Jie Yu

Abstract

To generate a plant-based multicomponent oral vaccine against cholera, rotavirus and enterotoxigenic E.coli (ETEC), we have constructed transgenic potato plants that synthesize the cholera toxin B and A2 subunits, the rotavirus enterotoxin NSP4 22 amino acid epitope and the ETEC fimbrial antigen CFA/L To use cholera toxin as a carrier molecule for delivery of multiple antigens to the gut-associated lymphoid tissues, the rotavirus NSP4 epitope was linked to the cholera toxin B subunit (CTB) and the ETEC fimbrial antigen CFA/I was linked to the cholera toxin A2 subunit. Cholera toxin-antigen fusion proteins were detected in transformed tuber tissues by immunoblot assay and quantitated by enzyme linked immunosorbent assay (ELISA). The CTB-NSP4 protein expression level was found to be approximately 0.01% of total soluble plant protein (TSP) and the CFA/I-CTA2 protein expression level was about 0.002% of TSP. Assembly of the CFA/I-CTA2 subunit into the CTBNSP4 pentamer was detected by immunoblot and Gmi ELISA assays. The CFA/ICTA2- CTB-NSP4 hetero-hexamer fusion protein complex retained its binding affinity for Gmi ganglioside enterocyte membrane receptors. Serum and intestinal antibodies against NSP4, CFA/I and CTB were detected in orally immunized mice. Analysis of IL-2, IL-4 and IFNy cytokine levels in splenocytes isolated from immunized mice indicated the presence of a strong Thl immune response to the plant synthesized antigens. Fluorescent antibody based flow cytometry analysis of immunized mouse spleen cells showed an increase in CD4+ T -helper cell populations. Following rotavirus challenge, passively immunized mouse pups showed a 50% reduction in diarrhea symptoms.

To generate a plant-based subunit vaccine against rotavirus, we have expressed the gene encoding rotavirus capsid protein VP6 in transgenic potato plants. The plant produced VP6 protein has a molecular mass similar to VP6 purified from virus and partially formed VP6 trimers. Mice fed transformed potato tubers containing VP6 generated serum and intestinal antibodies against VP6. The results of these experiments indicate that food plants can serve as safe and inexpensive vaccine production and distribution systems for simultaneous immunization against several diseases.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

William H. R. Langridge

Second Advisor

Alan P. Escher

Third Advisor

Istvan Fodor

Fourth Advisor

E. Clifford Herrmann

Fifth Advisor

Charles W. Slattery

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2001

Date (Title Page)

3-2001

Language

English

Library of Congress/MESH Subject Headings

Enterobacteriaceae; Bacterial Vaccines.

Type

Dissertation

Page Count

xii; 169

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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Biochemistry Commons

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