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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