Plant-made influenza
vaccines
Nathalie Landry, VP Product Development
Summer School on Influenza- Siena
July 18th 2012
© 2011 Medicago Inc.
All rights reserved
Agenda
• Plants as platform for recombinant protein production
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–
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Background
Technologies
Main advantages/disadvantages
Current status
• Vaccines produced in plants
• Manufacturing process for plant-based vaccines
• Plant-based influenza vaccines
– Preclinical results
– Clinical trials
• Concluding remarks
Plants for the production of recombinant
proteins
• First described by in 1989 by Hiatt et al
– Plant-made biopharmaceuticals produced in con, tobacco,
rice, Arabidopsis and others
• 23 years later, first FDA approval
• Why using plants?
– Cheap
– Complex metabolism
– Free of human pathogens
The different systems
• Seed-based
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–
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Developed for maize, rice , safflower, soybean
Suitable for storage
Less impurities during downstream processing
Not suitable for every protein
• Leaf tissues
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Developed for tobacco species
Maximize metabolic capacity
Not suitable for storage
More impurities in the upstream part
More adaptable to growth in greenhouses
• Cell culture
– Similar to cell culture systems (pro’s and con’s)
– Developed for carrot cell culture
Plants for the production of recombinant
proteins
• What are the difficulties?
– Plant growth in open fields
• Loss of containment by Prodigene (corn)
– Use of non-food crop
– More emphasis on greenhouse containment
– Regulatory background
• Guidance documents available although they do not apply to
all technologies
• Several clinical trials have been conducted in many countries
• Products approved as dietary sup. or drugs
– From proof-of-concept to GMP manufacturing
– $$$
6
Current status
Some examples
© 2011 Medicago Inc.
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Cell
culture
Transgenic
Transgenic
Expres
sion
Transient
Leaf tissue
Seed-based
System
Transgenic
Plant
Product
Status
Group
Maize
• Collagen
• Phase IIa
• Meristem
(discontinued)
Rice
• Lactoferrin
• Phase III
• Ventria
Safflower
• Insulin
• Phase I/II
• SemBioSys
(discontinued)
Tobacco
• CaroRx (mAb)
• Phase
II+EU
approval
• Planet
Biotechnology
Arabidopsis
• Human intrinsic
factor (food sup.)
• Phase II,
marketed
EU
• Cobento
Biotech AS
Lemna
• Interferon-alpha
• Phase IIb
• Biolex
Therapeutics
Tobacco
•
•
•
•
•
•
•
•
•
Carrot
• Glucocerebrosidase
Influenza VLP
Rec. HA
Non-Hodgkin’s
lymphoma
vaccine
Phase II
Phase I
Phase I
• FDA
approved
Medicago
Fraunhofer
Bayer
Innovation
• Protalix
Vaccines made in plants
Two options
• Edible vaccines
– Hep. B in transgenic potato
• Phase 1 (Thanavat aet al 2005)
– Hep. B in transgenic lettuce
• Phase 1 (Kapusta et al 1999)
– Rabies in spinach
• Phase 1 (Yusibov et al 2002)
– Norovirus vaccine in transgenic potato
• Phase 1 (Tacket et al 2000)
• Parenteral vaccines
– Recombinant hemagglutinin
• Phase 1 (Yusibov, data not published)
– Anti-idiotype antibodies for Non-Hodgkin’s lymphoma
• Phase 1 (Bendandi et al 2010)
Update on clinical trials with influenza VLP vaccines
• Seasonal vaccine – USA
– Phase 1 trial to study safety, tolerability and
immunogenicity of a single dose of H1 VLP (nonadjuvanted)
• 100 healthy adults (18-49 years of age)
– Phase 2 trial to be initiated in 2012
• Pandemic vaccine - Canada
– Phase 1 trial completed in 2009,
• 48 healthy adults (18-60 years of age)
– Phase 2 trial to evaluate immunogencity, safety and
tolerability of two adjuvanted doses of H5 VLP vaccine,
• 255 healthy adults (18-60 years of age)
– Phase 1 trial in 2012 for id administration with GLA (IDRI)
Vaccines made in plants
Challenges
• Edible vaccines
– Dose standardization
– Mucosal immunity
• Serologic marker
• Efficacy trials
• Parenteral vaccines
– Process robustness
– Product characterization
• General
– Safety issues (allergies)
Vaccines made in plants
Influenza vaccines
• Two main types
– Rec. Hemagglutinin
– HA VLPs
• Clinical data publicly available only for HA VLPs
Characteristics of the plant-made VLP vaccine
Medicago VLP
• Influenza virus-like particles in
plants using only one viral gene
(Hemagglutinin)
• No possibility of viral replication
• The VLP vaccine presents wildtype HA in an immunologicallyrelevant array as a membranebound protein
Influenza virus
Transient expression
• From gene sequence to flu VLP in 19 days
DNA sequence + plants
Extraction
Infiltration
Purification
Incubation
VLP
Flu virus
Plants: First responder solution in case
of severe pandemic
Number of H1N1 cases WW
• 19 days from gene sequence to first batch
April May June
Identification of
strain A/H1N1
July August September October
First wave
begins
November December
Second wave
13
begins
Greenhouse
15
Vacuum infiltration of the bacterial
inoculum in plant leaves
© 2011 Medicago Inc.
