The ecology of influenza viruses Understanding mortality from H1 pandemic and seasonal H2 H3 influenza H4 H5 Jonathan A. McCullers H6 H7 Associate Member H8 Department of Infectious Diseases H9 H10 St. Jude Children’s Research Hospital H11 H12 H13 H14 H15 H16

Timeline of viruses in humans and pigs Emergence of a pandemic strain

Novel N.A. Avian Novel “Classic” or H1N1H1N1 Segments Eurasian Swine H3N2 H3N2 1 PB2 H2N2H2N2 Human 2 PB1 H1N1H1N1 H1N1H1N1 3 PA 4 HA 5 NP H1N2H1N2 6 NA H1N1 7 M H3N2H3N2 8 NS H1N1H1N1

H1N1 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 H3N2

“Triple reassortant” novel pandemic H1N1 influenza virus

Pandemics in the last century Age Distribution

1918 – fully avian H1N1 virus enters humans and pigs simultaneously, 40-50 million deaths worldwide, endemic in pigs

1957 – reassortant virus takes H2N2 surface proteins and PB1 from avian sources, other genes from human H1N1, ~ 2 million deaths worldwide

1968 – reassortant virus takes H3 and PB1 from avian sources, other genes from human H2N2, ~ 1 million deaths worldwide

[1977 – H1N1 related to 1950 strain “re-emerges” from frozen source, has circulated together with H3N2 since]

2009 – “triple” reassortant H1N1 emerges from pigs, “novel H1N1”

Reichert TA…..McCullers JA. BMC Inf Dis 2010.

1 Clinical features of seasonal influenza Clinical manifestations of influenza

______Infants Children Adults Novel H1N1 * - Sudden onset of symptoms ______Fever ++ +++ +++ +++ Cough + ++ +++ +++ - Incubation period 1 to 7 days, typically 2-3 days Myalgias - + ++ ++ Sore throat - + ++ ++ - Infectious period varies by age: Headache - ++ ++ ++ - Adults shed virus typically 1 day before through Diarrhea ++ + - + 4-5 days after onset of symptoms Vomiting ++ + - + - Children shed virus longer, typically 2-3 days Rhinitis ++ + + + before through 7-10 days after onset of Maliase ++ + + + symptoms / lethargy Neurologic + - - + symptoms - Novel H1N1 thought to be similar, infectious period may be longer ______(more like naïve subjects) - rare, + uncommon, ++ common, +++ very common *Represents children and adult data combined McCullers JA. Infect in Med, November 2009.

Vaccines against H1N1 influenza Treatment of novel H1N1

- Standard vaccines targeting H1N1 have been made and tested - Novel H1N1 viruses are susceptible to neuraminidase inhibitors by same process we use every year (NAIs; oseltamivir, zanamivir) but resistant to adamantanes (amantadine, rimantadine) – limited published experience with - The vaccines are extremely safe and no problems were reported treatment outcomes

- Although plenty of vaccine was made, distribution problems - Prophylaxis has been undertaken with NAIs to prevent spread to limited access close contacts

- Children, young adults, pregnant women, first responders, and - Development of resistance in this clinical scenario has been persons with chronic illnesses were first priority documented (H275Y mutation), but these strains have not spread widely - Novel H1N1 will replace seasonal H1N1 in the trivalent vaccine for 2010-2011

WHO

Diagnosis of novel H1N1 Risk factors for severe disease

- Currently used antigen tests have 40-60% sensitivity for novel - Overall, similar to risk factors for seasonal influenza H1N1 compared to RT-PCR - Chronic medical conditions including cardiopulmonary disease - Most available RT-PCR assays cannot distinguish novel H1N1 and immunosuppression from seasonal strains - Pregnancy - Targeted antiviral use will require development and widespread utilization of PCR based assays that can distinguish type (A vs. - Neurodevelopmental delay B), subtype (H3N2 vs. H1N1), and strain (seasonal vs. pandemic) - Obesity (this is not recognized as a risk factor for seasonal - Sequencing based methods for rapid antiviral resistance influenza) determination could also be employed - Extremes of age have not been a risk factor for novel H1N1

