1. Lessons Learned from the Reconstruction of the 1918 Pandemic Adolfo García-Sastre, PhD Professor of Microbiology Mount Sinai School of Medicine

2. Armed Forces Institute of Pathology, Jeffery K. Taubenberger Washington DC Mount Sinai School of Medicine, Adolfo García-Sastre New YorkPeter Palese Christopher F. Basler CDCTerrence M. Tumpey USDA, Athens, Georgia David E. Swayne University of Washington, SeattleMichael G. Katze Scripps Research Institute, La JollaIan A. Wilson James Stevens NIH/NIAID support: P01 AI0581113 Lessons Learned from the Reconstruction of the 1918 Virus Collaborative effort among different research groups and institutions

3. U.S. Life Expectancy By Age 1900 ‘30 ‘50‘70‘90 70 60 50 40 30 1918 Flu Epidemic

4. Influenza and Pneumonia Deaths by Age Specific Death Rate

5. Why Study the 1918 Virus? The 1918 virus contains determinants responsible for its success as a pandemic virus The 1918 virus contains virulence determinants that are not understood A new 1918-like virus may evolve The knowledge of these determinants will allow us to better recognize the pandemic potential of circulating animal viruses and will provide us with novel targets for therapeutic and prophylactic intervention

6. Phenotypic characterization in: Tissue culture Animal models Signatures of Virulence of the 1918 Influenza Virus Gene sequencing Gene reconstruction Pathological specimen (circa 1918)..... Reverse genetics

7. 1918 Influenza AFIP Lung Block

8. Lung Tissue Sample (1918) Brevig Mission, AK

9. Reconstruction of 1918 Influenza Virus Genes Clone, Sequence and Repair Final Product

10. Plasmid-only Influenza A Virus Rescue vRNA expression plasmidsProtein expression plasmids Transfection POL IR R R R R PB2 PB1 PA HA NP NA M NS-1918 POL II POL IRPOL IIpA POL I RPOL IIpA POL IRPOL IIpA PB2 PB1 PA NP 6:2 reassortant virus

11. Texas/36/91 >6 Tx/91: PB2, PB1, PA, NP, M, NS 1918: HA, NA 4.75 Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M, NS 5.5 1918 “Spanish” flu MLD 50 ? Mouse Lethal Dose 50 (log) of Viruses Bearing 1918 Genes

12. M-1918 NS-1918 Plasmid-only Influenza A Virus Rescue Transfection POL IR R R R R R R R vRNA expression plasmids PB2-1918 PB1-1918 PA-1918 HA-1918 NP-1918 NA-1918 POL II pA Protein expression plasmids PB2 PB1 PA NP Spanish flu

13. Influenza A/CDC/1918 Virus

14. Intranasal Inoculation of Mice, 106 pfu 0 20 40 60 80 100 01234567891011121314 % Survival Days after infection Tx/91 1918 5:3 Tx/91 1918 1918 7:HA Tx/91 Viral titers in lungs, day 4 10 3 pfu 10 6 pfu 10 8 pfu 10 5 pfu

15. Virulence of the 1918 Virus: MLD50 Log pfu Texas/36/91 >6 Tx/91: PB2, PB1, PA, NP, M, NS 1918: HA, NA 4.75 Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M. NS 5.5 1918 “Spanish” flu 3.3 1918 Tx/91: HA >6

16. Virulence of the 1918 Virus: ELD50 Log pfu Texas/36/91 >7 Tx/91: PB2, PB1, PA, NP, M, NS 1918: HA, NA Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M. NS 1918 “Spanish” flu 1.5 1918 Tx/91: HA >7

17. The 1918 Virus Grows to High Titers in Human Bronchial Epithelial Cells (Calu-3) 1918 1918 5:3 Tx/91 Tx/91 Time (h post-infection) Log EID 50 /ml

18. The 1918 NA Allows Growth Without Trypsin Other genes NA Titer in MDCK cells + trypsin (pfu/ml) Titer in MDCK cells - trypsin (pfu/ml) Tx/91 1.4 x 10 7 - 1918 1.4 x 10 8 1.1 x 10 8 Tx/9119183.4 x 10 7 2.5 x 10 7

19. Oseltamivir Protects Mice from a Lethal Challenge with 1918 HA/1918 NA Virus Percent Survival

20. Rimantadine Protects Mice from a Lethal Challenge with 1918 M Virus Percent Survival

21. New Cal/99 PR8/34 Texas/91 Sw/Iowa/30 Protective Killed Inactivated Vaccines Against the 1918 HA/1918 NA Virus 0 20 40 60 80 100 01234567891011121314 % Survival PBS Days post-challenge with 1918HA/NA virus X-31(H3N2) 1918 HA/NA

22. 1.The 1918 virus is the only known human influenza virus lethal to mice and embryonated eggs 2.The glycoprotein and polymerase genes of the virus contribute to enhanced virulence 3.Alveolar macrophages and neutrophils have a protective role 4.A single amino acid change in HA changes receptor specificity 5.Viruses containing 1918 genes are sensitive to existing antivirals 6.H1N1 based vaccines are protective 1918 VIRUS What do we know now? Would a 1918-like HIN1 virus be today as lethal as in 1918?

23. H1N1 pre-existing immunity 1918 Influenza and Pneumonia Deaths by Age <11 to 45 to 1415- 2425- 3435- 4445- 5455- 6465- 7475- 84>85 Age Divisions Specific Death Rate 0 500 1000 1500 2000 2500 3000

24. Potential Ways to Fight a Highly Virulent 1918-like Pandemic Virus The existing antivirals and conventional vaccines will have beneficial effects (with the caveat that it will be difficult to have these products generated in large quantities at this moment) Consider HA and polymerase genes as targets for new antivirals Consider strategies that immunoregulate function of alveolar macrophages

25. Alicia Solorzano Luis Martinez Patricia AguilarJohn Kash Laurel GlaserDmitriy Zamarin Stacey Schultz-CherryHui Zeng Jacqueline KatzNancy Cox NIAID Biodefense Grants P01 AI58113 U54 AI57158 (NBC) U19 AI62623 (CIVIA)