1. By: Abigail Skorupski

2. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera Vibrio Cholerae

3. Construction of Plant Expression Vector Ppcv701FM4-CTB:SEKDEL -CTB gene fused with ER retention signal SEKDEL -Bacterial luciferase AB fusion gene -NPT-II expression cassette -β-lactamase cassette -Removed Shine-Delgarno

4. Transformation of Potato Plants Leaf explants placed in dish w/Agrobacterium Transformed plant cells moved to selective medium Shoot and root formation Plantlets grown into mature plants

5. Luciferase Activity #1-6 = Kanamycin resistant plants #7-12 = untransformed potato plants A&B = in duplicate C&D = in duplicate Plants #1, 3, 4 chosen

6. Analysis of CTB-SEKDEL fusion gene PCR amplification Gel electrophoresis Plant #4 chosen for Immunoblot analysis

7. Immunoblot Analysis Non-fat dry milk Rabbit anti-cholera antiserum Mouse anti-rabbit IgG conjugated to alkaline phosphatase Chemiluminescent substrate CSPD

8. Quantification of CTB protein ELISA assays Background blocked by BSA Rxn measured in plate luminometer 0.3% = max amt. 1 g = 30-35 µg recombinant CTB protein

9. Oral Immunization of CD-1 Mice 30 µg bacterial CTB 1 g transformed potato tissue 3 g transformed potato tissue chemiluminescent ELISA Neutralization titers

10. Reduction of CT-Induced Diarrhea Immunological protection due to prevention of binding to cellular GM1-ganglioside

11. Human Clinical Trials #TH110-51 potatoes 14 volunteers: 100 g transgenic 50 g transgenic 50 g wildtype

12. 91% = 4-fold rises in IgG anti-LT 73% = neutralization titers > 1:100 50% = 4-fold rises in sIgA

13. Issues with Transgenic Plants Level of expression of foreign gene can vary greatly Oral tolerance Greenpeace

14. Conclusions Edible vaccines = safe and inexpensive Bananas as vaccines

15. References Arakawa T, Chong D, Langridge W. Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nature Biotechnology [serial online]. March 1998;16(3):292-297. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012. Arakawa T, Chong D, Merritt J, Langridge W. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Research [serial online]. November 1997;6(6):403-413. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012. Tacket C, Mason H. A review of oral vaccination with transgenic vegetables. Microbes And Infection / Institut Pasteur [serial online]. August 1999;1(10):777-783. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012. Tacket C, Mason H, Losonsky G, Clements J, Levine M, Arntzen C. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Medicine [serial online]. May 1998;4(5):607-609. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012.