Transcriptional profiles and host-response biomarkers are used increasingly to investigate the severity, subtype and pathogenesis of disease. We now describe whole-blood mRNA signatures and concentrations of local and systemic immunological mediators in 131 adults hospitalized with influenza, from whom extensive clinical and investigational data were obtained by MOSAIC investigators. Signatures reflective of interferon-related antiviral pathways were common up to day 4 of symptoms in patients who did not require mechanical ventilator support; in those who needed mechanical ventilation, an inflammatory, activated-neutrophil and cell-stress or death (‘bacterial’) pattern was seen, even early in disease. Identifiable bacterial co-infection was not necessary for this ‘bacterial’ signature but was able to enhance its development while attenuating the early ‘viral’ signature. Our findings emphasize the importance of timing and severity in the interpretation of host responses to acute viral infection and identify specific patterns of immune-system activation that might enable the development of novel diagnostic and therapeutic tools for severe influenza.

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MOSAIC Study was supported by a joint award from the Wellcome Trust and the Medical Research Council (090382/Z/09/Z). We gratefully acknowledge the support of the MOSAIC administrative team (M. Cross, L.-A. Cumming, M. Minns, T. Ford, B. Cerutti, D. Gardner and Z. Williams) and the generosity of our patients and their families, healthy volunteers, and staff at participating National Health Service (NHS) hospitals (Alder Hey Children's Hospital; Brighton & Sussex University Hospitals NHS Trust; Central Manchester University Hospitals NHS Foundation Trust; Chelsea and Westminster Hospital NHS Foundation Trust; Imperial College Healthcare NHS Trust; Liverpool Women's NHS Foundation Trust; Royal Liverpool and Broadgreen University Hospitals NHS Trust; Royal Brompton and Harefield NHS Foundation Trust; University Hospitals Coventry and Warwickshire NHS Trust). In particular, we thank K. Alshafi, S. Ashton, E. Bailey, A. Bermingham, M. Berry, C. Bloom, A. Booth, E. Brannigan, S. Bremang, J. Clark, M. Cross, L. A. Cumming, S. Dyas, J. England-Smith, J. Enstone, D. Ferreira, N. Goddard, A. Godlee, S. Gormley, M. Guiver, M. O. Hassan-Ibrahim, H. Hill, P. Holloway, K. Hoschler, G. Houghton, F. Hughes, R. R. Israel, A. Jepson, K. D. Jones, W. P. Kelleher, M. Kidd, K. Knox, A. Lackenby, G. Lloyd, H. Longworth, S. Mookerjee, S. Mt-Isa, D. Muir, A. Paras, V. Pascual, L. Rae, S. Rodenhurst, F. Rozakeas, E. Scott, E. Sergi, N. Shah, V. Sutton, J. Vernazza, A.W. Walker, C. Wenden, T. Wotherspoon, A. D. Wright and F. Wurie. We also thank E. Anguiano and members of the Genomic Core, BIIR, Dallas, for assistance with the microarray analysis; and M. Berry for guidance. We especially thank the MOSAIC Data Team (L. Drumright, L. Garcia-Alvarez, J. Lieber, S. Mookerjee and B. Pamba) for assistance in collating and validating clinical data; R. Smyth for careful review of the manuscript; and K. Strimmer for statistical advice in revisions of the manuscript. F. Rozakeas helped with recruiting samples from all the healthy control subjects at NIMR (Mill Hill). The MOSAIC consortium (ClinicalTrials.gov identifier NCT00965354) was supported by UK National Institute for Health Research (NIHR) Comprehensive Local Research Networks (CLRNs), the Biomedical Research Centre (NIHR Imperial BRC) and Unit (NIHR Liverpool BRU), the Health Protection Research Unit in Respiratory Infections in partnership with Public Health England (PHE) at Imperial College London (NIHR HPRU RI), the Health Protection Agency (latterly PHE) Microbiology Services, Colindale and the staff of the Roslin Institute, Edinburgh, Scotland. A.O.G. and C.G. were supported by the Medical Research Council, United Kingdom (U117565642), The Francis Crick Institute, London (AOG10126, which receives its core funding from Cancer Research UK (FC001126)), the UK Medical Research Council (FC001126), the Wellcome Trust (FC001126) and the UK Medical Research Council (MR/U117565642/1). S.B. was in part jointly funded by the UK Medical Research Council (MRC) as above and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement (MR/J010723/1). C.B. was funded by an MRC CRTF. The views expressed are those of the authors and not necessarily those of the NHS, NIHR, Public Health England or the Department of Health (UK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

MOSAIC investigators:

Maximillian S. Habibi, Sebastian L. Johnston, Trevor T. Hansel, Mike Levin, Ryan S. Thwaites, John O. Warner, William O. Cookson, Brian G. Gazzard, Alan Hay, John McCauley, Paul Aylin, Deborah Ashby, Wendy S. Barclay, R. A. Elderfield, Simon Nadel, Jethro A. Herberg, Lydia N. Drumright, Laura Garcia-Alvarez, Alison H. Holmes, Onn M. Kon, Stephen J. Aston, Stephen B. Gordon, Tracy Hussell, Catherine Thompson, Maria C. Zambon, Kenneth J. Baillie, David A. Hume, Peter Simmonds, Andrew Hayward, Rosalind L. Smyth, Paul S. McNamara, Malcolm G. Semple, Jonathan S. Nguyen-Van-Tam, Ling-Pei Ho, Andrew J. McMichael, Paul Kellam, Walt E Adamson, William F Carman and Mark J. Griffiths

