Though much of the research for a link between Pandemrix and narcolepsy has focused on the flu vaccine’s adjuvant, a new study suggests the focus should actually be on the H1N1 viral proteins.

In their paper, “Antigenic Differences between AS03 Adjuvanted Influenza A (H1N1) Pandemic Vaccines: Implications for Pandemrix-Associated Narcolepsy Risk” published in PLOS One on December 15, Finnish researchers compare two different versions of the H1N1 flu vaccine—Pandemrix and Arepanrix—and find an interesting difference.

“What is more significant is that given that there were two pandemic vaccines against swine flu–one was Arepanrix, which was used in Canada, and the other was Pandemrix, which was used in Europe–Pandemrix caused narcolepsy,” first author Outi Vaarala told Finnish media outlet Yle. “The difference was that Pandemrix had one viral protein in a different form and there was more of it.”

In Vaarala and team’s study, they performed inhibition experiments in which the binding of plasma IgG-antibodies to H1N1 antigen of Pandemrix was inhibited with either H1N1 antigen of Pandemrix itself or H1N1 antigen of Arepanrix. The inhibition of IgG-antibodies to Pandemrix H1N1 viral antigen was significantly weaker with the Arepanrix H1N1 antigen than with the Pandemrix H1N1 antigen in both children with narcolepsy and healthy, vaccinated children (P <0.0001), which indicates antigenic differences between the viral components of Arepanrix and Pandemrix. To study the possible differences in the protein content of H1N1 viral antigen suspensions, the team performed high-resolution MES-polyacrylamide gel electrophoresis analysis. Pandemrix repeatedly showed a set of sharp bands at approximately 120 kDa molecular weight (reducing conditions), which could not be seen in the Arepanrix sample. Several other analyses are detailed in the paper.

In the paper’s discussion, the authors say:

We demonstrated that the viral protein NP was present in higher amount in Pandemrix than in Arepanrix, particularly structurally altered NP [nucleoprotein]. Given that Pandemrix vaccine antigen was produced under different conditions than Arepanrix, the purification process of viral proteins has likely resulted in the differential enrichment of NP. Moreover, it seems that these production differences have led to a yet unknown cascade of detergent influenced protein polymerization. Polymeric proteins are known to be more immunogenic than monomeric proteins, and polymerization may also result in the changes in the antigenic epitopes in NP as reported earlier.

The paper’s conclusion states:

This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06:02 risk allele of narcolepsy.