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15
15
16
Infiltration/infection
TDNA
1.
2.
3.
© 2011 Medicago Inc.
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Docking onto the plant cell
Excision of the T-DNA segment
Transport of the T-DNA to the nucleus of the plant cell
Binary plasmid
16
16
Vacuum Infiltration
After infiltration, 5-6 days incubation then
harvesting of biomass
18
19
HA accumulation on lipid rafts and VLP
formation
Virus-like particle
Plasma membrane
HA homotrimer
Host proteins
20
HA-VLP accumulation in plants
Cell wall
Plasma
membrane
(indented)
© 2011 Medicago Inc.
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20
Protein Expression and Harvest
• Plants moved to controlled environments after infiltration
to allow expression of protein and accumulation of
particles
• Plants harvested manually and biomass is diced
• Biomass is digested to allow release of particles
• S:\Videos of processes\Harvest movie 1.MOV
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22
VLP Purification
© 2011 Medicago Inc.
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23
Product testing
Lot-release assays
Test
Information
Test
Information
Appearance
Content
Nicotine content
HPLC-UV
pH
Content
HPLC-UV
Western blot
Identity
Anabasine
content
SDS-PAGE
analysis
Ratio
lipids/proteins
SRID
Tween-80
Residual DNA
© 2010 Medicago Inc.
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Endotoxins
(rFC)
Purity
Content
Potency assay
(E.P.)
Residual HCP
(cellulases)
Residual
Rubisco
Chromogenic
end-point
Western blot
Bioburden
Sterility
HPLC-ELSD
Purity
ELISA
GST
<USP 71> and
E.P.
21 CFR 610.11
2 animal species
24
Product testing Analytical methods (characterization)
Information
Potency
Precision ± 1
dilution
NanoSight
Tracking
Analysis
Particle size
distribution
Integrity of
VLPs
≥ 2000 kDa,
free HA
MicroFlow
Imaging
Presence of
aggregates
Information
Agglutination of
RBC
SEC-HPLC
Electron
microscopy
Circular
dichroism
© 2010 Medicago Inc.
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Test
Test
Dynamic light
scattering
Particle sizing
Polydispersity
index (PDI)
Protein
impurities by
LC/MS/MS
Identity of protein
impurities and
relative abundance
Morphology
Secondary and
tertiary
structure
Lipid identity by
LC/MS/MS
Identification of
lipids
Glycan analysis
by LC/MS/MS
Total glycans and
glycopeptides
25
Correlates of protection – H5N1
Lethal challenge in ferrets
Medicago VLPs
based on
Indonesia strain
(H5 Clade 2.1)
Challenge done with
Vietnam strain
(H5 clade 1)
Cross-clade challenge
Design of the experiment
Part A:
Challenge after a single vaccine dose
Part B:
Challenge after two vaccine doses
1.9 µg VLP +
alum
1.9 µg VLP +
alum
7.5 µg VLP +
alum
7.5 µg VLP +
alum
7.5 µg VLP
Placebo +
alum (control)
© 2010 Medicago Inc.
