Lessons Learnedfrom the Reconstruction

of the 1918 Pandemic

Adolfo García-Sastre, PhDProfessor of Microbiology

Mount Sinai School of Medicine

• Armed Forces Institute of Pathology, Jeffery K. TaubenbergerWashington DC

• Mount Sinai School of Medicine, Adolfo García-SastreNew York 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. WilsonJames Stevens

NIH/NIAID support: P01 AI0581113

Lessons Learned from the Reconstruction of the 1918 Virus

Collaborative effort among different research groups and institutions

U.S. Life Expectancy

By Age

1900 ‘30 ‘50 ‘70 ‘90

70

60

50

40

30

1918Flu Epidemic

Influenza and Pneumonia Deaths by Age

0

500

1000

1500

2000

2500

3000

<1 1 to 4 5 to 14 15-24 25-34 35-44 45-54 55-64 65-74 75-84 >85

Age Divisions

1911-1917

1918

Spe

cific

Dea

th R

ate

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

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

1918 Influenza AFIP Lung Block

Lung Tissue Sample (1918)

Brevig Mission, AK

Reconstruction of 1918 InfluenzaVirus Genes

Clone, Sequence and Repair Final Product

Plasmid-only InfluenzaA Virus Rescue

vRNA expression plasmids Protein expression plasmids

Transfection

POL I R

POL I R

POL I R

POL I R

POL I R

PB2

PB1

PA

HA

NP

NA

M

NS-1918

POL II

POL I RPOL II pA

POL I RPOL II pA

POL I RPOL II pA

pA

PB2

PB1

PA

NP

6:2 reassortant virus

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

MLD50?

Mouse Lethal Dose 50 (log) of Viruses Bearing 1918 Genes

M-1918

NS-1918

Plasmid-only Influenza A Virus Rescue

Transfection

POL I R

POL I R

POL I R

POL I R

POL I R

POL I R

POL I R

POL I R

vRNA expression plasmids PB2-1918

PB1-1918

PA-1918

HA-1918

NP-1918

NA-1918

POL II

POL II

POL II

POL II

pA

pA

pA

pA

Protein expression plasmids PB2

PB1

PA

NP

Spanish flu

Influenza A/CDC/1918 Virus

Intranasal Inoculation of Mice, 106 pfu

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

%

Su

rviv

al

Days after infection

Tx/91

1918 5:3 Tx/911918

1918 7:HA Tx/91

Viral titers in lungs, day 4

103 pfu

106 pfu108 pfu

105 pfu

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

3.31918

Tx/91: HA

>6

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

1.51918

Tx/91: HA

>7

>7 >7

The 1918 Virus Grows to High Titers inHuman Bronchial Epithelial Cells (Calu-3)

0

1

2

3

4

5

6

7

8

9

2 12 16 24

1918

1918 5:3 Tx/91Tx/91

Time (h post-infection)

Log

EID

50/

ml

The 1918 NA Allows GrowthWithout Trypsin

Othergenes 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

Oseltamivir Protects Mice from a Lethal Challenge with 1918 HA/1918 NA Virus

0

20

40

60

80

100

0 5 10

Days Post-Infection

Oselt. only

1918 HA/1918NA & Oselt.

1918 HA/1918NA & PBSP

erc

en

t Su

rviv

al

Rimantadine Protects Mice from a Lethal Challenge with 1918 M Virus

Pe

rce

nt S

urv

ival

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

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

% S

urv

ival

PBS

Days post-challenge with 1918HA/NA virus

X-31(H3N2)

1918 HA/NA

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 VIRUSWhat do we know now?

Would a 1918-like HIN1 virus be todayas lethal as in 1918?

H1N1 pre-existing immunity

1918 Influenza and Pneumonia Deaths by Age

<1 1 to 4 5 to 14 15- 24 25- 34 35- 44 45- 54 55- 64 65- 74 75- 84 >85

Age Divisions

Sp

ecifi

c D

ea

th R

ate

0

500

1000

1500

2000

2500

3000

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 Luis MartinezPatricia Aguilar John KashLaurel Glaser Dmitriy ZamarinStacey Schultz-Cherry Hui ZengJacqueline Katz Nancy Cox

NIAID Biodefense GrantsP01 AI58113U54 AI57158 (NBC)U19 AI62623 (CIVIA)