Abstract

The B subunit of cholera toxin (CTB) functions as an effective mucosal carrier molecule of chemically or genetically conjugated antigens. Orally delivered CTB or CTB fusion protein binds to GM1-ganglioside receptor molecules located on cells in the gutassociated lymphoid tissues, the site of immunological induction. We have generated transgenic plants synthesizing CTB conjugated with vaccine antigens for food plant-based prevention of cholera and rotavirus toxin-induced diarrhea and for autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM).

Mice fed with transgenic potato tubers synthesizing 0.3% of CTB as total tuber protein generated serum and mucosal antibodies with toxin neutralizing capability. The immunized mice were partially protected from diarrhea induced by intraileal inoculation of cholera holotoxin. A 22-amino acid rotavirus enterotoxin NSP4 epitope was fused at the C-terminus of the CTB subunit. Transgenic potato tubers synthesized up to 0.1% of total soluble protein as the pentameric CTB-NSP4 fusion peptide.

To prevent IDDM, we generated transgenic potato plants synthesizing the IDDM autoantigens, insulin and glutamic acid decarboxylase and their conjugate proteins with CTB. Transgenic potato plants synthesizing the fusion proteins significantly reduced pancreatic inflammation (insulitis) and clinical diabetes when fed to nonobese diabetic mice. Potato tissues producing comparable amounts of insulin or glutamic acid decarboxylase alone conferred only low levels of protection against insulitis, suggesting that conjugation with CTB markedly increases the tolerogenicity of autoantigens.

Since autoimmune diabetes is often linked to the consumption of bovine milk and its products, we took a novel approach to prevention of DDDM in non-breast fed infants and children by producing transgenic food plants synthesizing human milk proteins. We have synthesized human p-casein and lactoferrin in food plants as these are prototypical casein and whey proteins in human milk. In addition to nutritional enhancement, lactoferrin may provide antimicrobial activity against a variety of enteropathogens including rotavirus. Construction of food plants synthesizing vaccine antigens as well as antimicrobial human milk proteins may be a safe, inexpensive, and convenient method for the prevention of infectious and autoimmune diseases for people living in developing countries.

LLU Discipline

Microbiology

Department

Microbiology

School

Graduate School

First Advisor

William H. R. Langridge

Second Advisor

Alan P. Escher

Third Advisor

Istvan Fodor

Fourth Advisor

Benjamin H. S. Lau

Fifth Advisor

Giuseppe Molinaro

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1998

Date (Title Page)

6-1998

Language

English

Library of Congress/MESH Subject Headings

Cholera Toxin -- genetics; Vibrio cholerae -- immunology; Plants, Transgenic -- pharmacology; Glutamic Acid -- metabolism; Rotovirus; Plant Proteins -- genetic; Adjuvants, Immunologic -- physiology.

Type

Dissertation

Page Count

x; 288

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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