The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

The coat protein (CP) of alfalfa mosaic virus was used as a carrier molecule to express antigenic peptides from rabies virus and HIV. The antigens were separately cloned into the reading frame of alfalfa mosaic virus CP and placed under the control of the subgenomic promoter of tobacco mosaic virus CP in the 30BRz vector. The in vitro transcripts of recombinant virus with sequences encoding the antigenic peptides were synthesized from DNA constructs and used to inoculate tobacco plants. The plant-produced protein (virus particles) was purified and used for immunization of mice. Both antigens elicited specific virus-neutralizing antibodies in immunized mice.Recent studies have shown that plants and plant viruses can be effective tools for antigen production and delivery (1-4). Plants offer several advantages for the production of vaccine antigens and other therapeutic proteins, including lack of contamination with animal pathogens, relative ease of genetic manipulation, eukaryotic protein modification machinery, and economical production. Like liposomes and microcapsules, plant cells and plant viruses are expected to provide natural protection for the passage of antigens through the gastrointestinal tract.A variety of important protein antigens have been expressed in transgenic plants, including hepatitis B surface antigen (1), Escherichia coli heat-labile enterotoxin (2), rabies virus glycoprotein (3), and Norwalk virus capsid protein (4). However, the level of antigenic protein produced by transgenic plants is relatively low, suggesting the need for new approaches to express foreign proteins in plants.One approach is to engineer virus coat proteins (CPs) to function as carrier molecules for fused antigenic peptides. Such carrier proteins may have the potential to self-assemble and form recombinant virus particles displaying the desired antigenic epitopes on their surfaces. A commonly used carrier molecule is the CP of viruses that infect bacteria (5-7), animals (8-10), and plants (11-16). The CP of tobacco mosaic virus (TMV) was among the first plant virus proteins to be used as a carrier molecule for antigenic epitopes from other sources (11). Fitchen et al. (14) and McLain et al. (15) showed that antigens produced in plants as a result of infection with engineered TMV and cowpea mosaic viruses can elicit specific antibodies when injected into mice. A limitation of this approach is the failure of virus assembly when even moderately sized peptides are incorporated into the CP. Only peptides less than 25 amino acids in length have been successfully introduced into the TMV CP. This is a significant limitation for the production of various molecules of biomedical importance using plant viruses. For example, a chimeric peptide antigen that successfully protects mice against rabies virus and is therefore suitable for the use in a plant based vaccine has been described (17). However, this peptide is 38 amino acids long, far in excess of capacity of TMV CP. We have overcome this size limitation by using the ...

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.

No abstract