A Potato a Day Keeps the Cholera Away

By: Abigail Skorupski

Background Info What is cholera?

Vibrio Cholerae

Symptoms Cholera Toxin

A subunitB subunit

Vaccination

Countries Affected by Cholera

Vibrio Cholerae

Construction of Plant Expression VectorPpcv701FM4-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

Transformation of Potato PlantsLeaf explants placed in dish w/AgrobacteriumTransformed plant cells moved to selective

mediumShoot and root formationPlantlets grown into mature plants

Luciferase Activity#1-6 = Kanamycin

resistant plants#7-12 =

untransformed potato plants

A&B = in duplicateC&D = in duplicate

Plants #1, 3, 4 chosen

Analysis of CTB-SEKDEL fusion gene

PCR amplification

Gel electrophoresis

Plant #4 chosen for Immunoblot analysis

Immunoblot AnalysisNon-fat dry milkRabbit anti-cholera

antiserumMouse anti-rabbit

IgG conjugated to alkaline phosphatase

Chemiluminescent substrate CSPD

Quantification of CTB proteinELISA assaysBackground blocked

by BSARxn measured in

plate luminometer0.3% = max amt.1 g = 30-35 µg

recombinant CTB protein

Oral Immunization of CD-1 Mice30 µg bacterial CTB1 g transformed potato tissue3 g transformed potato tissuechemiluminescent ELISANeutralization titers

Reduction of CT-Induced Diarrhea

Immunological protection due to prevention of binding to cellular GM1-ganglioside

Human Clinical Trials#TH110-51

potatoes14 volunteers:

100 g transgenic

50 g transgenic50 g wildtype

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

Issues with Transgenic PlantsLevel of expression of

foreign gene can vary greatly

Oral toleranceGreenpeace

ConclusionsEdible vaccines = safe and inexpensiveBananas as vaccines

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.