Bacterial super-infections: The other side of influenza

pathogenesis Jon McCullers, M.D.

Associate MemberDepartment of Infectious Diseases

St. Jude Children’s Research Hospital

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.

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.

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.

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.

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.

Secondary pneumococcal pneumonia

Bioluminescent pneumococcus expressing luciferase

Iverson AR…McCullers JA, J Inf Dis., In Press Lee MH et al., J Inf DIs 2010;201(4):508-15.

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

*

*

*

Mice received influenza virus followed 7 days later by 1x105 CFU of pneumococcus

Different pneumococcal clinical strains

McCullers JA, et al., J Inf Dis., In Press

Mice received influenza virus followed 7 days later by 1x108 CFU of S. aureus

Different staphylococcal strains

Iverson AR…McCullers JA, J Inf Dis., In Press

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

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 functionChanges in tropism of virus (ability to access the lower lung)

McCullers JA, Antiviral Ther 2010, In Press

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 functionChanges 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

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 functionChanges 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 bacteriaCleavage of influenza virus hemagglutinin by bacterial

proteasesComplementation of PB1-F2 by bacterial cytotoxins

McCullers JA, Antiviral Ther 2010, In Press

0 2 4 6 8 10 12 14 16 18 20

ViralLung Titer

Total AirwayCells

Antibody

Innate ImmunityPro-inflammatory stateOnset of acute lung injuryInflux of macrophages, neutrophils

Transition to adaptive immunityAcute lung injury peaks then begins to resolveInflux of T-cells

Wound healingAnti-inflammatory stateTransition to memoryAnergy of innate responses

Adapted from Hussell T & Cavanaugh MM, Biochem Soc Trans, 2009;37:811-3.

Timing of secondary infections

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?

Gibbs JS et al., J Vir 2003;77:7214-24.

Jon Yewdell, NIH

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.

…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.

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.

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.

PB1-F2 and S. aureus

- PB1-F2 is important in secondary staphylococcal pneumonia

Iverson AR…McCullers JA, J Inf Dis., In Press

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

Histopathology

Much of PB1-F2’s contribution to virulence appears to be mediated through enhanced inflammation, particularly in concert with bacterial pathogens

PR8 ΔPB1-F2 WT PR8 1918 PB1-F2 / PR8

H5N1 ΔPB1-F2 H5N1 PB1

McAuley JL…McCullers JA, Cell Host & Microbe, 2007;2:240-9 and unpublished data

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!

Overall Summary – PB1-F2

H1N1

1920 201019701930 1940 1950 1960 1980 1990 2000

90 a.a. 56 a.a. 56 a.a.

H2N2H3N2 90 a.a. Non-functional by mid-’80s

pH1N1

Non-functional protein taken from human H3N2 lineage and truncated during circulation in swine prior to emergence as a

pandemic strain

11 a.a.

Human strains

Avian strains

H5N1, H9N2, etc.Nearly all PB1-F2s from avian strains are full-length and highly inflammatory Swine strains

Broad diversity in both length and functional capacity of PB1-F2s in swine

H1, H3, etc.

Are other swine viruses a greater threat?

Clear differences in support for bacterial superinfections among swine viruses

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)

Non-inflammatory

Pro-inflammatory

Iverson AR…McCullers JA, Unpublished data

Streptococcus pyogenes

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

Non-inflammatory

Pro-inflammatory

*

Huber VC, Unpublished data – Please see Victor Huber’s Poster (#P-569) for more information!

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

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!

AcknowledgementsContributors 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