Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk - PubMed (original) (raw)

. 2014 Dec 15;9(12):e114361.

doi: 10.1371/journal.pone.0114361. eCollection 2014.

Arja Vuorela 1, Markku Partinen 2, Marc Baumann 3, Tobias L Freitag 4, Seppo Meri 4, Päivi Saavalainen 4, Matti Jauhiainen 5, Rabah Soliymani 3, Turkka Kirjavainen 6, Päivi Olsen 7, Outi Saarenpää-Heikkilä 8, Juha Rouvinen 9, Merja Roivainen 10, Hanna Nohynek 1, Jukka Jokinen 1, Ilkka Julkunen 11, Terhi Kilpi 1

Affiliations

Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk

Outi Vaarala et al. PLoS One. 2014.

Abstract

Background: Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated.

Methods and findings: Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06:02 risk allele and in DQB1*06:02 transgenic mice immunized with Pandemrix when compared to controls.

Conclusions: 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. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.

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Conflict of interest statement

Competing Interests: HN received honoraria for technical consultancy from GlaxoSmithKline (GSK), and Pfizer for development of pneumococcal conjugate vaccines. JJ is co-investigator of a nationwide effectiveness study of the ten-valent pneumococcal conjugate vaccine mainly funded by GlaxoSmithKline. MP has been consultant for Bioprojet and UCB Pharma and received funding support and honoraries for lecturing from Bioprojet, GSK, Cephalon, and Leiras/Takeda. T. Kilpi is principal investigator of a nationwide effectiveness study of the ten-valent pneumococcal conjugate vaccine mainly funded by GlaxoSmithKline, and her unit received funding for a clinical trial on the safety and immunogenicity of a prototype pandemic influenza vaccine from Solvay Pharmaceuticals. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. All other authors confirm they have no conflicts of interest. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. The IgG-antibody levels to H1N1 antigen suspension of Pandemrix (A), tetanus toxoid (B) and neutralizing antibody levels to Polio Sabin vaccine virus (C) in 47 children with narcolepsy (N) and 57 healthy vaccinated children with and without HLA DQB1*06∶02 risk allele of narcolepsy (C/DQ6+ and C/DQ6−).

Antibodies to Pandemrix H1N1 antigen and tetanus toxoid were determined by EIA and the results expressed as optical density units (y-axis).

Figure 2

Figure 2. The in vitro inhibition of IgG-antibodies binding to H1N1 antigen of Pandemrix is evaluated using H1N1 antigen suspension of Pandemrix, Arepanrix or detergents present in Pandemrix as liquid-phase inhibitors pre-incubated with plasma samples from study subjects. A.

The inhibition of IgG-antibodies binding to solid-phase bound H1N1 antigen of Pandemrix (% of inhibition in y-axis) is lower when Arepanrix antigen (1/10) is used as inhibitor compared to Pandemrix (1/10) as an inhibitor both in children with narcolepsy (N; n = 47) and in healthy vaccinated children (C; n = 57). B. The inhibition of IgG-antibodies binding to solid-phase bound H1N1 antigen of Pandemrix (% of inhibition in y-axis) with Arepanrix antigen(1/10) is weaker in the children with narcolepsy who are homozygotic for HLA DQB1*06∶02 risk allele than in the children with narcolepsy or healthy children who are heterozygotic for HLA DQB1*06∶02 risk allele of narcolepsy. C. Dose-dependent inhibition of IgG-antibodies binding to solid-phase bound H1N1 antigen of Pandemrix (% of inhibition in y-axis) using as a liquid phase inhibitor H1N1 antigen suspension of Arepanrix or Pandemrix at the final dilutions of 1/250, 1/50 and 1/10 (0.4, 2, 10 on x-axis) or the detergents present in the H1N1 antigen suspension of Pandemrix, Triton X and polysorbate 80, at the final concentrations 0.8, 4, and 20 µg/mL (0.4, 2, 10 on axis). The inhibition curves represent mean value of the % of inhibition in plasma samples from 3 children with narcolepsy.

Figure 3

Figure 3. High-resolution MES-PAGE analysis of Arepanrix (lane A) and Pandemrix (lane B) H1N1 antigen suspensions under reducing conditions, and immunostaining of the separated proteins in Pandemrix by western blotting using anti-NP antibody (C).

A set of sharp bands at approximately 120 kDa molecular weight could be seen in Pandemrix (B) but not in Arepanrix sample (A). These bands in Pandemrix were identified by mass spectrometry to be polymeric forms of the influenza virus nucleoprotein (NP) and by western blotting with anti-NP antibody (C).

Figure 4

Figure 4. High-resolution MES-PAGE analysis of Pandemrix and Arepandrix vaccines under reducing and non-reducing conditions.

