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Corn as a production and delivery system for oral
vaccines
Kan WangJanuary 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
Illustration by Electronic Illustrators Group
B. The antibodies are produced to fight the weakened or dead viruses in the vaccine.
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).
How vaccines work?
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.
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
Modified from Fischer et al., 1999.
Propagation Easy Easy Hard Feasible
Production cost
Low Medium High High
Multi Protein asse
Yes No No Yes
Glycosylation Correct? Incorrect/absent Correct Correct
Folding accuracy
High? Medium/low High High
Protein yield High Medium/low Medium-high High
Risk (residual) Unknown Yes Yes Yes
Safety High Low Medium High
Scale up costs Low High Medium High
Which plant ? Which tissue ?
Tissue Plant Advantages
Green tissues
Tobacco, alfalfa Large productivity Multiple crops per year Ease of genetic manipulation
Seed, tuber
Corn, rice, wheat, soybean, canola, potato
etc
Edible Large productivity Ease in purification Long term storage
Fruit Banana, tomato, apple
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
Altered bacterial gene is spliced into Agrobacterium
Agrobacterium delivers the bacterial gene into plant cells and integrated into
plant’s own chromosome
Transformed plant cells divide rapidly and form plantlets
Transgenic Maize plants are grown in greenhouse
to maturity
How vaccine corn is made?
Title: Development of Corn-based Edible Vaccine for Livestock
USDA-NRI Award ID # 99-35504-7799
ObjectiveObjectiveLong 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 LT-B – Mice Feeding
0
1000
2000
3000
4000
5000
6000
7000
8000
Day -1 Day 6 Day 13 Day 20 Day 27
Bleed Day
IgG concentration (
μ/ )g ml
Negative
Spiked
Transgenic
0
50
100
150
200
250
300
350
Day -1 Day 6 Day 13 Day 20 Day 27
Collection Day
μ
Negative
Spiked
Transgenic
0
100
200
300
400
500
Day -1 Day 6 Day 13 Day 20 Day 27
Collection Day
μ
Negative
Spiked
Transgenic
Serum IgG Fecal IgA Serum IgA
Negative
Spiked
Transgenic
Immunization dates: days 0, 3, 7 and 21
Sampling dates: days –1, 6, 13, 20, 27
Anti-LT-B antibody analysis in orally immunized mice
Patent mouse assay for toxin challenge of mice
0.08
0.09
0.10
0.11
0.12
0.13
0.14
0.15
Negative Spiked Transgenic PBS
Feed catagory
Gut : Carcass ratio
LT
Mouse gavaged with 25 μg 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
0
0.5
1
1.5
2
2.5
3
3.5
4
2 3 7 9 10 11 15 17 18 20 24
P77 Event # (F1)
% LT-B in TAEP
0
0.51
1.52
2.5
33.5
4
2-1 2-2 3-1 7-1 7-2 9-1 9-2 10-
1
10-
2
11-
1
11-
2
17-
1
18-
1
18-
2
P77 Event # (F2)
% LT-B of Soluble protein
LT-B expression increased by 53-fold over 2 generations
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)
Sequence similarity
Stability to Digestion/processing
Market
Consult withReg. Agency
Label as to source
DBPCFC(IRB)
Skin Prick
Less commonlyAllergenic
CommonlyAllergenic
Solid Phase Immunoassay
Yes No
Yes
No
No
No
Yes
Yes
No
Yes
No
Yes
No
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
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)
Containment and restricted field release
Summer, 2002
Demonstration of corn pollen flow
30 inch
60 inch
120 inch
Courtesy Dr. P. Peterson (Department of Agronomy, ISU)
Molecular Pharming
“Do not put forward anything that you cannot prove by experimentation.”
Louis Pasteur
Chemist
1822-1895
“Chance favors only the prepared mind.”