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Transcript of TOWARD A UNIVERSAL INFLUENZA VIRUS VACCINE Icahn School of Medicine at Mount Sinai PETER PALESE...
![Page 1: TOWARD A UNIVERSAL INFLUENZA VIRUS VACCINE Icahn School of Medicine at Mount Sinai PETER PALESE Department of Microbiology Munich, May 30, 2013.](https://reader038.fdocuments.net/reader038/viewer/2022110320/56649cac5503460f9496ee4b/html5/thumbnails/1.jpg)
TOWARD A UNIVERSAL INFLUENZA VIRUS VACCINE
Icahn School of Medicine
at Mount Sinai
PETER PALESEDepartment of Microbiology
Munich, May 30, 2013
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>1 in 5 hospitalizations in the U.S. influenza-related (Jan. 2013)
https://login.medscape.com/login/sso/getlogin?urlCache=aHR0cDovL3d3dy5tZWRzY2FwZS5jb20vdmlld2FydGljbGUvNzc4MjMyX3ByaW50&ac=401
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Influenza virus vaccine formulations (2000 – 2010)
Vaccine Recommendations
H1N1 H3N2 B
2000 – 2001 A/NEW CALEDONIA/20/99 A/MOSCOW/10/99 B/BEIJING/184/93
2001 – 2002 A/NEW CALEDONIA/20/99 A/MOSCOW/10/99 B/SICHUAN/379/99
2002 – 2003 A/NEW CALEDONIA/20/99 A/MOSCOW/10/99 B/HONG KONG/330/2001
2003 – 2004 A/NEW CALEDONIA/20/99 A/MOSCOW/10/99 B/HONG KONG/330/2001
2004 – 2005 A/NEW CALEDONIA/20/99 A/FUJIAN/411/2002 B/SHANGHAI/361/2002
2005 – 2006 A/NEW CALEDONIA/20/99 A/CALIFORNIA/7/2004 B/SHANGHAI/361/2002
2006 – 2007 A/NEW CALEDONIA/20/99 A/WISCONSIN/67/2005 B/MALAYSIA/2506/2004
2007 – 2008 A/SOLOMON ISLANDS/3/2006 A/WISCONSIN/67/2005 B/MALAYSIA/2506/2004
2008 – 2009 A/BRISBANE/59/2007 A/BRISBANE/10/2007 B/FLORIDA/4/2006
2009 – 2010 A/BRISBANE/59/2007 A/BRISBANE/10/2007 B/BRISBANE/60/2008
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Source: CDC ILI and Vaccine Distribution Data
Percentage of Visits for ILI and H1N1 Vaccine Distribution, Sep 2009 – May 2010
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HOW CAN WE DO BETTER?
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HEMAGGLUTININ STALK-SPECIFIC ANTIBODIES
• HUMAN STALK-SPECIFIC MONOCLONAL ANTIBODIES HAVE BEEN IDENTIFIED
• WE CAN GENERATE MONOCLONAL ANTIBODIES OF SUCH SPECIFICITIES IN THE MOUSE
• NATURE USES THIS MECHANISM TO ELIMINATE “EARLIER” STRAINS – EXTINCTION OF sH1N1 BY pH1N1 VIRUSES
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Sui, J.,Hwang, W. C., Perez, S., Wei, G., Aird, D., Chen, L. M., Santelli, E., Stec, B., Cadwell, G. Ali, M., Wan, H., Murakami, A., Yammanuru, A., Han, T., Cox, N. J., Bankston, L. A., Donis, R. O., Liddington, R. C., Marasco, W. A. (2009) Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273.
