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Lessons Learned from the Reconstruction of the 1918 Pandemic Adolfo García-Sastre, PhD Professor of Microbiology Mount Sinai School of Medicine Lessons Learned from the Reconstruction of the 1918 Virus Collaborative effort among different research groups and institutions • Armed Forces Institute of Pathology, Washington DC Jeffery K. Taubenberger • Mount Sinai School of Medicine, New York Adolfo García-Sastre Peter Palese Christopher F. Basler • CDC Terrence M. Tumpey • USDA, Athens, Georgia David E. Swayne • University of Washington, Seattle Michael G. Katze • Scripps Research Institute, La Jolla Ian A. Wilson James Stevens NIH/NIAID support: P01 AI0581113 U.S. Life Expectancy By Age 70 60 50 1918 Flu Epidemic 40 30 1900 ‘30 ‘50 ‘70 ‘90 Influenza and Pneumonia Deaths by Age 3000 Specific Death Rate 2500 1911-1917 1918 2000 1500 1000 500 0 <1 1 to 4 5 to 14 15-24 25-34 35-44 45-54 55-64 65-74 75-84 Age Divisions >85 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 Signatures of Virulence of the 1918 Influenza Virus . ... . Pathological specimen (circa 1918) Gene sequencing Gene reconstruction Reverse genetics Phenotypic characterization in: Tissue culture Animal models 1918 Influenza AFIP Lung Block Lung Tissue Sample (1918) Brevig Mission, AK Reconstruction of 1918 Influenza Virus Genes Clone, Sequence and Repair Final Product Plasmid-only Influenza A Virus Rescue Protein expression plasmids PB2 POL II pA vRNA expression plasmids PB2 POL I PB1 PB1 POL II pA POL II PA pA R POL I PA POL I NP POL II R R HA pA POL I R NP POL I R NA POL I R M POL I R NS-1918 POL I R Transfection 6:2 reassortant virus Mouse Lethal Dose 50 (log) of Viruses Bearing 1918 Genes Texas/36/91 Tx/91: PB2, PB1, PA, NP, M, NS 1918: >6 HA, NA Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M, NS 4.75 5.5 1918 “Spanish” flu MLD50? Plasmid-only Influenza A Virus Rescue Protein expression plasmids vRNA expression plasmids PB2 PB2-1918 POL II pA PB1 POL II POL II POL II pA PA NP pA pA POL I R PB1-1918 POL I R PA-1918 POL I R HA-1918 POL I R NP-1918 POL I R NA-1918 POL I R M-1918 POL I R NS-1918 POL I R Transfection Spanish flu Influenza A/CDC/1918 Virus Intranasal Inoculation of Mice, 106 pfu Viral titers in lungs, day 4 105 pfu 1918 7:HA Tx/91 Tx/91 100 103 pfu % Survival 80 60 1918 5:3 Tx/91 1918 108 pfu 40 106 pfu 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Days after infection 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 1918 “Spanish” flu 3.3 Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M. NS 5.5 1918 Tx/91: HA >6 Virulence of the 1918 Virus: ELD50 Log pfu Texas/36/91 Tx/91: PB2, PB1, PA, NP, M, NS 1918: >7 HA, NA >7 1918 “Spanish” flu 1.5 Tx/91: PB2, PB1, PA 1918: HA, NP, NA, M. NS >7 1918 Tx/91: HA >7 Log EID50/ml The 1918 Virus Grows to High Titers in Human Bronchial Epithelial Cells (Calu-3) 9 1918 8 7 6 5 Tx/91 1918 5:3 Tx/91 4 3 2 1 0 2 12 16 Time (h post-infection) 24 The 1918 NA Allows Growth Without Trypsin NA Titer in MDCK cells + trypsin (pfu/ml) Titer in MDCK cells - trypsin (pfu/ml) Tx/91 Tx/91 1.4 x 107 - 1918 1918 1.4 x 108 1.1 x 108 Tx/91 1918 3.4 x 107 2.5 x 107 Other genes Oseltamivir Protects Mice from a Lethal Challenge with 1918 HA/1918 NA Virus Percent Survival 100 80 Oselt. only 60 1918 HA/1918 NA & Oselt. 40 1918 HA/1918 NA & PBS 20 0 0 5 10 Days Post-Infection Rimantadine Protects Mice from a Lethal Challenge with 1918 M Virus Percent Survival 100 80 1918 M + PBS 60 1918 M + rimantadine 40 20 0 0 5 10 Days Post-Infection Protective Killed Inactivated Vaccines Against the 1918 HA/1918 NA Virus 1918 HA/NA Sw/Iowa/30 100 PR8/34 % Survival 80 Texas/91 60 New Cal/99 40 X-31(H3N2) 20 PBS 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Days post-challenge with 1918HA/NA virus 14 1918 VIRUS What do we know now? 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 Would a 1918-like HIN1 virus be today as lethal as in 1918? 1918 Influenza and Pneumonia Deaths by Age Specific Death Rate 3000 H1N1 pre-existing immunity 2500 2000 1500 1000 500 0 <1 1 to 4 5 to 14 15- 24 25- 34 35- 44 45- 54 55- 64 65- 74 75- 84 >85 Age Divisions 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 Alicia Solorzano Patricia Aguilar Laurel Glaser Stacey Schultz-Cherry Jacqueline Katz Luis Martinez John Kash Dmitriy Zamarin Hui Zeng Nancy Cox NIAID Biodefense Grants P01 AI58113 U54 AI57158 (NBC) U19 AI62623 (CIVIA)