The protein concentration was determined by a Bradford assay

e density fraction corresponding density between 1.0631.21 g/mL, and soluble HA, NA and NP were detected in the density fraction above 1.21 g/mL. doi:10.1371/journal.pone.0114361.g008 Fig. 9. Antibodies to untreated and detergent treated recombinant viral proteins studied by EIA. IgG-antibodies against untreated recombinant viral proteins, rHA and rNP, and against rHA and rNP exposed to non-ionic detergents, Triton X and polysorbate 80, used in the manufacturing process of Pandemrix H1N1 antigen, but not Arepanrix, in six plasma samples from vaccinated children with narcolepsy C. Children with narcolepsy had higher levels of IgG-antibodies to polysorbate 80 treated recombinant NP but not to untreated NP than healthy vaccinated children. Children with narcolepsy who all carry HLA get AVE8062A DQB106:02 allele and healthy vaccinated children with HLA DQB106:02 allele ) had higher levels of antibodies to untreated NP in comparison to the children without HLA DQB106:02 allele. D. Children with narcolepsy showed higher levels of IgG-antibodies to untreated recombinant HA and polysorbate 80 treated HA than healthy vaccinated children. No difference was found in the antibody levels to treated or untreated HA between healthy children with or without HLA DQB106:02 allele. doi:10.1371/journal.pone.0114361.g009 16 / 23 Influenza A Vaccine Antigen and Narcolepsy Risk children PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681699 with DQB106:02 risk allele showed higher levels of antibodies to rNP than children without DQB106:02 allele, but interestingly the levels of antibodies to rNP were higher in the children with narcolepsy even when compared to the children with DQB106:02 risk allele. The antibody levels to untreated rHA and detergent treated rHA were higher in children with narcolepsy than in healthy vaccinated children, but no association of antibodies to HA was found with the presence of DQB106:02 risk allele. The antibodies to detergent treated and untreated rHA showed a strong correlation with antibodies to H1N1 antigen suspension of Pandemrix PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683642 in both children with narcolepsy and healthy children as expected. Instead, the antibodies to rNP correlated with antibodies to H1N1 antigen of Pandemrix only in children with narcolepsy, but not in the healthy children. We then immunized with Pandemrix NOD mice transgenic for the narcolepsy related human DQB106:02 allele or the type 1 diabetes related DQB103:02 allele and studied the antibody response to detergent exposed rHA and rNP. We observed that IgG-antibodies induced by Pandemrix vaccination to rNP were higher in the HLA DQB106:02 transgenic mice when compared HLA DQB103:02 transgenic mice, whereas no difference was seen in the antibodies to rHA. Discussion The aim of the current study was to explore the possible antigenic differences between two AS03 adjuvanted H1N1 vaccines, Pandemrix and Arepanrix, in order to elucidate the mechanisms behind Pandemrix-associated narcolepsy. Importantly, we provide the first evidence of significant antigenic differences between the Pandemrix and Arepanrix H1N1 antigen suspensions. Based on these immunological findings and the epidemiological observations of Pandemrixassociated narcolepsy, we suggest that the H1N1 viral antigen suspension of Pandemrix is in a key role in the triggering vaccine-induced narcolepsy. The presence of an antigen-specific trigger of narcolepsy in the Pandemrix vaccine is also suggested by the observations that no increased risk of other autoimmune diseases has been observed in association with Pa