CROSS-REACTIVE ANTIBODY BINDS TO STALK REGION OF HEMAGGLUTININ
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Strategy for boosting the antibody response against the conserved regions (grey) of the influenza virus hemagglutinin
Wang et al., Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathogens 2010
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Seasonal H1N1(sH1N1/pre-2009) sH1N1/pre-2009
sH1N1/pre-2009Pandemic H1N1
(pH1N1/2009)
Elimination of seasonal H1N1 viruses by anti-stalk antibodies
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ASSAY FOR STALK SPECIFIC ANTIBODIES USING CHIMERIC HEMAGGLUTININS
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REASSORTANT VIRUSES EXPRESS CHIMERIC HEMAGGLUTININ cH9/1
Rong Hai
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PATIENTS INFECTED WITH pH1N1 HAVE HIGHER TITERS OF ANTIBODIES THAT NEUTRALIZE cHA-EXPRESSING VIRUS
Natalie Pica and Dirk Eggink
cH9/1N3 virus
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SUMMARY
We have developed analytical tools that allow us to detect and quantify antibodies that bind the hemagglutinin protein and neutralize influenza virus but do not have hemagglutination inhibition activity
Influenza virus pH1N1 infected patients have higher titers of stalk-specific antibodies than uninfected controls
Increased titers of stalk antibodies in the population infected with pH1N1 serve as an explanation of why sH1N1 viruses have disappeared from circulation
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY
YYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY YYY
Y
YYYYYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY YYY
Y
YY Y
YY
YYY
YYYY
YY
YYYYYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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A/Netherlands/602/09 pH1N1 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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H5N1 and H6N1 challenge
positive control (matched inactivated)cH9/1 DNA + H1 protein/cH6/1 protein + cH5/1 protein/H1 proteincH9/1 DNA + BSA +BSA
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A more realistic approach – mimicking pre-existing immunity as it is present in the human population
• A/cHA expressing influenza B virus as tool to mimic pre-existing immunity in the mouse model
• No background immunity against internal viral proteins and neuraminidase
Control groups: BcH9/1+ BSA + BSABwt + BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)
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Mechanism of action
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Alternative mechanisms of neutralization
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CD8 T-cell depletion and PR8 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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Targeting group 2 HAs
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Targeting group 2 HAs
H7N9
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Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge
Phil82 (H3N2) ELISA Phil82 (H3N2) challenge
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Group 2 proof of concept
cH4/3 DNA cH5/3 proteinboost
cH7/3 proteinboost(H3 protein for the H7 challenge)
ma Phil82 (H3) ma X31 (H3) Rhea (H7)challenge
Control groups: cH4/3 DNA + BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)
4 weeks3 weeks3 weeks
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A/Philippines/2/82 H3N2 challenge
***, p=0.0008
N=1
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X-31 H3N2 and H7N1 challenge
***, p=0.0001**, p=0.0088
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Conclusions from proof of principle studies
• A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice
• Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains
• The observed protection is antibody mediated
• Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks
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Ferrets (Mustela putorius furo)
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Vaccination scheme
pre-prime bleed post-prime bleed post-boost bleed
~ 3 weeks ~ 3 weeks ~ 5 weeks
A/Netherlands/602/09 challenge
AdV cH6/1
VSV cH5/1B cH9/1 infection
Control groups:B wild type infectionVSV-GFPAdenovirus-GFP
Prime: 107 PFU/ferretChallenge: 104 PFU/ferret
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Challenge virus titers
A/Netherlands/602/09 (pH1N1) virus
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Conclusions• Immunization with chimeric hemagglutinins
induces broadly protective antibodies directed against the stalk domain
• Vaccinated animals are protected against homologous, heterologous as well as heterosubtypic challenge
Natalie Pica, Florian Krammer, Matthew Miller, Adolfo García-Sastre Taia Wang, Rong Hai, Irina Margine, Peter Palese Randy Albrecht
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Induction of a broadly neutralizing immune response against influenza HA by novel
universal influenza virus vaccine constructs
YY
YY
YYY Y
YY
Glo
bu
lar h
ea
d d
om
ain
(im
mu
no
-d
om
ina
nt)
Previous exposurePre-existing memory
Annual vaccination with similar strain
Chimeric HA
Y
Y YY
YYY Y
YYY Y
Y
YYY Y
YY
YY
YY
Y Y
YY
YY
YY Y
Y
YYY Y
YY
Y
Vaccination with universal vaccine constructs
Sta
lk d
om
ain
Boost of a
nti-globular
head antibodies
Boost of anti-stalk antibodies
Y
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A proof of principle:Protective levels of stalk-reactive antibodies can be induced
by chimeric HA constructs in mice
cH9/1 DNA cH6/1 protein cH5/1 protein PR/8/34 H1N1FM/1/47 H1N1 orpH1N1 (2009)challenge
Controls: cH9/1 DNA plus 2x BSA (neg. contr.)inactivated challenge virus (pos. contr.)
