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
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.
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.
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.
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
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
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 function
Changes 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 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
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
Timing of secondary infections
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
Viral
Lung
Titer
0
2
Wound healing
Anti-inflammatory state
Transition to memory
Anergy of innate responses
4
6
8
10
Antibody
12
Adapted from Hussell T & Cavanaugh MM, Biochem Soc Trans, 2009;37:811-3.
14
16
18
20
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
PR8 ΔPB1-F2
WT PR8
H5N1 ΔPB1-F2
1918 PB1-F2 / PR8
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
Conclusions - Inflammation
- PB1-F2 has immunostimulatory activity – C-terminal portion of PB1F2 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
H1, H3, etc.
Broad diversity in both length
and functional capacity of
PB1-F2s in swine
H5N1, H9N2, etc.
Nearly all PB1-F2s from avian strains are
full-length and highly inflammatory
Non-functional protein taken from
human H3N2 lineage and truncated
during circulation in swine prior to
emergence as a pandemic strain
pH1N1
Swine strains
11 a.a.
Avian strains
90 a.a.
Human strains
H3N2
Non-functional by mid-’80s
H2N2
90 a.a.
1920
H1N1
1930
56 a.a.
1940
1950
56 a.a.
1960
1970
1980
1990
2000
2010
Are other swine viruses a greater threat?
Noninflammatory
Proinflammatory
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
Streptococcus pyogenes
Noninflammatory
*
Proinflammatory
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!
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!
Acknowledgements
Contributors from
the McCullers lab:
Amy Iverson
Our Collaborators:
Support:
Julie McAuley
Nick van de Velde
Kelly Zhang
Elaine Tuomanen
Keith English
Kelli Boyd
Robert Webster
Doug Green
Jerry Chipuk
Jon Yewdell
Victor Huber
NIH (AI-49178, AI-54802, AI-66349)
ALSAC