Bacterial super-infections: The other side of influenza pathogenesis Jon McCullers, M.D.

1. Bacterial super-infections: The other side of influenza pathogenesis Jon McCullers, M.D. Associate Member Department of Infectious Diseases St. Jude Children’s Research Hospital

2. Secondary bacterial infections - R.T.H. Laennec was the first to describe secondary bacterial infections following influenza - He noted that the prevalence of pneumonia increased during an epidemic of “la grippe” in 1803 in Paris - Today it is well-appreciated that many influenza-related deaths are due to secondary invaders such as Streptococcus pneumoniae and Staphylococcus aureus Laennec, R. T. H. 1923., p. 88-95. In Translation of selected passages from De l'Auscultation Mediate.

3. Bacterial pneumonia and pandemics - It is estimated that 95% of all deaths during the 1918 pandemic were complicated by secondary bacterial pneumonia (primarily S. pneumoniae) - Estimated at 50-70% in 1957 and 1968 - This has been a key concern for pandemic planning - The emergence of the novel pandemic H1N1 strain has led to increased opportunities to study the epidemiology and pathogenesis of secondary bacterial infections following influenza Morens DM, et al., J Infect Dis 2008;198:962-70. McCullers JA. J Infect Dis 2009;198:945-7.

4. Influenza and Staphylococcus aureus - S. aureus was the primary secondary invader in 1957 - In recent decades, however, it had not been a prominent cause of pneumonia - With the emergence of USA300 strains, necrotizing pneumonia, particularly in association with influenza, has become much more common - In the 2008-2009 season, 44% of pediatric deaths from influenza (of those tested) had bacterial super-infection, 75% of the etiologic agents were S. aureus Finelli L et al. Pediatrics 2008;122:805--11. Centers for Disease Control, MMWR 2009;58:369-74.

5. Bacterial pneumonia and pH1N1 - Few reports of bacterial superinfections in initial descriptions of severe pandemic related disease - However, most critically ill patients were treated with broad spectrum antibiotics, and invasive assays (e.g., pleural taps) were not commonly done - Recent evaluations of severe and fatal cases show 25-56% have evidence of bacterial super-infection (S. pneumoniae, S. aureus, S. pyogenes), with 14-46% mortality - 4 deaths in healthy children in Memphis from S. aureus super-infections during the H1N1 pandemic Dominguez-Cherit G, et al. JAMA 2009;302:1880-7. Gill JR, et al., Arch Pathol Lab Med 2010;134:235-43. CDC. MMWR 2009;58(38):1071-4. Mauad T, et al., Am J Respir Crit Care Med 2010;181:72-9. Esstensoro E, et al., Am J Respir Crit Care Med 2010, doi:10.1164/201001-0037OC.

6. Secondary pneumococcal pneumonia Bioluminescent pneumococcus expressing luciferase Pneumococcus = 0.002 MLD50 D39 Influenza = 0.05 MLD50 PR8 Mock = PBS (diluent) 10 mice per group pictured Second challenge was 7 days after primary infection McCullers JA et al., J Inf Dis 2002;186:341-50.

7. Secondary staphylococcal pneumonia * * * Influenza = 0.03 MLD50 PR8 NRS-193 = USA400 strain PBS = mock infection 6 mice per group pictured Second challenge was 7 days after primary infection * = p < 0.05 by Log-Rank test on Kaplan-Meier survival data vs. other groups Iverson AR…McCullers JA, J Inf Dis., In Press Lee MH et al., J Inf DIs 2010;201(4):508-15.