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Placebo +
alum (control)
26
H5N1 Challenge results after one vaccine dose
104.5 EID or 75 LD50 administered
intranasal 30 days after 1st immunization
3,00
HI titers before challenge
Strong HI antibody
response for the
homologous strain
(Indo) but low to
undetectable for
challenge strain (VN)
HI titer (GMT; log 10)
2,50
GMT 35
(13/16)
2,00
GMT 113
(15/16)
GMT 12
(4/16)
(0/16)
1,50
(0/16) (0/16)
A/Indonesia/5/05
A/Vietnam/1203/04
1,00
0,50
0,00
Placebo
1.9 µg H5 VLP 7.5 µg H5 VLP
vaccine +
vaccine +
Alhy.
Alhy.
100,0
80,0
Survival rate
Survival rate
60,0
Placebo + Alh.
40,0
Vaccine + Alh.
1.9µg
Vaccine + Alh.
7.5µg
20,0
0,0
0
© 2010 Medicago Inc.
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5
10
Days after challenge
15
27
H5N1 Challenge results after one vaccine dose
Viral titers in upper respiratory
tract
Mean Virus Titer (Log10
TCID50/ml)
6
5
Placebo + Alhy.
4
**
3
**
**
2
1.9 µg H5 VLP
vaccine + Alhy.
7.5 µg H5 VLP
vaccine + Alhy.
***
***
*p<0.01, ** p<0.001, and
*** p<0.0001 vs Placebo group
1
0
0
1
3
5
7
Day after challenge
9
Viral titers in nasal turbinates and lungs of 3 animals pre-assigned for
sacrifice at Day 5 post-challenge
Treatment group
Nasal turbinates
Lungs
1.9 µg H5 VLP + Alhydrogel
<1.7 **
<1.7 *
7.5 µg H5 VLP + Alhydrogel
<1.7 **
<1.7 *
5.2
3.1
Control group (buffer +
Alhydrogel)
Data presented as reciprocal of log10 TCID50 per gram of tissue
* p< 0.001 and **p < 0.0001 vs Placebo group
Safety of plantmade influenza
VLP vaccines
© 2011 Medicago Inc.
All rights reserved
General safety profile
Phase 1 H1 VLP and Phase 2 part A H5 VLP
29
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
Non-adjuvanted H1 VLP
Placebo Phase 1
Adjuvanted H5 VLP
Non-adjuvanted H5 VLP
Placebo Phase 2 part A
% of incidence
General safety profile
Phase 1 H1 VLP and Phase 2 part A H5 VLP
30
25
20
15
10
5
0
Severe
Fatigue
Headache
Body or
muscle ache
General
discomfort
Solicited systemic AEs
Chills
Joint ache
Swelling at
axilla
30
Swelling in
the neck
Moderate
Mild
Type I hypersensitivity reactions
Degranulation of mast cells require the binding of at least two epitopes to
two adjacent IgE antibody molecules. This cross-linking may be achieved by
two peptide epitopes, by one glycan and one peptide epitope, but also two
glycan epitopes.