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pH1N1(2009) challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + ch5/1 proteincH9/1 DNA + BSA +BSA
challenge dose: 10 LD50
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positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + ch5/1 proteincH9/1 DNA + BSA +BSA
FM/1/47 and PR/8/34 challenge
CD8 T-cell depletion and PR/8/34 challenge
challenge dose: 5 LD50
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NaïvePositive controlWT fluB +BSA+BSAfluB cH9/1 + cH6/1 + cH5/1 (or PR8)fluB cH9/1 + BSA + BSA
pH1N1(2009) and H5N1 challenge
challenge dose: 250 (pH1N1) and 5 LD50 (H5N1)
Passive transfer and PR/8/34 challenge
passive transfer challenge dose: 5 LD50
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What about H3/group 2 HA vaccines in mice?
cH4/3 DNA cH5/3 proteinboost
cH7/3 proteinboost(PerthH3 protein for the H7 challenge)
5 LD50 ma Phil82 (H3) 5 LD50 ma X31 (H3) 5 LD50 Rhea (H7)challenge
Control groups: cH4/3 DNA + BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)
4 weeks3 weeks3 weeks
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Phil82 H3N2 challenge (5 LD50)
Day post infection Day post infection
% w
eig
ht
loss
% s
urv
ival
N=1
SurvivalWeight loss
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X31(1968) H3N2 challenge (5
LD50)
Day post infection
% s
urv
ival
A/Rhea/NC/39482/93 H7N1 challenge (5 LD50)
Day post infection
% s
urv
ival
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cH5/3 N1 virus challenge (100 LD50)
Day post infection Day post infection
% w
eig
ht
loss
% s
urv
ival
Weight loss Survival
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Cross-protection in vivo by passive transfer of anti-H3 stalk mAbs
0
5
10
15
20
25
mAb 12D1 mAb 39A4 IgG
Lu
ng
tit
er (
4Lo
g10
pfu
/ml)
Mice were given 30mg/kg mAb 12D1, 39A4 or isotype control by
intraperitoneal injection 1 hour prior to infection with A/Georgia/1981. Data
represent lung titers from groups of 5 mice, 2 days post infection.
Taia Wang
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A
C
E
B
D
F
Prophylactic treatment of mice with anti-H3 mAbs diminishes lung
damage associated with viral pneumonia
Lung tissue taken 4 days post
infection with the A/HK/68
reassortant virus.
A,B: Untreated
C,D: Mice treated with mAb 39A4
E,F: Mice treated with mAb 12D1
Taia Wang
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Mechanism of action:Red blood cell fusion assay
0
20
40
60
80
100
150 100 10 1
[mAb] ug/ml
% F
us
ion
in
hib
itio
n
7A7
12D1
39A4
1A7
1) Incubate virus + mAb.
2) Add chicken red blood cells.
3) Add low pH buffer.
4) Evaluate supernatant for presence of NADPH (340nm).
HK/68 virus
Taia Wang
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Reactivity of group 2 HA stalk vaccinated mice to Perth09 H3 and
Shanghai13 H7 HA protein
Florian Krammer and Irina Margine5/1/13
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Proteins used as substrate have different trimerization domains and purification tags than proteins used for vaccination.
