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Re-engineering HA as a strategy to develop universal influenza vaccines

Harry Kleanthous, Ph.D.Discovery ResearchJanuary 24th 2013

WHO Integrated Meetingon Development & Clinical Trials of

Influenza Vaccines

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Topics

Background

Target Product Profile

Universal strategies

Mapping the HA universe

Consensus-based clustering (CBC’s)

SMARt

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Background

● Universal influenza vaccine approaches have paradigm-shifting potential ● Goal is replacement of the seasonal vaccines

● Recent developments in the field offering protection against disease● Promising targets ● Disruptive (change the influenza vaccination business model)

● By contrast, the clinical benefit of the majority of ‘universal vaccine’candidates in development (>40 candidates) limited to disease modulation● ‘Adjunct’ positioning● M2e development efforts halted – expensive and incremental benefit

● Approaches to universal flu vaccination have re-focused to sub-type universality

Project established focusing on universal vaccine candidates offering protection against influenza disease

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● Strategy: Universal vaccines based on novel antigen constructs.● Opportunity: A universal vaccine that protects against disease represents a significant

opportunity to alter the current influenza vaccine landscape.

NP

M1

M2e

ConservedHA-stem

sequences

Conservedepitopes

Re-engineered HA

Background

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Universal Influenza vaccine targeting both A (H1 and H3) and B strains: Top-line TPP

● A (pre-)pandemic indication will also be targeted as a second step● Production should be high-yielding, readily-scalable using recombinant technologies

permitting rapid and cost-effective production (‘state-of-the-art’ manufacturing systems)● The TPP fulfils a ‘True Universal’ vaccine, covering all virus types (‘optimal TPP’), and a ‘Sub-

type Universal’ covering circulating virus types (i.e. H1, H3 and two B lineages; ‘base case’)

Other Considerations

● Systemic and local reaction profiles comparable to marketed adjuvanted seasonal vaccines● No clinically significant interference on co-administration with routine vaccinations

Safety & Interactions

● IM (ID) administration● Two or more initial doses, followed by repeat vaccination at intervals of several years

Dosage & Administration

● Global vaccineTarget Markets

● All age groups as for regular influenza vaccineTarget Popln.

● Non-inferiority to ‘standard-of-care’ flu vaccine at time of launch (replace seasonal vaccine)● ‘Standard-of-care’ at launch in the elderly segment is expected to be defined by ‘improved’ flu

vaccines (e.g. HDIM), possibly necessitating modifications e.g. adjuvant● Protection against infection by a broad range of influenza virus subtypes and strains

Indications

● Coverage of virus subtypes contained in regular QIV vaccines, ● One or more antigens may be required per virus subtype● Assumption is that an adjuvant will be required

Composition

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Universal Influenza strategies

● Antigen● Re-engineered & immune-refocused HA ● Sequence diversity● Validation of designs

• Expression, folding & assessing breadth● Alternate antigens (tetrameric NA)

● Adjuvants & delivery● Breadth, magnitude and durability ● Route● Antigen presentation (VLP, INV)

● Immunogenicity / Efficacy● Role of pre-existing immunity ● Transmission

● Clinical Research

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Strategy: HA as a Principal ComponentGenerating novel, re-engineered antigens that induce a neutralizing antibody response against a broad-range of viruses (H1 POC)

ANTIGENS

Vaccine with improved

breadth of neutralization

(POC)

NA tetramer(Univ. Ghent)

HA-stemHeadless-stem, chimeric HA,

immune refocused HA

Modified Full-Length HA(Re-engineered heads)

Split vaccinefrom Reverse

Genetics

Virus-Like Particles (VLPs)

DELIVERY SYSTEMS(Proprietary vectors)

PROPRIETARY ADJUVANT

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Scope of the problem: Breadth of Sequence Space for H1N1

0.1 H5N1:263 unique sequences

Pandemic – current:1956 unique sequences H1N1

H5N1

H1N1

% Sequence Identity

Overall Sequence Variation

1918 – 2009:1032 unique sequences

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Universe of H1N1 Hemagglutinin SequencesH1 Strains to assess breadth (PRNT)

1

2

3 4

5

6

78

A/NewCaledonia/20/1999

A/Texas/36/1991

A/Puerto Rico/8/1934

A/New Jersey

A/California/07/2009

A/Brisbane/59/2007A/Solomon Islands/3/2006

PRNT assays established for each cluster

A/Brazil/11/1978

A/Fort Monmouth/1/1947

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Strategy 1: Cluster-Based Consensus (CBC)

1

Tier 3

1

2

3 4

5

6

78

*

***

* Denotes where cluster consensus matches circulating virus sequence

● Identification of most common amino acids at each position within and between H1N1 clusters followed by structure-guided optimization

Cluster-basedConsensus (CBC)

Tier 1 Tier 2

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● Re-engineered HA sequence retains proper folding of head and stem, as shown by binding of neutralizing mAbs (FACS) (and sialic acid on RBCs)

