ProImmune REVEAL® MHC Peptide Binding Assays used by team at Novartis and Stanford University to investigate immunological insights into the causation of narcolepsy associated with pandemic H1N1 influenza vaccine Pandemrix®


Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2

Ahmed, S.S. et al., Science Translational Medicine 7, 294ra105 (2015) PubMed DOI: 10.1126/scitranslmed.aab2354



Seasonal outbreaks of influenza A pose a serious threat to world public health as they can cause serious epidemics that may result in severe illness and death. Preventative flu vaccination programs are often aimed at specific groups of individuals, such as key workers in the health services for example, or patient groups such as the elderly, young people or the chronically ill. Publicity campaigns reinforce the message of disease prevention, but this is dependent upon maintaining the confidence of the public in the safety of vaccination.

Swine flu vaccine linked to onset of narcolepsy

In the H1N1 swine-flu outbreak of 2009, two H1N1 vaccines with different formulations were administered, Focetria and Pandemrix. About 30.5 million people across the EU/EEA region received the Pandemrix and 6.5million the Focetria vaccines.  Alarm bells sounded when monitoring revealed over 1300 cases across the immunized region of narcolepsy – a serious sleep disorder – was associated specifically with the Pandemrix vaccine. Typically, adjuvanted influenza vaccine preparations many contain varying proportions of sequences derived from the matrix, hemagglutinin (HA), NP or neuraminidase proteins.

Figure 1. A representation of the influenza A nucleoprotein NP at 3.2A resolution (PDB structure 2IQH) as modelled in PyMol. Shown in green is a surface helix having homology to hypocretin receptors 1 and 2. The NP peptide sequence showing I116(red) derived from the influenza A (H1N1)/California/07/2009 strain and M variant (orange) from the X-181 vaccine reassortant stain derived from A/California/07/2009 are enlarged. Stronger binding to the HLA-DQB1*06:02 allele as determined using the ProImmune REVEAL® assay was seen for the NP (I116) peptide that resembles both HCRT1 and HCRT2. The M116 variant of the NP peptide bound less strongly, but was similar to the native hypocretin ligand. Substitution of the threonine (T) across this region residue with either I or M dramatically increased the REVEAL® score – emphasizing the importance of both amino acids in the differential binding to HLA-DQB1*06:02.

Studies have revealed that narcolepsy patients display a deficiency of the neuropeptide hypocretin (HCRT) due to loss of hypothalamic cells.While questions in medical journals were raised that the use of different adjuvants was implicated in the increased incidence of narcolepsy observed in connection with the use of Pandemrix, Ahmed and colleagues took a different view. Strikingly, in humans, narcolepsy is linked strongly to the HLA-DQB1*06:02 haplotype.  Ahmed et al suspected mimicry between a vaccine component and a hypocretin-related protein found in the brain. In silico analysis revealed a sequence from the influenza NP protein that was found to be similar to a sequence in the surface-exposed N-terminal domain of HCRT receptor 2, and the corresponding domain of HCRT receptor 1. Analysis of serum from HLA-DQB1*06:02 narcoleptic patients revealed the presence of antibodies that bound to both H1N1 NP and HCRT receptor 2.  Importantly, this antibody response was restricted to patients who had received the Pandemrix vaccine that contains a higher proportion of NP compared to Focetria.

Molecular Mimicry

To characterize the anti-HRCT receptor 2 antibodies further, using patient derived sera the team showed that the reactivity of anti-HCRT receptor 2 antibodies in ELISA experiments was blocked by NP derived peptides containing the mimic sequence. A sequence variant in NP, isoleucine/methionine, was identified in the vaccine reassortant strain X181 when compared to the sequence of the A/California/07/2009 H1N1 pandemic flu virus. A similar degree of blocking activity of the two peptides was observed in the ELISA assays, suggesting that the methionine or isoleucine change was not contributing towards a differential B cell receptor recognition that lead to the generation of cross-reactive antibodies in vaccine-associated narcolepsy.

Molecular modelling suggested that peptides containing either I or M may differentially bind to the HLA-DQB1*06:02 protein product. To test this hypothesis, Ahmed et alturned to the ProImmune REVEAL® MHC Peptide Binding Assays. Using ProImmune REVEAL® technology, stronger binding of the isoleucine compared to the methionine substituted peptide was demonstrated. Although the methionine-containing peptide bound less strongly, this was at a similar level to the native physiologically active hypocretin ligand. Furthermore, substitution of the threonine residue in the hypocretin ligand with either isoleucine or methionine dramatically increased binding, showing the relevance of both amino acids in the differential binding to HLA-DQB1*06:02.


Future Perspectives

The implications of the findings by Ahmed et al are important and far reaching. Many important outstanding questions remain to be answered concerning the link between the development of antibodies and the onset of the disease.  Nevertheless, this research draws attention to the protein composition of a particular vaccine, and shows that, in rare cases, an adjuvanted vaccine might accidentally trigger the production of cross-binding antibodies – in this case that can lead to the induction of a serious brain disorder in individuals who were genetically susceptible. Clearly the benefits of flu vaccination outweigh the associated risk of complications. Nonetheless, a reduction in adverse events associated with vaccines differing in composition that could be made possible through in-depth research strategies used here, will contribute towards a deeper understanding of the innate and adaptive immune responses to infections and their associated vaccines.


About the authors

Lawrence Steinman is Professor of neurology and neurological sciences, paediatrics and genetics at Stanford University. Steinman’s research interests centre on the better understanding of immune disorders and immunological responses that may underlie the development of important human diseases such as multiple sclerosis, type I diabetes and neuromyelitis optica. He has developed several new therapies for autoimmune diseases, including MS and type I diabetes, some of which are in Phase 2 trials. He had been awarded several major prizes and in 2015 was elected to the National Academy of Sciences.

Sohail Ahmed is currently Head of Clinical Sciences at GSK Vaccines. Previously, he earned his MD in 1998 at The University of Texas Medical School where he specialized in internal medicine and rheumatology (including clinical immunology, inflammation and autoimmunity) and was Global Head of Clinical Sciences at Novartis. His translational research interests currently span several areas, including leading programs aimed at guiding future vaccine antigen discovery and targeted approaches to mitigating risks associated with novel vaccine delivery platforms.