Author information

Author notes

    • Jake Dunning

    Present address: National Infection Service, Public Health England, London, UK

  1. A list of MOSAIC members and affiliations appears at the end of the paper.


  1. Respiratory Infection Section, National Heart and Lung Institute, Imperial College London, London, UK

    • Jake Dunning
    • , Long T. Hoang
    • , Maximillian S. Habibi
    • , Sebastian L. Johnston
    • , Trevor T. Hansel
    • , Mike Levin
    • , Ryan S. Thwaites
    • , John O. Warner
    • , Onn M. Kon
    •  & Peter J. M. Openshaw
  2. Laboratory of Immunoregulation & Infection, The Francis Crick Institute, London, UK

    • Simon Blankley
    • , Christine M. Graham
    • , Chloe I. Bloom
    •  & Anne O’Garra
  3. Genomic Medicine, National Heart and Lung Institute, Imperial College London, London, UK

    • Mike Cox
    • , Philip L. James
    • , William O. Cookson
    •  & Miriam F. Moffatt
  4. Sidra Medical and Research Centre, Doha, Qatar

    • Damien Chaussabel
  5. The Jackson Laboratory for Genomic Medicine, Farmington, USA

    • Jacques Banchereau
  6. Surgery and Cancer, National Heart and Lung Institute, Imperial College London, London, UK

    • Stephen J. Brett
  7. Chelsea and Westminster NHS Foundation Trust, London, UK

    • Brian G. Gazzard
  8. Francis Crick Institute, World Influenza Centre, London, UK

    • Alan Hay
    •  & John McCauley
  9. School of Public Health, Imperial College London, London, UK

    • Paul Aylin
    •  & Deborah Ashby
  10. Department of Virology, Imperial College London, London, UK

    • Wendy S. Barclay
    •  & Ruth A. Elderfield
  11. Department of Paediatrics, Imperial College London, London, UK

    • Simon Nadel
    •  & Jethro A. Herberg
  12. Department of Respiratory Medicine, Imperial College Healthcare Trust, London, UK

    • Simon Nadel
    •  & Onn M. Kon
  13. Centre for Infection Prevention, Imperial College London, London, UK

    • Lydia N. Drumright
    • , Laura Garcia-Alvarez
    •  & Alison H. Holmes
  14. Respiratory Infection Group, Liverpool School of Tropical Medicine, Liverpool, UK

    • Stephen J. Aston
    •  & Stephen B. Gordon
  15. Manchester Collaborative Centre for Inflammation Research (MCCIR), Manchester, UK

    • Tracy Hussell
  16. Public Health England (formerly Health Protection Agency), London, UK

    • Catherine Thompson
    •  & Maria C. Zambon
  17. The Roslin Institute, University of Edinburgh, Edinburgh, UK

    • Kenneth J. Baillie
    • , David A. Hume
    •  & Peter Simmonds
  18. University College London, London, UK

    • Andrew Hayward
  19. UCL Institute of Child Health, London, UK

    • Rosalind L. Smyth
  20. University of Liverpool, Liverpool, UK

    • Paul S. McNamara
    •  & Malcolm G. Semple
  21. University of Nottingham, Nottingham, UK

    • Jonathan S. Nguyen-Van-Tam
  22. University of Oxford, Oxford, UK

    • Ling-Pei Ho
    •  & Andrew J. McMichael
  23. Wellcome Trust Sanger Institute, London, UK

    • Paul Kellam
  24. West of Scotland Specialist Virology Centre, Glasgow, UK

    • Walt E Adamson
    •  & William F Carman
  25. Royal Brompton and Harefield NHS Foundation Trust, London, UK

    • Mark J. Griffiths


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  1. MOSAIC Investigators


J.D., A.O.G. and P.J.M.O. conceived of and designed the study, with input from D.C. and J.B.; J.D., S.B., L.T.H. and C.I.B. analyzed the microarray data, with supervision by A.O.G. and P.J.M.O. and input from J.B. and D.C.; M.C., P.L.J. and M.F.M. performed and analyzed the microbiome experiments; C.M.G. performed the microarray experiments; J.D., S.J.B. and P.J.M.O. developed clinical protocols, recruited subjects and collated clinical data; and J.D., S.B., L.T.H., A.O.G. and P.J.M.O. wrote and revised the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Anne O’Garra or Peter J. M. Openshaw.

Integrated supplementary information

  1. Supplementary Figure 1 Interferon signaling pathway.

    IPA canonical pathway for interferon signaling, identified as one of the top five canonical pathways for upregulated transcripts shown in Figure 1d (2010/11 cohort). Red shading represents up-regulated genes, blue represents down-regulated genes.

