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.”