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Corn as a production and delivery
system for oral vaccines
Kan Wang
January 10, 2003
Pasteur and invention of vaccine
• Attenuation of virulent microorganisms
– Chicken cholera
– Anthrax
– Swine erysipelas
Louis Pasteur
Chemist
1822-1895
• Prevention of infectious diseases
• Therapeutic vaccines
– Anti-tumor (cancer)
– Anti-HIV
How vaccines work?
A. Vaccine contain antigens (weakened or
dead virus, bacteria that cause disease).
When enter the body, antigens stimulate
the immune system (B cells to produce
antibodies with help of T cells).
B. The antibodies are produced to fight the
weakened or dead viruses in the vaccine.
C. The antibodies “practice” on the
weakened viruses, preparing the immune
system to destroy real and stronger viruses
in the future.
D. When new virulent strain enter the body,
white blood cells called macrophages
engulf them, recognize the antigens, and
send it to the T-cells so that the immune
system response can be mobilized.
Illustration by Electronic Illustrators Group
Vaccination: benefits and risks
• Conventional vaccines:
– Three types:
• Killed whole organisms
• Live but severely weakened (attenuated) organisms
• Inactive specific parts or products of infectious organisms
– Not always effective
– Can cause allergic reactions or even death
• Modern vaccines:
– DNA vaccines
– Subunit vaccines
Why oral vaccine?
• First line of defense
• Effective and economical in
inducing systemic immunity
• Vertical transmission
• Safer compare to injectable
vaccines
Current limitations in subunit vaccine
production
 Chemical synthesis
 Size
 Cost
 Microbial fermentations
 Protein processing
 Downstream purification
 Animal tissue cultures
 Cost
 Possible pathogen contamination
Farm-aceuticals:
why use plant as bioreactor ?
 Safer
 Plants carries no common
mammalian pathogenes
 Animal viruses – Bovine
viral diseases
 Microbes – E. coli 0157,
Salmonella
 Prions – Mad cow diseases
SV40 contamination in
polio vaccine in 1960’s
Farm-aceuticals:
why use plant as bioreactor ?
 Safer
 High-volume production
 Seeds allow long-term protein storage and stability
• 11 million children each year die from
– Pneumonia, Diarrhea, Measles, Malaria, Malnutrition
Source: WHO Fact Sheet No 178, 1998
Plant process vs other process:
Transg plants
Yeast/bacteria
Animal culture
Transg animal
Time required
High
medium/low
High
High
Cost/storage
Cheap/RT
Cheap/-20oC
Expensive/N2
Expensive/N2
Distribution
Easy
Feasible
Difficult
Difficult
Folding accuracy
High?
Medium/low
High
High
Correct?
Incorrect/absent
Correct
Correct
Multi Protein asse
Yes
No
No
Yes
Production cost
Low
Medium
High
High
Propagation
Easy
Easy
Hard
Feasible
Protein yield
High
Medium/low
Medium-high
High
Unknown
Yes
Yes
Yes
Safety
High
Low
Medium
High
Scale up costs
Low
High
Medium
High
Glycosylation
Risk (residual)
Modified from Fischer et al., 1999.
Which plant ? Which tissue ?
Tissue
Plant
Green
tissues
Tobacco, alfalfa
Seed,
tuber
Corn, rice, wheat,
soybean, canola,
potato etc
Fruit
Banana, tomato,
apple
Advantages
 Large productivity
 Multiple crops per year
 Ease of genetic manipulation




