Preliminary Studies Mason, Arntzen and colleagues have conducted fusion protein engineering studies using transgenic plants, with a goal of designing mucosally targeted antigens. Much of this effort has been directed to the use of either enterotoxigenic E. coli (ETEC) toxin's binding subunit (LIB) or the analogous binding subunit of the Cholera toxin (CTB), which assemble pentamers that bind to ganglioside receptors on mucosal cell surfaces. Oral immunization with plant-derived LIB induced strong systemic and mucosal antibody responses and afforded protection to LT challenge in mice [103, 124] and a strong immune response in humans ([125]). Because of its unique mucosal-targeting property, LIB has been widely used as a carrier to present small peptides or epitopes (which are often poorly immunogenic when administered alone) to develop mucosal vaccines. Members of this center have explored the use of both CTB and LIB as mucosal targeting agents for antigens of differing sizes. The CTB gene was linked to the coding sequence of the 35 aa P1 peptide of HIV-1 MN isolate (Genbank AF075722) and an ER retention signal (SEKDEL). This P1 peptide,, the conserved GalCer binding domain of the HIV-1 gp41 envelope protein, which mediates the transcytosis of HIV-1 across the mucosal epithelia, had been very difficult to express in other systems [126]. Chimeric protein expressed in bacteria or plants assembled into oligomers that were capable of binding GalCer and GMi gangliosides. Mucosal (i.n.) administration in mice of the purified chimeric protein followed by an i.p. boost resulted in transcytosis-blocking serum IgG and mucosal IgA responses and induced immunological memory. Plant-derived HBsAg is correctly folded to form the 'a'antigenic determinant and assemble virus-like particles (VLPs), and was found to be orally immunogenic. We have also explored the use a fusion between LTB and the HBsAg middle protein (MHBs). The plantexpressed LTB:MHBs fusion accumulated as full-length product, retained ganglioside-binding activity, formed HBsAg 'a'determinant and, more importantly, co-sedimented with yeast-derived HBsAg VLPs. Immunization of mice with the LTB:MHBs fusion stimulated the production of serum antibodies against both LTB and MHBs, indicating that the fusion is a bivalent immunogen. Our results demonstrate that HBsAg fusions with extended foreign protein segments can be produced in plants without altering their major antigenic and immunogenic properties, and thus open a way for the development of multicomponent HBsAg vaccines. Moreover, fusion of a large (280 aa HBsAg M protein) polypeptide to LT-B still allowed assembly of ganglioside-binding oligomers by the LT-B subunits, even thought the fusion protein was embedded in the membrane of a VLP.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program--Cooperative Agreements (U19)
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Arizona State University-Tempe Campus
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Diamos, Andrew G; Rosenthal, Sun H; Mason, Hugh S (2016) 5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves. Front Plant Sci 7:200
Chen, Qiang; He, Junyun; Phoolcharoen, Waranyoo et al. (2011) Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin 7:331-8
Huang, Zhong; Phoolcharoen, Waranyoo; Lai, Huafang et al. (2010) High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng 106:9-17