A Potato a Day Keeps the Cholera Away

A Potato a Day Keeps the Cholera Away By: Abigail Skorupski

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

Construction of Plant Expression Vector Ppcv 701 FM 4 -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 Plants �Leaf explants placed in dish w/Agrobacterium �Transformed plant cells moved to selective medium �Shoot and root formation �Plantlets grown into mature plants

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

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

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

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

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

Reduction of CT-Induced Diarrhea Immunological protection due to prevention of binding to cellular GM 1 -ganglioside

Human Clinical Trials �#TH 110 -51 potatoes � 14 volunteers: � 100 g transgenic � 50 g wildtype

• 91% = 4 -fold rises in Ig. G anti-LT • 73% = neutralization titers > 1: 100 • 50% = 4 -fold rises in s. Ig. A

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

Conclusions �Edible vaccines = safe and inexpensive �Bananas as vaccines

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