Edible
Large productivity
Ease in purification
Long term storage
 Edible
 Ease in distribution
 Ease in processing
Why corn ?
•
•
•
•
•
•
Major staple food and feed worldwide
Most tolerated plant for both humans and animals
Can be fed raw
Yield
Seeds allow long-term protein storage and stability
Established infrastructure for production and
protein extraction
• Possible low-cost production and administration of
proteins
• Genetic transformation is routine in ISU
How vaccine corn is made?
Altered bacterial gene is
spliced into Agrobacterium
Transformed plant cells divide
rapidly and form plantlets
Agrobacterium delivers the bacterial
gene into plant cells and integrated into
plant’s own chromosome
Transgenic Maize plants
are grown in greenhouse to
maturity
Title: Development of Corn-based Edible Vaccine for
Livestock
USDA-NRI Award ID # 99-35504-7799
Objective
Long term goal:
to produce effective vaccines in corn to protect
livestock against viral pathogens.
Specific objective:
to produce high levels of vaccines against
1) human diarrhea disease and 2) porcine transmissible gastroenteritis
(TGE) in transgenic maize plants.
Ubiquitin
promoter
27 kD  -zein
promoter
Mouse feeding trial
Dry corn pellet
BALB/c mice
LT-B corn
Functional analyses of maize generated LTB – Mice Feeding
Anti-LT-B antibody analysis in orally immunized mice
IgG concentration ( m g/ml)
8000
70 0 0
6000
N e ga t i v e
S pi k e d
T r a n sg e n i c
50 0 0
4000
3000
2000
10 0 0
0
D ay - 1
D ay 6
D ay 13
D ay 2 0
D ay 2 7
B leed D ay
Serum IgG
Negative
Spiked
Transgenic
Fecal IgA
Serum IgA
Immunization dates: days 0, 3, 7 and 21
Sampling dates: days –1, 6, 13, 20, 27
Patent mouse assay for toxin challenge of mice
Gut : Carcass ratio
0 . 15
0 . 14
LT
0 . 13
0 . 12
0 . 11
0 . 10
0.09
0.08
N e ga t i v e
S pi k e d
T r a n sg e n i c
P BS
Feed catagory
Mouse gavaged with 25 mg of LT or PBS at day 28 and
gut/carcass ratio determined 3 h after euthanasia.
Comparing level and stability of seed
expression of LT-B
% LT-B of Soluble protein
4
% LT-B in TAEP
3.5
3
2.5
2
1.5
1
0.5
0
4
3.5
3
2.5
2
1.5
1
0.5
0
2-1 2-2 3-1 7-1 7-2 9-1 9-2 10- 10- 11- 11- 17- 18- 18-
2
3
7
9 10 11 15 17 18 20 24
P77 Event # (F1)
1
2
1
2
1
1
P77 Event # (F2)
LT-B expression increased by 53-fold over 2 generations
2
I am wondering if
it is a transgenic
apple?
Plant-Made Pharmaceuticals
- points to consider:

Scientific considerations:
–
–

Proof of concept
Genetic stability
Regulatory considerations:
 Product comparability
 Purity

Safety considerations:
 Environmental impact
 Product protection
 Gene flow

Commercialization considerations:
 Legal issues
Product comparability
Potential problems:
 Different glycosylation pattern
What is Glycosylation:
- Adding sugar moieties to protein
Why is it bad?



Can affect activity of products
Can affect optimal dose
Can cause allergic reactions in some patients
What should we do?
 Structure, function, bio-activity tests
Purity
Potential problems:
Contamination of
– plant alkaloids
– plant macromolecules (DNA, polysaccharides,
lipids etc)
– pesticides, herbicides
– bacterial and fungi endotoxin
– other protein products
Purity (cont’n)
Why is it bad?



direct toxic effects on the recipient
effects on product stability and biodistribution
allergic reactions
What should we do?



effective purification process
rigorous testing and/or validation protocols
identity preservation
Assessment of the allergenic potential of
GM food (decision tree)
Source of Gene
(Allergenic)
Yes
Solid Phase Immunoassay
Commonly
Allergenic
No
Sequence
similarity
Yes
Less commonly
Allergenic
No
No
No
Yes
Skin Prick
Stability to
Digestion/processing
Yes
No
Yes
DBPCFC
(IRB)
Yes
Label as
to source
No
Market
No
Consult with
Reg. Agency
Environmental impact
Potential problems:
 horizontal gene transfer to soil
microorganisms
 recombinant toxin may contaminate soil
or affect wildlife eating the plant
What should we do?
 Containment or restricted field release
 Risk assessment
Containment and restricted field release
1.
Physical isolation: one mile (radius) away from any corn field
2.
Temporary isolation: three weeks delayed planting
3.
Biological isolation: male sterile (not this project)
Summer, 2002
Demonstration of corn pollen flow
60 inch
30 inch
120 inch
Courtesy Dr. P. Peterson (Department of Agronomy, ISU)
Molecular Pharming
“Do not put forward anything that you
cannot prove by experimentation.”
“Chance favors only the prepared mind.”
Louis Pasteur
Chemist
1822-1895