CBC designs recognized by neutralizing mAb’s (analogs)

0

500

1,000

1,500

2,000Ca09

NC9

9

PR8/34

HAco1M

HAco2M

HAco3M

HAco4M

HAco5M

HAco6M

HAco7M

HAco8M

HAcb12

M

HAcb34

5M

HAcb67

M

HAcb1‐

8_01

HAcb1‐

8_02

HAcb1‐

8_03

HAcb1‐

8_04

HAcb1‐

8_05

MFI

C179

0

1,000

2,000

3,000

Ca09

NC9

9

PR8/34

HAco1M

HAco2M

HAco3M

HAco4M

HAco5M

HAco6M

HAco7M

HAco8M

HAcb12

M

HAcb34

5M

HAcb67

M

HAcb1‐

8_01

HAcb1‐

8_02

HAcb1‐

8_03

HAcb1‐

8_04

HAcb1‐

8_05

MFI

CH65

0

1,000

2,000

3,000

Ca09

NC9

9

PR8/34

HAco1M

HAco2M

HAco3M

HAco4M

HAco5M

HAco6M

HAco7M

HAco8M

HAcb12

M

HAcb34

5M

HAcb67

M

HAcb1‐

8_01

HAcb1‐

8_02

HAcb1‐

8_03

HAcb1‐

8_04

HAcb1‐

8_05

MFI

5J8

0

1,000

2,000

3,000

4,000

Ca09

NC9

9

PR8/34

HAco1M

HAco2M

HAco3M

HAco4M

HAco5M

HAco6M

HAco7M

HAco8M

HAcb12

M

HAcb34

5M

HAcb67

M

HAcb1‐

8_01

HAcb1‐

8_02

HAcb1‐

8_03

HAcb1‐

8_04

HAcb1‐

8_05

MFI

4K8

STEM

HEA

D (E

XPRE

SSION‐CORR

ECTED)

WT CBC Tier 1 CBC Tier 2 CBC Tier 3

Epitope shared with neutralized strains

Okuno et al. 1993

Whittle et al. 2011

Krause et al. 2011

Krause et al. 2011

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Viable Influenza viruses rescued containing Consensus (CBC) HA’s

HA PB1 PB2 PA NP

PB1 PB2 PA NP

NS M NAConsensus/SMARt HA

Rescued virus with Consensus HA

Tier 1Cluster 4

Tier 1Cluster 5

Tier 2Cluster 345

+

Res

cued

viru

ses

HA titer

32

64

128

Plaque Assay

>1x10^6

4.7x10^5

6.6x10^5

1:1000

1:1000

1:1000

PR8 Backbone

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Breadth of neutralization (PRNT) induced by Tier1 (cluster 4) virus similar to NC

** P<0.01 Tukey-Kramer HSD test

Tier viruses immunization (IN; 2x) day 35

Calif

orni

aNew

Jer

sey

Puer

to R

ico

Texa

s

New C

aled

onia

Bris

bane

Solo

mon

Isla

nds

10 1

10 2

10 3

10 4

10 5

influenza strains

3 4 81 2 5cluster

Cut Off

50%

PR

NT

abov

e pl

aceb

o tit

er **

NS

New Caledonia WTHAco4M tier 1

Placebo

% difference

NC1999 reference seq.

Heat map of sequence differences at key epitopes/antigenic regions

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Breadth of neutralization induced by Tier2 (clusters 345) virus similar to NC

50%

PR

NT

abov

e pl

aceb

o tit

er

*** P<0.001 Tukey-Kramer HSD test

*****

Tier viruses immunization (IN; 2xl day 35)

Califo

rnia

New J

erse

yPue

rto R

ico

Texas

New C

aled

onia

Brisba

ne

Solom

on Is

land

s

10 1

10 2

10 3

10 4

10 5

influenza strains

3481 2 5cluster

Cut Off

HAcb345M tier 2

New Caledonia WTBrisbane WTSolomon Islands WT

Texas WT

Placebo

% difference

Heat map of sequence differences at key epitopes/antigenic regions

NC1999 reference seq.

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HAI confirms limited breadth of initial prototypes

L iv e W T v ir u s (IN ) v e r s u s liv e C B C T ie r v ir u s e s (IN ) d a y 3 5 (2 x )

PBS

Texas

New C

aledonia

Brisban

e

Solomon Isl

HAco4M

HAco5M

HAcb34

5M

1 0 0

1 0 1

1 0 2

1 0 3

a nti-N e w C a le d o nia(p o o ls )

a nti-B ris b a ne (p o o ls )

a nti-S o lo m o n Is la nd(p o o ls )

a nti-P R (p o o ls )

Plac

ebo

New

Cal

Bris

bane

L iv e v ir u s (IN ; 1 x 1 0 ^ 5 p fu )

C u t o ff

Solo

mon

Isl

a nti-T e xa s (p o o ls )