Substrate: Perth/09 H3 protein
Substrate: Shanghai/13 H7 protein
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Conclusions • Immunization with chimeric hemagglutinins
induces broadly protective antibodies directed against the stalk domain
• Vaccinated animals are protected against homologous, heterologous as well as heterosubtypic challenge
Florian Krammer, Irina Margine, Rong Hai, Adolfo García-Sastre Matthew Miller, Taia Wang, Natalie Pica, Peter Palese
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FUTURE
• Precise mechanism of how stalk-specific anti-hemagglutin antibodies work
• Hemagglutinin-specific binding antibodies and effector functions such as phagocytosis, complement activation and antibody-dependent cellular cytotoxicity (ADCC)
• Efficay of chimeric hemagglutinin-based vaccines in humans
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Toward a universal influenza virus vaccine
Project 1: Design of new immunogens based on conserved epitopes in the influenza virus hemagglutinin
PI Peter Palese
1st Scientific Advisory Board meeting5/24/13
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Conclusions from proof of principle studies
• A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice
• Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains
• The observed protection is antibody mediated
• Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks
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Toward a universal influenza virus vaccine
Project 1: Design of new immunogens based on conserved epitopes in the influenza virus hemagglutinin
PI Peter Palese
1st Scientific Advisory Board meeting5/24/13
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Aims
1) Characterization of broadly neutralizing antibodies
2) Development of novel broad spectrum influenza virus vaccines
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A universal influenza virus vaccine based on chimeric hemagglutinin
constructs
Florian Krammer, Irina Margine, Natalie Pica, Rong Hai and Peter Palese
5/24/13
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Influenza A virus hemagglutinin
adapted from Krammer and Grabherr, 2010, Trends Mol Med.
Globular head domain: mediates binding to host receptors
Stalk domain: mediates fusion of viral and endosomal membranes
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Antibodies against the influenza virus HA stalk domain
Ekiert et al., 2009, Science
•Rare and not induced/boosted upon regular seasonal vaccination
•Have been isolated from humans and mice
•Cross-reactive between HAs of different subtypes
• Have mostly conformational epitopes
•Broad neutralizing activity
•in vitro
•in passive transfer studies in animals (ferrets, mice)
•HI negative!!
Imm
unod
omin
ant
head
dom
ain
Stal
k do
mai
n
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Alternative mechanisms of neutralization
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The HA stalk is conserved among group 1 and among group 2 HAs
Shaw and Palese, 2011, Field’s Virology
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Chimeric hemagglutinins (cHAs)
Conserved stalk dom
ainG
lobular head dom
ain
H6
H1
chimeric H6/1Hai et al., 2012, JVI
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY
YYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY YYY
Y
YYYYYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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cH9/1 DNA cH6/1 protein(replace by H1 full length protein for H6N1 challenge)
cH5/1 protein (replaced by H1 full length protein for H5N1 challenge)
PR8 H1N1FM1 H1N1 orpH1N1H5N1H6N1challengeControl groups:
cH9/1 DNA + BSA + BSAmatched vaccine (pos. contr.)
YY YYY
Y
YY Y
YY
YYY
YYYY
YY
YYYYYY
Induction of protective levels of stalk-reactive antibodies using chimeric HA constructs in mice
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A/Netherlands/602/09 pH1N1 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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H5N1 and H6N1 challenge
positive control (matched inactivated)cH9/1 DNA + H1 protein/cH6/1 protein + cH5/1 protein/H1 proteincH9/1 DNA + BSA +BSA
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How does pre-existing immunity to the HA stalk impact on the vaccine
regimen?