  2. Supplementary Figure 2 Validation of transcriptional signatures in an independent cohort.

    a) 2009/2010 cohort clustered on individuals and transcripts (Pearson’s uncentered with averaged linkage) using 1255 transcript list (from Figure 1). (b) 2009/2010 cohort clustered on individuals and transcripts (Pearson’s uncentered with averaged linkage) using 25 transcript list (from Figure 1). (c) 2009/2010 cohort clustered on individuals and transcripts (Pearson’s uncentered with averaged linkage) using 231 transcript list of severity (from Figure 2, transcripts retained if >2FC between severity 3 and severity 1&2). GO Terms analysis of 3 major branches of the transcripts dendrogram was undertaken and listed next to the heat-map. (d) Using 51 and 112 transcripts lists (from Figure 3) ‘viral response’ and ‘bacterial response’ molecular scores were calculated and plotted for each influenza patient (relative to healthy controls). Cases were coded according to severity of illness, indicated by the colour of the respective dots (severity 1, black; severity 2, blue; severity 3, red).

  3. Supplementary Figure 3 Change of ‘viral’ and ‘bacterial’ molecular scores over time and association with influenza viral load.

    (a) ‘Viral’ molecular scores plotted for 59 influenza patients (2010/11 cohort) who provided T1 and T2 samples, plotted against respective day of illness at time of sampling. (b) Change in ‘viral’ molecular score between first (T1) and precise second time point (48 hours after T1) in 41 patients with appropriate samples available (P= 0.0002, Mann-Whitney test, two-tailed). (c) ‘Bacterial’ molecular score plotted for 59 influenza patients who had both a T1 and a T2 sample, shown plotted against respective day of illness. (d) Change in ‘bacterial’ molecular score between T1 and precise T2 (48h post T1), in 41 patients with appropriate samples available (NS, Mann-Whitney test, two-tailed). (e) Influenza viral load estimation (pfu/ml) in nasopharyngeal samples obtained at T1 (n=42) and T2 (n=40). Bars show the median and interquartile range. Zero values were reassigned a value of 0.001 for display purposes. Mann Whitney test (two-tailed); ** P=0.0094. (f) Relationship between influenza viral load (pfu/ml) at T1 or T2 and the simultaneous ‘viral’ molecular score on whole blood (n=82).

  4. Supplementary Figure 4 Administration of antibiotics does not affect ‘bacterial’ or ‘viral’ molecular scores.

    (a) Influenza patients (2010/11 cohort) presenting within the first 14 days of illness grouped by administration of any antibiotic (n=35) or no administration of antibiotics (n=35) in the 24 hours prior to T1 sampling. There was no difference (NS, Mann-Whitney test, two tailed) in either bacterial or viral molecular scores between the two groups. Bars show the median and interquartile range. (b) Prescription of antibiotics after T1 did not significantly influence ‘bacterial’ molecular score (P=0.9616, Kruskal Wallis test). Fifty-nine influenza patients who had both T1 and T2 samples were grouped by those who did not receive antibiotics (n=7), those whose antibiotics were stopped at T1 (n=1), those who had antibiotics prescribed after T1 but before T2 (n=24), and those who were receiving antibiotics at both T1 and T2 (N=27). Bars show median with interquartile range. (c) Total 16S rRNA copies at T1 in throat swabs and NP aspirate in patients adjudicated to be with or without bacterial co-infection. Those with confirmed bacterial infections (Bac +) had greater levels of total 16S rRNA copies in NP aspirate than those deemed to be without co-infection (Bac -) (Mann- Whitney test, P = 0.036). Throat swab Bac -, n=44; throat swab bac +, n=53; NP aspirate Bac -, n=17; NP aspirate Bac +, n=41.

  5. Supplementary Figure 5 Correlation of serum cytokines and bacterial load in nasophaynx with ‘viral’ and ‘bacterial’ molecular distance to health.

    (a) Levels of IL-17 in the serum of healthy controls (HC, n=36) and influenza infected patients (severity 1-3; n=59, n=43, and n=31, respectively). (b) Concentration of IL-17 in broncoalveolar lavage (BAL) of HC (n=11) and from influenza patients’ BAL (n=8), NPA (n=8), nasadsoprtion fluid (SAM; n=8) and serum (n=8). (c) Correlation of levels of IL-17 in serum (n=165) with the bacterial MDTH (Spearman R =0.39, P<0.001). (d) Correlation of levels of TNF-α in serum (n=165) with bacterial MDTH (Spearman R = 0.4, P<0.01). (e) Total 16S rRNA gene copies in NP aspirate samples (n=58) are inversely correlated with the viral MDTH (Spearman R = -0.28, P value < 0.05). (f) Total 16S rRNA gene copies in NP aspirate samples (n=58) are positively correlated with the bacterial MDTH (Spearman R = 0.37, P value < 0.05).

Supplementary information

  1. Supplementary Figures and Tables

    Supplementary Figures 1-5; Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Dataset

    Excel spreadsheet providing clinical data corresponding to raw and normalized microarray data deposited in GEO, Data Accession Code GSE111368.

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