toward a universal influenza virus vaccine icahn school of medicine at mount sinai peter palese...

TOWARD A UNIVERSAL INFLUENZA VIRUS VACCINE

Icahn School of Medicine

at Mount Sinai

PETER PALESEDepartment of Microbiology

Munich, May 30, 2013

>1 in 5 hospitalizations in the U.S. influenza-related (Jan. 2013)

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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

Source: CDC ILI and Vaccine Distribution Data

Percentage of Visits for ILI and H1N1 Vaccine Distribution, Sep 2009 – May 2010

HOW CAN WE DO BETTER?

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

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

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

Seasonal H1N1(sH1N1/pre-2009) sH1N1/pre-2009

sH1N1/pre-2009Pandemic H1N1

(pH1N1/2009)

Elimination of seasonal H1N1 viruses by anti-stalk antibodies

ASSAY FOR STALK SPECIFIC ANTIBODIES USING CHIMERIC HEMAGGLUTININS

REASSORTANT VIRUSES EXPRESS CHIMERIC HEMAGGLUTININ cH9/1

Rong Hai

PATIENTS INFECTED WITH pH1N1 HAVE HIGHER TITERS OF ANTIBODIES THAT NEUTRALIZE cHA-EXPRESSING VIRUS

Natalie Pica and Dirk Eggink

cH9/1N3 virus

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

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





YY

YYY






YY YYY

Y

YYYYYY






YY YYY

Y

YY Y

YY

YYY

YYYY

YY

YYYYYY


A/Netherlands/602/09 pH1N1 challenge

positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + cH5/1 proteincH9/1 DNA + BSA +BSA

A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge


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

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.)

Mechanism of action

Alternative mechanisms of neutralization

CD8 T-cell depletion and PR8 challenge


Targeting group 2 HAs


H7N9

Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge

Phil82 (H3N2) ELISA Phil82 (H3N2) challenge

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

A/Philippines/2/82 H3N2 challenge

***, p=0.0008

N=1

X-31 H3N2 and H7N1 challenge

***, p=0.0001**, p=0.0088

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

Ferrets (Mustela putorius furo)

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

Challenge virus titers

A/Netherlands/602/09 (pH1N1) virus

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

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

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.)

pH1N1(2009) challenge

positive control (matched inactivated)cH9/1 DNA + cH6/1 protein + ch5/1 proteincH9/1 DNA + BSA +BSA

challenge dose: 10 LD50

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

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

What about H3/group 2 HA vaccines in mice?


cH7/3 proteinboost(PerthH3 protein for the H7 challenge)

5 LD50 ma Phil82 (H3) 5 LD50 ma X31 (H3) 5 LD50 Rhea (H7)challenge



Phil82 H3N2 challenge (5 LD50)

Day post infection Day post infection

% w

eig

ht

loss

% s

urv

ival

N=1

SurvivalWeight loss

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

cH5/3 N1 virus challenge (100 LD50)

Day post infection Day post infection

% w

eig

ht

loss

% s

urv

ival

Weight loss Survival

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

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

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

Reactivity of group 2 HA stalk vaccinated mice to Perth09 H3 and

Shanghai13 H7 HA protein

Florian Krammer and Irina Margine5/1/13

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

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

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

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

Aims

1) Characterization of broadly neutralizing antibodies

2) Development of novel broad spectrum influenza virus vaccines

A universal influenza virus vaccine based on chimeric hemagglutinin

constructs

Florian Krammer, Irina Margine, Natalie Pica, Rong Hai and Peter Palese

5/24/13

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

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

Alternative mechanisms of neutralization

The HA stalk is conserved among group 1 and among group 2 HAs

Shaw and Palese, 2011, Field’s Virology

Chimeric hemagglutinins (cHAs)

Conserved stalk dom

ainG

lobular head dom

ain

H6

H1

chimeric H6/1Hai et al., 2012, JVI





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YYY






YY YYY

Y

YYYYYY






YY YYY

Y

YY Y

YY

YYY

YYYY

YY

YYYYYY


A/Netherlands/602/09 pH1N1 challenge


A/Fort Monmouth/1/47 and A/Puerto Rico/8/34 H1N1 challenge


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

How does pre-existing immunity to the HA stalk impact on the vaccine

regimen?

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.)

A/Puerto Rico/8/34 H1N1 challenge

NaïvePositive controlWT fluB +BSA+BSAfluB cH9/1 + cH6/1 + cH5/1fluB cH9/1 + BSA + BSA

A/Fort Monmouth/1/47 and A/Netherlands/602/09 H1N1 challenge


H5N1 challenge

Mechanism of action

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

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

Passive transfer (PR8 H1N1 challenge)


IgG ug/ml


H2 pseudotyped particle neutralization assay

Protection is mediated by humoral immunity

CD8 T-cell depletion and PR8 challenge



H7N9

Group 1-Group 2 cross-reactivity is not sufficient to protect from virus challenge

Phil82 (H3N2) ELISA Phil82 (H3N2) challenge

Group 2 proof of concept


cH7/3 proteinboost(H3 protein for the H7 challenge)

ma Phil82 (H3) ma X31 (H3) Rhea (H7)challenge



A/Philippines/2/82 H3N2 challenge

***, p=0.0008

N=1

X-31 H3N2 and H7N1 challenge

***, p=0.0001**, p=0.0088

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

cH5/3N1 challenge(A/Perth/16/09 H3 stalk)

**, p=0.0082

N=2

A/Philippines/2/82 H3N2 and X-31 H3N2 challenge

****, p<0.0001 ****, p<0.0001

Lung titers

Mechanism of action

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

Mucosal IgA and Ig subtype distribution

Reciprocal nasal wash dilution

Passive transfer and entry inhibition assay

Passive transfer (Phil82 challenge)Pseudotyped particle entry assay (Vic11)

Ferrets (Mustela putorius furo)

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

ELISA data after vaccination

Challenge virus titers

A/Netherlands/602/09 (pH1N1) virus

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

toward a universal influenza virus vaccine icahn school of medicine at mount sinai peter palese...

Documents

circulation slide

stalk specific antibodies

titers of stalk antibodies

antistalk antibodies

ph1n1 viruses

h3 influenza viruses

higher titers of antibodies

human influenza