Pandemrix H1N1 antigen suspension run under reducing conditions (A) with a major band including a mixture of HA and NP, and under non-reducing conditions (B) with HA shown in the upper area and NP at its original place identical to RF position of the reducing gel (A). Recombinant NP from H1N1/A/Puerto Rico 1934 (Sino Biologicals Inc., China) alone run under reducing conditions (C). HA stained with monoclonal mouse anti-HA antibody (Sino Biologicals Inc., China) using western blotting of the separated proteins of Pandemrix H1N1 antigen suspension under non-reducing conditions (D) and under reducing conditions (E). Arepanrix H1N1 antigen suspension run under reducing conditions (F) with a major band including a mixture of HA and NP (F), and run under non-reducing conditions (G) with HA shown in the upper area and NP at its original place identical to RF position of the reducing gel (F).

Figure 5

Figure 5. Western-blot analysis of the presence of NP in Pandemrix and Arepanrix H1N1 antigen suspension.

Pandemrix and Arepanrix H1N1 antigen suspensions were run under reducing conditions and NP stained with monoclonal mouse NP-antibody (Sino Biologicals Inc., China).

Figure 6

Figure 6. Antibody levels to HA as antibody titers determined with the hemagglutination inhibition (HI) test.

Antibody titers against influenza A/California/07/2009 vaccine strain (A), epidemic A/Finland/814/01 (H1N1) virus strain (B) and influenza A/Finland/715/00 (H3N2) virus strain (C) in plasma samples from 47 children with narcolepsy and in 57 healthy vaccinated children.

Figure 7

Figure 7. A sandwich EIA for the detection of IgG-antibodies to H1N1 proteins captured from H1N1 antigen of Pandemrix with solid-phase bound antibodies to HA, NA and NP.

A. Capture of H1N1 viral proteins from Pandemrix antigen in a sandwich EIA revealed that HA and NA are found in a complex in the Pandemrix H1N1 antigen, whereas NP does not complex with HA or NA. H1N1 viral proteins from Pandemrix vaccine were captured with rabbit monoclonal anti-HA, anti-NA and anti-NP antibodies bound to EIA plate and mouse monoclonal anti-HA, anti-NA and anti-NP antibodies were used as the detection antibodies. B. IgG-antibodies binding to Pandemrix derived NP were higher in children with narcolepsy (N; n = 47) than in vaccinated healthy children(C; n = 57) when studied with sandwich EIA using anti-NP antibody to capture NP from Pandemrix H1N1 antigen suspension. The children with narcolepsy (N) had higher levels of IgG-antibodies to Pandemrix derived NP than the healthy, vaccinated children without HLA DQB1*06∶02 risk allele (C/DQ6−; n = 37), whereas no difference was seen in comparison with the healthy, vaccinated children with HLA DQB1*06∶02 risk allele (C/DQ6+; n = 20).

Figure 8

Figure 8. Dot blot detection of viral proteins with mouse monoclonal anti-HA, anti-NA and anti-NP antibodies (Sino Biologicals Inc., China) in the density fractions isolated with gradient ultracentrifugation from Pandemrix H1N1 antigen suspension.

HA and NA was detected as lipid-protein micelles in the density fraction corresponding density between 1.063–1.21 g/mL, and soluble HA, NA and NP were detected in the density fraction above 1.21 g/mL.

Figure 9

Figure 9. Antibodies to untreated and detergent treated recombinant viral proteins studied by EIA.

IgG-antibodies against untreated recombinant viral proteins, rHA (A) and rNP (B), and against rHA and rNP exposed to non-ionic detergents, Triton X and polysorbate 80 (PS80), 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 (N; n = 47) had higher levels of IgG-antibodies to polysorbate 80 treated recombinant NP (PS80) but not to untreated NP (PBS) than healthy vaccinated children (C; n = 57). Children with narcolepsy who all carry HLA DQB1*06∶02 allele (N; n = 47) and healthy vaccinated children with HLA DQB1*06∶02 allele (C/DQ6+; n = 20)) had higher levels of antibodies to untreated NP in comparison to the children without HLA DQB1*06∶02 allele (C/DQ6−; n = 37). D. Children with narcolepsy (N; n = 47) showed higher levels of IgG-antibodies to untreated recombinant HA (PBS) and polysorbate 80 treated HA (PS80) than healthy vaccinated children (C; n = 57). No difference was found in the antibody levels to treated or untreated HA between healthy children with or without HLA DQB1*06∶02 allele (C/DQ6+; n = 20 and C/DQ6−; n = 37).

Figure 10

Figure 10. IgG-antibody levels to detergent treated recombinant HA (A) and NP (B) in the Pandemrix immunized mice transgenic for HLA DQB1*06∶02 (DQ6 Pand) or HLA DQB1*03∶02 (DQ8 Pand) and in the HLA DQB1*06∶02 transgenic mice immunized with PBS (DQ6 ctrl).

IgG-antibodies to rNP are higher in the immunized HLA DQB1*06∶02 (DQ6 Pand) than in the immunized HLA DQB1*03∶02 transgenic mice (DQ8 Pand), whereas no difference is seen in the antibody levels to rHA in the EIA.

References

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