in 2012 and b) the Narcolepsy Family Study from 2015-2017 including 31 Pandemrix®-vaccinated NT1 patients and 66 of their first-degree relatives as controls. Influenza nucleoproteins were expressed by in vitro transcription translation. Antibody levels were determined using radiobinding assay (RBA). Antibody affinities were determined using competitive assays with the addition of increasing concentrations of non-labelled proteins and defined using Kd50 as the concentration necessary to displace 50% of maximum radiolabelled binding. High-resolution sequencing with Illumina Miseq technology was used to type HLA class II alleles. Extended HLA haplotypes and genotypes were computed based on inheritance within the narcolepsy families. Identification of immune-markers from whole blood was assessed with complete blood count. PBMC analyses for the identification of immunocyte signatures were performed with CyTOF® Mass Cytometry, enabling the simultaneous analysis of 25 cell-surface markers. Screening for the identification of novel NT1 autoantigens was performed using protein array. Validation was performed using RBA. Previously suggested candidate autoantigens were reinvestigated using RBA; included were Prepro-hypocretin, Hypocretin peptides 1 and 2, Hypocretin Receptor 2, Tribbles Homolog 2, Pro-opiomelanocortin/ Alpha-melanocyte-stimulating-hormone, Prostaglandin D2 Receptor DP1, ATP-Dependent Inwardly Rectifying Potassium Channel Kir4.1 and Calciumactivated chloride channel Anoctamin 2. Increased NP antibody levels were demonstrated among child patients compared to age-matched controls. IgG antibody affinity to Pandemrix®-vaccine NP1934 was lower among child patients, while affinity to pandemic NP2009 was comparable. HLA sequencing conferred the high risk HLADQB1'06:02:01 allele but also revealed a risk genotype as 23% of the NT1 patients were associated with the HLA-DRB5'01:01:01-DRB1'15:01:01-DQA1'01:02:01-DQB1'06:02:01/ DRB4'01:03:01-DRB1'04:01:01//'04:07:01//'04:08:01-DQA1'03:02//'03:03:01-DQB1'03:01:01 genotype. Increased frequency of basophils and a lower count of CD8+CD27+ T cells were related to NT1 patients compared to family controls. Autoantibodies to novel antigens as well as to retested previous candidates could not be validated, as none of these targets could distinguish patients from controls in the RBA. In this thesis, we have studied the autoimmunogenetic aspects of vaccine-induced NT1. Mechanistic explanations related to immunological processes were outlined, first through alterations in the humoral immune response to Pandemrix®-vaccine NPs, second through determination of genetic HLA predispositions and third through identification of immunocyte signatures in the blood associated with NT1. Additional studies are warranted to explain what might cause the immune system to eliminate the hypocretin neurons and how this relates to vaccination.