Texa

s

Tier

1co5

M

Tier

2cb3

45

C B C T ie r v ir u s

Tier

1co4

M

a nti-C a lif o rn ia (p o o ls )

a nti-N e w J e rs e y (p o o ls )

HA

I tite

r

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Expression and folding screen with mAbs have yielded multiple SMARtcandidates

● Broadly neutralizing anti-stem & anti-head mAbs demonstrate surface localization and folding

● VLPs prepared of SMARt HA candidates and assessing breadth of neutralizing antibody response with and without adjuvant

Neutralizing Epitope #1Influenza strain 1 Neutralizing Epitope #3

Influenza strain 3

SMARt DESIGN

Single HA molecule containing repertoires of neutralizing epitopes. Epitopes computationally assembled from diverse strains and carefully selected to collectively elicit broadly neutralizing antibodies

Neutralizing Epitope #2Influenza strain 2 Neutralizing Epitope #4

Influenza strain 4

Strategy 2: Structural Mapping of Antigenic Repertoires

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Mosaic SMARt designs: Differences to NC99 template sequence

CH65

HC45

FI6

• Surface representation of trimeric HA with monomers colored blue green and gray. Amino acid differences between the designed molecule and the New Caledonia reference are highlighted in red. Fab fragments are docked onto the locations of three known neutralizing epitopes on the surface of HA: CH65, HC45 and FI6

SMARt_NC_CH2a SMARt_NC_CO1a SMARt_NC_DO1a SMARt_NC_RA3a(maximal strain coverage) (consensus epitope patterns) (dominant epitope patterns) (random combination of

epitope patterns)

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● Modification of HA sequence by SMARt method still retains proper folding of head and stem, as shown by binding of neutralizing mAbs (FACS)

STEM

HEA

D (E

XPRE

SSION‐CORR

ECTED)

0500

1,0001,5002,0002,500

Ca09

NC9

9

PR8/34

SMARtCH

2a

SMARtCH

3a

SMARtCO

1a

SMARtDO1a

SMARtDO2a

SMARtRA

3a

MFI

C179

0500

1,0001,5002,0002,500

Ca09

NC9

9

PR8/34

SMARtCH

2a

SMARtCH

3a

SMARtCO

1a

SMARtDO1a

SMARtDO2a

SMARtRA

3a

MFI

CH65

0500

1,0001,5002,0002,500

Ca09

NC9

9

PR8/34

SMARtCH

2a

SMARtCH

3a

SMARtCO

1a

SMARtDO1a

SMARtDO2a

SMARtRA

3a

MFI

5J8

0500

1,0001,5002,0002,5003,0003,500

Ca09

NC9

9

PR8/34

SMARtCH

2a

SMARtCH

3a

SMARtCO

1a

SMARtDO1a

SMARtDO2a

SMARtRA

3a

MFI

4K8

WT SMARt (NC99 backbone)

SMARt designs appear well-folded and are functional

Epitope shared with neutralized strains

NC1999 reference seq.

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0

50

100

150

Moc

k

A/N

ew C

aled

onia

A/C

alifo

rnia

/07/

2009

SMA

RtC

H2a

SMA

RtC

H3a

SMA

RtC

O1a

SMA

RtD

O1a

SMA

RtD

O2a

SMA

RtR

A3a

SMARt HA proteins bind sialic acid on RBCs

Assay performed using Guinea Pig RBCs

SMARtNC

Cal

if

% R

BC

Bin

ding

Com

pare

d to

New

C

aled

onia

● Immunogenicity and breadth of neutralization studies on-going

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● Implemented strategy to evaluate re-engineered HA for increased breadth● Mapped the evolution of influenza viruses● Established bioinformatics approach to screen novel HA candidates● Generated full-length consensus and mosaic HA that are properly folded

& functionally active● Viable viruses rescued with CBC HA’s validates modeling approach ● Initial CBC designs (closely grouped clusters) did not increase breadth

over wild type but approaches looking to increase breadth acrossmultiple clusters appear promising (SMARt)

• NC99 is a broadly protective virus

● Test novel SMARt and Tier 3 CBC’s in-vivo (+/- adjuvant)

Conclusions

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Acknowledgements

Bioinformatics: Tod Strugnell, Eliud Oloo, Ray Oomen

Bacteriology: Tim Alefantis (PL), Guadalupe Cortes-Garcia, Qing-Sheng Gao

Protein Chem: Fuqin Ma, Sophia Mundle, Jit Ray, Svetlana Stegalkina, JixZhang Steve Anderson

Viral Immunology: Josh Dinapoli, Xiaochu Duan, Tim Farrell, Svetlana Pougatcheva, Darren Smith, Irina Ustyugova, Claire Ventura, Thorsten Vogel

Animal Center: Johan Guerrero, Michael Howard, Lori Jasinski

Management: Mark Parrington, Jeff Almond