MMWR 2009 / 58(30);826-829

2 Complications of novel H1N1 Bacterial pneumonia and H1N1

- Few reports of bacterial superinfections in initial descriptions of - Viral pneumonia – most common cause of death severe pandemic related disease

- Most severe disease in persons with no underlying risk factors - However, most critically ill patients were treated with broad has been in older children and young adults – immunopathology? spectrum antibiotics, and invasive assays (e.g., pleural taps) were not commonly done - Bacterial super-infections – otitis media, pneumonia, sinusitis – are being found more commonly with novel H1N1 - Several pathology series have shown 30-50% of all fatal cases had evidence of bacterial super-infection (S. aureus, S. pneumoniae, Group A Streptococcus)

Dawood FS, et al. NEJM 2009;360(25):2605-15. Dawood FS, et al. NEJM 2009;360(25):2605-15. Gill JR et al., Arch Pathol Lab Med 2010;134:235-43. th Perez-Padilla R, et al. NEJM 2009;June 29 electronic publication. Mauad T et al., Am J Respir Crit Care Med 2010;181:72-9. CDC. MMWR 2009;58(38):1071-4.

Bacterial pneumonia and pandemics Questions about Mortality

- It is estimated that 95% of all deaths during the 1918 pandemic We understand about half the deaths – they are in persons with were complicated by secondary bacterial pneumonia chronic medical conditions and the deaths are what we expect from seasonal influenza - Estimated at 50-70% in 1957 and 1968 - Why is the clinical attack rate low in older adults? - This has been a key concern for pandemic planning - Why are bacterial superinfections only accounting for ~30% of - Now that the novel H1N1 has emerged, we need to understand deaths? influenza-bacterial interactions to better predict outcomes and more effectively treat cases - Why are healthy, older children and young adults disproportionately affected by viral pneumonia?

- Why is obesity a new risk factor?

Morens DM, et al., J Infect Dis 2008;198:962-70. McCullers JA. J Infect Dis 2009;198:945-7.

Questions about Mortality Natural immunity in older adults

We understand about half the deaths – they are in persons with Study Design: chronic medical conditions and the deaths are what we expect Lee-Ann from seasonal influenza - Enrolled 126 adult employees Van de Velde of SJCRH who were ≥ 55 y.o. - Why is the clinical attack rate low in older adults? - Asked about receipt of 1976 - Why are bacterial superinfections only accounting for ~30% of swine flu vaccine deaths? - Determined antibody titers to the 2009 - Why are healthy, older children and young adults novel H1N1, the 2008-2009 seasonal disproportionately affected by viral pneumonia? H1N1, and the 1976 swine flu vaccine strain

- Why is obesity a new risk factor? Hypothesis: Persons who recall receiving swine flu vaccine in 1976 will have higher antibody titers against the 2009 S-OIV than those who do not recall receiving vaccine

3 2000s Antibody titers

1990s Phylogenetic relationships 1980s 97% of subjects received between human 2008-2009 seasonal 1970s H1N1 viruses influenza vaccine; 36.5% recalled receiving swine flu vaccine in 1976 1947-1950s

1930s – 1940s 1976 “Swine flu” and 2009 pandemic H1N1 1918 Reichert TA…..McCullers JA. 2010 BMC Inf Dis McCullers et al., Clin Inf Dis 2010 In Press.

Antibody titers Effect of seasonal vaccine

- Some advantage to prior vaccination in 1976 (not statistically Some advantage for age against all 3 viruses significant); some advantage to age in a highly vaccinated cohort

McCullers et al., Clin Inf Dis 2010 In Press. McCullers et al., Clin Inf Dis 2010 In Press.