8. Different pneumococcal clinical strains Mice received influenza virus followed 7 days later by 1x105 CFU of pneumococcus McCullers JA, et al., J Inf Dis., In Press

9. Different staphylococcal strains Mice received influenza virus followed 7 days later by 1x108 CFU of S. aureus Iverson AR…McCullers JA, J Inf Dis., In Press

10. Mechanisms of viral-bacterial synergism Factors enhancing bacterial adherence Epithelial damage enhancing bacterial adherence Alteration of epithelium through sialidase activity Upregulation of receptors for bacterial adherence McCullers JA, Antiviral Ther 2010, In Press

11. Mechanisms of viral-bacterial synergism Factors enhancing bacterial adherence Epithelial damage enhancing bacterial adherence Alteration of epithelium through sialidase activity Upregulation of receptors for bacterial adherence Factors facilitating bacterial access to normally sterile sites Mechanical alterations to airway or Eustachian tube function Changes in tropism of virus (ability to access the lower lung) McCullers JA, Antiviral Ther 2010, In Press

12. Mechanisms of viral-bacterial synergism Factors enhancing bacterial adherence Epithelial damage enhancing bacterial adherence Alteration of epithelium through sialidase activity Upregulation of receptors for bacterial adherence Factors facilitating bacterial access to normally sterile sites Mechanical alterations to airway or Eustachian tube function Changes in tropism of virus (ability to access the lower lung) Factors altering innate immune responses Increased inflammation through expression of cytotoxins Anergy of responses to bacteria during resolution of inflammation Dysregulation of protective immune pathways Alteration of bacterial clearance through effects on immune cells McCullers JA, Antiviral Ther 2010, In Press

13. Mechanisms of viral-bacterial synergism Factors enhancing bacterial adherence Epithelial damage enhancing bacterial adherence Alteration of epithelium through sialidase activity Upregulation of receptors for bacterial adherence Factors facilitating bacterial access to normally sterile sites Mechanical alterations to airway or Eustachian tube function Changes in tropism of virus (ability to access the lower lung) Factors altering innate immune responses Increased inflammation through expression of cytotoxins Anergy of responses to bacteria during resolution of inflammation Dysregulation of protective immune pathways Alteration of bacterial clearance through effects on immune cells Complementation of the virus by bacteria Cleavage of influenza virus hemagglutinin by bacterial proteases Complementation of PB1-F2 by bacterial cytotoxins McCullers JA, Antiviral Ther 2010, In Press

14. Timing of secondary infections Wound healing Anti-inflammatory state Transition to memory Anergy of innate responses Innate Immunity Pro-inflammatory state Onset of acute lung injury Influx of macrophages, neutrophils Transition to adaptive immunity Acute lung injury peaks then begins to resolve Influx of T-cells Total Airway Cells Antibody Viral Lung Titer 0 2 4 6 8 10 12 14 16 18 20 Adapted from Hussell T & Cavanaugh MM, Biochem Soc Trans, 2009;37:811-3.

15. PB1-F2: newly identified protein - 87 aa peptide with predicted highly cationic, amphipathic helix at C-terminal end - sequence spanning aa 63-75 targets peptide to mitochondria - resembles some anti-microbial peptides - What is the role of PB1-F2 in pathogenesis? Jon Yewdell, NIH Gibbs JS et al., J Vir 2003;77:7214-24.

16. PB1-F2s from pandemic strains promote inflammation BAL fluid cell counts 3 days post exposure to PB1-F2 McAuley JL…McCullers JA. PLoS Pathog, 2010;6(7):e10011014.

17. …and this corresponds to morbidity Weight loss groups of 5 mice, 50 μM PB1-F2 i.n. McAuley JL…McCullers JA. PLoS Pathog, 2010;6(7):e10011014.

18. Inflammation from the full virus 50 TCID50, day 3 post infection, n = 5 per group BALB/c mice McAuley JL…McCullers JA. PLoS Pathog, 2010;6(7):e10011014.

19. KO mutant does not prime for pneumonia - mice infected with wt or KO then 7 days later challenged with pneumococcus A66.1 (type 3) - KO virus did not prime for bacterial pneumonia McAuley JL…McCullers JA, Cell Host & Microbe, 2007;2:240-9.

