Transgenic Corn as an Edible Vaccine Joan Cunnick, Ph.D. Dept. Animal Science Iowa State University

Overview Vaccine history & types Transgenic plants Edible vaccines at ISU Future work

Small Pox 1250AD – a “childhood disease” Edward Jenner 1796-observation pays off Observation: Testing? Jenner: Smallpox is Stemmed_____ "The first vaccination against smallpox was performed by Edward Jenner, English rural physician, in his apartment in the Chantry House, Berkeley Gloucestershire. Exudate from a cowpox pustule on the hand of a dairymaid, Sarah Nelmes, was inserted into scratches on the arm of eight-year-old James Phipps, May 14, 1796. The vaccination was effective, for two later attempts to induce infection with smallpox were unsuccessful. Proving and reproving his discovery, Jenner published his vaccination findings in 1798. Dispite opposition, vaccination became accepted practice during Jenner's lifetime." This painting by Robert A. Thom appeared in "Great Moments in Medicine" published by Parke Davis & Company, in 1966. The text quoted is from the same source. Both are reproduced with the permission of Parke Davis & Company.

One of the top 10 Medical Advances of 20th century James Phipps - first person Edward Jenner vaccinated against smallpox Ali Maow Maalin - last known case of naturally acquired smallpox (Somalia, 1977) Luis Fermin - last person in the Western hemisphere to acquire paralytic poliomyelitis from wild poliovirus

The Golden Age of Microbiology Louis Pasteur 1822-1895 “Did you ever observe to whom the accidents happen? Chance favors only the prepared mind.” Attenuated vaccines

Important Milestones of 1900’s “Its time we close the book on infectious disease” -US Surgeon General, 1967 Small pox declared eradicated 1967 “Its time we close the book on infectious disease” US Surgeon General 1975-2004 Emerging Diseases

Some Current Vaccines Attenuated vaccines Killed/inactivated vaccines oral polio (Sabin), MMR Killed/inactivated vaccines rabies, polio (Salk), Influenza Subunit vaccines HIB-Haemophilus influenzae type B Hepatitis B

Some Current Vaccines Attenuated vaccines Killed/inactivated vaccines oral polio (Sabin), MMR Killed/inactivated vaccines rabies, polio (Salk), Influenza Subunit vaccines HIB-Heamophilus influenzae type B Hepatitis B

Injected Vaccines Induce Serum antibodies (Ab) IgM IgG Protect from organisms in the blood and tissues

Why oral vaccine? Some organisms infect via mucosal tract Gut pathogens Respiratory pathogens Ig A First line of defense-at mucosal surfaces inhibit binding of bacteria neutralize toxin

Why oral vaccine? Immunizing in mucosa favors IgA ex: gut or intra-nasal Cross protection of mucosal surfaces Induces systemic immunity Safer compared to injectable vaccines

Problems with oral vaccine Ability to survive digestive enzymes need for encapsulation Large quantity of antigen needed or Need for an appropriate adjuvant 1990’s - development of CT (Cholera toxin) & LT (E. coli heat labile toxin)

Biotech crops 1st generation: 2nd generation: 3rd generation: Input-traits (agronomic benefits) - Bt corn, RR soybean 2nd generation: Output-traits (health and nutritional benefits) - Vit A rice, Lycopene tomato 3rd generation: New industry and pharmaceutical properties - Vaccine corn, plastic or ethanol corn

Farm-aceuticals: why use plant as bioreactor ? Safer Plants carry no common mammalian pathogens Animal viruses – Bovine viral diseases 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

Rationale for using 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

Targeting of protein expression Gene construction Promoter determines site of protein expression Constitutive Callus/Leaves Seed specific Germ Starch storage Natural encapsulation? Ubiquitin promoter 27 kD  -zein promoter

E. coli heat labile toxin (LT): A-B Toxin B=binding A= active toxin http://www.bmsc.washington.edu/WimHol/figures/figs2/Pickens-Moon.JPG

E. coli heat labile toxin (LT): A-B Toxin B=binding Remove A subunit LT-B 82% homologous with CT-B

Rationale for using LT-B Ideal model antigen Strong immunogen Induces strong mucosal and serum responses when orally administered. Potent adjuvant

Immunogenicity of corn expressed LT-B BALB/c mice fed with …. ….corn meal pellets expressing 0.2-20 μg LTB/g corn at days 0, 3, 7, 21.

ELISA to measure Ab Enzyme linked immunosorbent assay LT-B protein from T. plants Ganglioside GM1

ELISA to measure Ab Enzyme linked immunosorbent assay Source of antibodies: serum mucosal (fecal pellets) Mouse Antibodies LT-B protein from T. plants Ganglioside GM1

ELISA to measure Ab Enzyme linked immunosorbent assay Anti-mouse IgG-HRP ENZYME Mouse Antibodies LT-B protein from T. plants Ganglioside GM1

ELISA to measure Ab Enzyme linked immunosorbent assay Anti-mouse IgG-HRP Substrate ENZYME Mouse Antibodies Product LT-B protein from T. plants Ganglioside GM1

ELISA to measure Ab more Ab = more enzyme = more color Anti-mouse IgG-HRP Substrate ENZYME Mouse Antibodies ENZYME Product ENZYME Product LT-B protein from T. plants ENZYME Product Product Ganglioside GM1

Does Corn LT-B induce a serum & mucosal Ab response Sample collection -3 6 13 20 27 34 41 48 Feed 0 7 21 4 mice per group Transgenic groups 20 µg LT-B 2 µg LT-B 0.2 µg LT-B 0.02 µg LT-B Non-transgenic groups Non-transgenic (nt) Collected serum and fecal samples weekly

Effect of CT and LT on gut:carcass ratio of mice Patent mouse assay: Effect of CT and LT on gut:carcass ratio of mice

Antibodies protect from whole toxin Mice challenged with 25 μg LT or CT

Anti-LT-B IgA is produced at distant sites: Lung

Future Promise of LT-B vaccine B=binding Replace A subunit with protein of interest Influenza? SARS? PRRS? ?

Future Promise of LT-B vaccine B=binding Replace A subunit with protein of interest Influenza? SARS? PRRS? GFP

If biotech crops contaminate corn used for food… Will the vaccine still be effective?

Daily vs. intermittent LT-B feeding Affect of exposure on vaccine response Treatments: Intermittent Daily 20 µg LT-B 20 µg LT-B 2 µg LT-B 2 µg LT-B 0.2 µg LT-B 0.2 µg LT-B non-transgenic (nt) non-transgenic (nt) 4 mice were included in each group for a total of 32 mice.

Serum IgG Daily vs. intermittent LT-B feeding

Serum IgG Affect of exposure on vaccine response

Summary Vaccines are important public health initiatives Plant derived vaccines can utilize the best of subunit vaccines, but are safer Transgenic corn LT-B is a potent immunogen Safety considerations need to be examined as vaccine is being developed

Colleagues Kan Wang (ISU, Agronomy) Rachel Chikwamba Sule Karaman April Beyer Sarah Rahn

Opportunities for High School & Middle School Students Microbiology High School Workshop Nov 14, 2008 8:45AM - 3:10PM Joan Cunnick (jcunnick@iastate.edu) Science in Iowa (Middle School; April date) & Visiting Scientist Program (All Grades-throughout year) College of Agriculture Topics change each year Mickie Franks (mjfranks@iastate.edu)