Effect of seasonal vaccine Neutralization titers are low

Unvaccinated persons in 2008-2009 on the lower end of responses in the 55-59 group

McCullers et al., Clin Inf Dis 2010 In Press. McCullers et al., Clin Inf Dis 2010 In Press.

4 Answer: older adults What about bacterial pneumonia?

- Older adults may have some cross- Should we be seeing more than we are? protective immunity against the novel H1N1

- Immunity increases with age, although neutralization responses are modest and do not increase with age Julie McAuley - Receipt of the 1976 swine flu likely enhanced these responses (23.9% vs. 14.3% by neutralization)

McCullers et al., Clin Inf Dis 2010 In Press.

Secondary pneumococcal pneumonia PB1-F2: newly identified protein

Mouse model

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 - 87-90 aa peptide with predicted amphipathic helical domain at C-terminal end in the +1 reading frame of the PB1 gene segment - sequence spanning aa 63-75 targets peptide to mitochondria

McCullers JA et al., J Inf Dis 2002;186:341-50. Gibbs JS et al., J Virol 2003;77:7214-24. Bruns K. et al., J Biol Chem 2007;282(1):353-63.

PB1-F2 supports bacterial super-infections Decreased necrosis and inflammation

WT PB1-F2-

- mice infected with wt or KO virus then challenged 7 days later with pneumococcus A66.1 10X

- KO virus did not prime for bacterial pneumonia

40X

Lungs of mice with secondary bacterial pneumonia

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

5 PB1-F2 from pandemic strains Conclusions - Inflammation promotes inflammation

- PB1-F2 has immunostimulatory activity – C-terminal portion of PB1- F2 from 20th century 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

- strain specific differences in PB1-F2 are important

BAL fluid cell counts 3 days post exposure to PB1-F2

McAuley JL…McCullers JA. Submitted for publication. McAuley JL…McCullers JA. Submitted for publication.

What about the novel H1N1 PB1-F2? Are other swine viruses a greater threat?

PB1-F2 Motif

Non- Inflammatory

Highly Inflammatory

- has 2 stop codons at positions 12 and 58 Groups of 5 mice infected i.n. with 0.1 MLD of selected viruses followed 5 days - has attenuating mutations at 75 and 79 50 later with 1000 CFU of S. pneumoniae strain A66.1 (type 3) - unlikely to be functional through reversion Clear differences in support for bacterial superinfections among swine viruses

Implications for novel H1N1 Conclusions…Clinical

The current pandemic shares common features with past - PB1 gene segment derived from human H3N2 precursors, but pandemics, it: has 2 stop codons in PB1-F2 ORF - has a high clinical attack rate due to lack of immunity - Could acquire full length PB1-F2 by mutation or reassortment - transmits easily enabling worldwide spread with seasonal H3N2 strains - causes severe disease in some risk groups (but fortunately is milder than was anticipated) - However, sequence analysis of C-terminal region predicts that in either scenario, PB1-F2 would be non-functional Children have occupied a central role in the pandemic: - highest clinical attack rate - Reassortment with avian or swine viruses to gain a full length - main vectors of transmission functional PB1-F2 could herald enhanced secondary bacterial - most severe disease in patients without chronic disease medical conditions

6 Conclusions…Research Acknowledgements

Contributors from Julie McAuley - The sparing of older of older adults is likely due to exposure to the McCullers lab: Keith Wanzeck antigenically similar viruses in childhood Lee-Ann van de Velde Amy Iverson - The novel H1N1 pandemic strain lacks one of the key virulence Irina Alymova factors that have helped previous pandemic strains cause severe disease and death in association with bacterial pneumonia Our Collaborators: Pat Flynn Richard Webby - Other viruses in the avian and swine reservoirs are a much Kim Allison Kris Branum Kelli Boyd Jacco Boon greater threat to cause a severe pandemic Robert Webster Tom Reichert Gerardo Chowell Hiroshi Nishuri Paul Thomas Julia Hurwitz

Support: NIH (AI-49178, AI-54802, AI-66349) ALSAC

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