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A more realistic approach – mimicking pre-existing immunity as it is present in the human population
• A/cHA expressing influenza B virus as tool to mimic pre-existing immunity in the mouse model
• No background immunity against internal viral proteins and neuraminidase
Control groups: BcH9/1+ BSA + BSABwt + BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)
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A/Puerto Rico/8/34 H1N1 challenge
NaïvePositive controlWT fluB +BSA+BSAfluB cH9/1 + cH6/1 + cH5/1fluB cH9/1 + BSA + BSA
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A/Fort Monmouth/1/47 and A/Netherlands/602/09 H1N1 challenge
NaïvePositive controlWT fluB +BSA+BSAfluB cH9/1 + cH6/1 + cH5/1 (or PR8)fluB cH9/1 + BSA + BSA
H5N1 challenge
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Mechanism of action
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Binding to PR8 (H1N1) virus Binding to Cal09 (pH1N1) protein
ELISA reactivity to H1 HA and H1N1 virus
cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSAnaïve serum
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Binding to H2N2 virusBinding to H5N1 virus
(altered vaccination regimen with H1 instead of cH5/1 HA)
ELISA reactivity to H2N2 and H5N1 virus
cH9/1 DNA + cH6/1 protein + cH5/1 protein/H1 proteincH9/1 DNA + BSA +BSAnaïve serum
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Passive transfer (PR8 H1N1 challenge)
NaïvePositive controlWT fluB +BSA+BSAfluB cH9/1 + cH6/1 + cH5/1 (or PR8)fluB cH9/1 + BSA + BSA
IgG ug/ml
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
H2 pseudotyped particle neutralization assay
Protection is mediated by humoral immunity
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CD8 T-cell depletion and PR8 challenge
positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA
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Targeting group 2 HAs
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Targeting group 2 HAs
H7N9
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Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge
Phil82 (H3N2) ELISA Phil82 (H3N2) challenge
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Group 2 proof of concept
cH4/3 DNA cH5/3 proteinboost
cH7/3 proteinboost(H3 protein for the H7 challenge)
ma Phil82 (H3) ma X31 (H3) Rhea (H7)challenge
Control groups: cH4/3 DNA + BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)
4 weeks3 weeks3 weeks
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A/Philippines/2/82 H3N2 challenge
***, p=0.0008
N=1
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X-31 H3N2 and H7N1 challenge
***, p=0.0001**, p=0.0088
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Mimicking a pre-existing anti-H3 stalk response in the mouse model
A/cH7/3
BcH7/3 exposure cH5/3 proteinboost(H3 protein for the cH5/3 challenge)
cH4/3 proteinboost
cH5/3N1A/Philippines/82 (H3N2) X31 (H3N2)
Control groups: Bwt+ BSA + BSAnaïve (neg. contr.)matched vaccine (pos. contr.)BcH7/3 + BSA + BSA
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cH5/3N1 challenge(A/Perth/16/09 H3 stalk)
**, p=0.0082
N=2
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A/Philippines/2/82 H3N2 and X-31 H3N2 challenge
****, p<0.0001 ****, p<0.0001
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Lung titers
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Mechanism of action
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ELISA reactivity to H3N2 and H7N1 viruses and two HA proteins of H3N2 and H7N9 origin
Substrate: A/rhea/NC/39482/93 virus (H7N1)virus
Substrate: A/Victoria/361/11 (H3N2) virus
Substrate: A/Perth/16/09 H3 protein Substrate: A/Shanghai/1/13 H7 protein
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Mucosal IgA and Ig subtype distribution
Reciprocal nasal wash dilution
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Passive transfer and entry inhibition assay
Passive transfer (Phil82 challenge)Pseudotyped particle entry assay (Vic11)
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Conclusions from proof of principle studies
• A broadly protective immune response to the stalk domain can be induced by vaccine constructs in mice
• Chimeric HA constructs protect mice from challenge with heterologous and heterosubtypic virus strains
• The observed protection is antibody mediated
• Good protection against the new Chinese H7N9 strain is expected, data will be generated in the coming weeks
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Ferrets (Mustela putorius furo)
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Vaccination scheme
pre-prime bleed post-prime bleed post-boost bleed
~ 3 weeks ~ 3 weeks ~ 5 weeks
A/Netherlands/602/09 challenge
AdV cH6/1
VSV cH5/1B cH9/1 infection
Control groups:B wild type infectionVSV-GFPAdenovirus-GFP
Prime: 107 PFU/ferretChallenge: 104 PFU/ferret
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ELISA data after vaccination
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Challenge virus titers
A/Netherlands/602/09 (pH1N1) virus
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Influenza viruses circulating in the human population
A
H1N1 (Group1)
H2N2 (Group1)
19601918 1940 20001980
B
?H3N2 (Group2)
pH1N1
A
H1N1 (Group1)
H2N2 (Group1)
19601918 1940 20001980
BB
?H3N2 (Group2)
pH1N1