Transcript W01_MONTO_WHITLEY

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)