20. PB1-F2 and S. aureus - PB1-F2 is important in secondary staphylococcal pneumonia Iverson AR…McCullers JA, J Inf Dis., In Press

21. Secondary bacterial pneumonia - PR8, H5N1, and 1918 PB1-F2 proteins support secondary bacterial infections, H3N2 from 1995 does not McAuley JL…McCullers JA, Cell Host & Microbe, 2007;2:240-9 and unpublished data

22. Histopathology PR8 ΔPB1-F2 WT PR8 1918 PB1-F2 / PR8 H5N1 ΔPB1-F2 H5N1 PB1 Much of PB1-F2’s contribution to virulence appears to be mediated through enhanced inflammation, particularly in concert with bacterial pathogens McAuley JL…McCullers JA, Cell Host & Microbe, 2007;2:240-9 and unpublished data

23. Conclusions - Inflammation - PB1-F2 has immunostimulatory activity – C-terminal portion of PB1-F2 from pandemic strains and H5N1 cause inflammation, recent H3N2 does not - inflammatory lung damage appears to play a role in both induction and severity of bacterial pneumonia following influenza - PB1-F2s from 1918 and H5N1 viruses contribute to virulence in mice and to secondary bacterial pneumonia, 1995 H3N2 PB1-F2 does not Please see Julie McAuley’s poster (P-495) for more information!

24. Overall Summary – PB1-F2 H1, H3, etc. Non-functional protein taken from human H3N2 lineage and truncated during circulation in swine prior to emergence as a pandemic strain H5N1, H9N2, etc. Broad diversity in both length and functional capacity of PB1-F2s in swine pH1N1 Nearly all PB1-F2s from avian strains are full-length and highly inflammatory Swine strains 11 a.a. Avian strains H3N2 90 a.a. Non-functional by mid-’80s Human strains H2N2 H1N1 90 a.a. 56 a.a. 56 a.a. 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010

25. Are other swine viruses a greater threat? Non- inflammatory Pro- inflammatory Groups of 5 mice infected i.n. with 0.1 MLD50 of selected viruses followed 5 days later with 1000 CFU of S. pneumoniae strain A66.1 (type 3) Clear differences in support for bacterial superinfections among swine viruses Iverson AR…McCullers JA, Unpublished data

26. Streptococcus pyogenes Non- inflammatory Pro- inflammatory * Groups of 5 mice infected i.n. with 0.1 MLD50 of selected viruses followed 5 days later with 1000 CFU of Group A Streptococcal strain MGAS (type M1) * CA409 = pandemic H1N1, does not express PB1-F2 Huber VC, Unpublished data – Please see Victor Huber’s Poster (#P-569) for more information!

27. Synergism is strain dependent - Animal model data demonstrate that different strains of bacteria can differentially participate in secondary bacterial superinfections - Similar data show that viruses differ in their capacity to prime for bacterial super-infection, and virus-bacteria pairs may differ by strain - This implies that specific virulence factors of both the virus (e.g., PB1-F2) and the bacteria (e.g., cytotoxins) differentially contribute to outcome depending on the strain combinations

28. Overall Summary - Enhanced inflammation, particularly in the setting of bacterial superinfection, appears to be a general function of PB1-F2 proteins from pandemic strains, but is generally lost through mutation / truncation during adaptation in mammals - Most avian PB1-F2s have molecular signatures suggestive of high pathogenicity – correlates with higher mortality of 20th century pandemics than the 2009 H1N1 pandemic - PB1-F2s from swine vary greatly in length and functionality; some are likely to be a greater pandemic threat than others Please see Nick Van De Velde’s poster (P-406) for insight into the mechanism!

29. Acknowledgements Contributors from Julie McAuley the McCullers lab: Nick van de Velde Kelly Zhang Our Collaborators: Elaine Tuomanen Doug Green Keith English Jerry Chipuk Kelli Boyd Jon Yewdell Robert Webster Victor Huber Support: NIH (AI-49178, AI-54802, AI-66349) ALSAC Amy Iverson