31
Cross-reactive glycans found in plant
allergens
Complex Nglycan not
associated
with allergy
(Altmann F. 2007)
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N-glycans found in HA VLP vaccines
N-glycans found in H1 VLP vaccine (lot PDD-20101108A)
Structure
Relative abundance
Gn2M3FGn2
21.4%
GnM3FGn2
9.6%
Gn2M3XFGn2
43.5%
GnM3XFGn2
9.2%
GnM4XGn2
8.6%
Lewis
7.8%
and alike
• Glycan structures confirmed by 3 laboratories: Medicago, Univ. Of Rouen,
Proteodynamics
• Glycans in H5 VLP vaccine are of similar structure (data not shown) but
33
relative abundance not assessed
Safety
IgE to plant glycans
Clinical trial
Group
Phase 1 with
H1 VLP (one
dose)
Non-adjuvanted
VLP (n=58)
Number of
subjects with
IgEs ≥grade 1
to bromelain at
screening
Fluzone (trivalent,
n=20)
Placebo (n=20)
Phase 2 with
H5 VLP (two
doses)
•
Adjuvanted VLP
(n=192)
Number of
subjects that
showed an IgE
increase after
vaccination
Number of
subjects that
showed detectable
IgEs 6 months
after vaccination
3.5%
0%
1.8%
(2/57)
(0/57)
(1/56)
0%
0%
0%
(0/20)
(0/20)
(0/20)
0%
0%
0%
(0/20)
(0/20)
(0/20)
3%
0%
3%
(6/191)
(0/188)
(6/191)
Non-adjuvanted
VLP (n=29)
7%
0%
4%
(2/29)
(0/29)
(1/27)
Placebo (n=27)
0%
0%
0%
(0/28)
(0/28)
(0/28)
No onset of allergic reactions correlating with in vitro assay
(manuscript in preparation)
Cell-mediated
immune response to
plant-made influenza
VLP vaccines
© 2011 Medicago Inc.
All rights reserved
36
Cells of the immune system
Measured in
clinical trials
The quality of innate response dictates the quality
of adaptive immune response
© 2011 Medicago Inc.
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How the plant-made can stimulate innate
immunity?
• Because it is a particle of the size and shape of a virus
– better uptake by APCs
• Lipids and glycolipids
– TLR agonists
• Plant glycans
– Lewis carbohydrates are C-lectin type agonists
Rapid Innate Response to H5-VLP
Expression of Activation Markers at 6 Hours (B
Ward’s lab)
B Lymphocyte
Monocyte
B cells, monocytes
and NK cells are all
activated by VLP
CD3 T cells show
no response to
VLP stimulation
NK Cell
CD3 T Cell
CD4+
H1 VLP Vaccine Results in Durable
T cell Responses against HA (H1N1)
Phase I
H1 VLP
(USA)
Responding Cells
per 106 CD4+ T cells
Total CD4+ T cell response against HA (H1N1) :
• PBMC at +6 month post-vaccine
• In vitro stimulation with H1 VLP
Placebo
H1 VLP
Fluzone
* P<0.05 (MannWhitney)
Phase I
H1 VLP
(USA)
H1 VLP Vaccine Results in Durable CD4+
T cell Responses against HA (H1N1)
Responding Cells
per 106 CD4+ T cells
Total CD4+ T cell response against HA (H1N1) :
• PBMC at +6 month post-vaccine
• In vitro stimulation with 15-mer peptide pool (BEI
resources)
Placebo
H1 VLP
Fluzone
* P<0.05 (MannWhitney)
H1 VLP Vaccine Induced a Long Lasting
Memory Multifunctional T cell Response
Memory CD8+ T Cells
H1 (VLP) Response
Per 106 CD45RA- CD8+ T cells
H1 (VLP) Response
Per 106 CD45RA- CD4+ T cells
Memory CD4+ T Cells
Polyfunctional T cells
Main conclusions from clinical trials
• Safety
–
–
–
–
–
–
safe and well tolerated
More than 400 subjects dosed
1 or 2 doses
With or without alum
Dosages up to 45 µg
No onset of allergic reactions
• Immunogenicity
– The HA-VLP vaccine induces antibody titers comparable
to licensed vaccines
• Antibody levels detectable 6 months after administration
• Plant-made VLP induces innate immune response and
and T-cell response
Conclusions
© 2011 Medicago Inc.
All rights reserved
Industry Impact of plant made vaccines
• Advantages of the plant-based technology:
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–
–
–
Overall costs of a manufacturing facility
Speed (21 days from sequence to candidate)
Target any HA sequence including wild-type
Affordability for emerging markets
Plant-made vaccines: local facilities
Large production centers
Facilities distributed globally
Smaller less complex
Low capital investment and fixed costs
Accessible to emerging countries
Requiring complex infrastructure and
organization (egg supply)
High capital and fixed costs
Mainly located in North American and
Europe
Construction, automation, start-up
September 2010
January 2011
March 2011
Construction
August 2011
February 2012
September 2011
Start-up
47
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