TRENDS PREVIEW 3

Transgenic Plants for Vaccine Production
Expectations and Limitations

by Daniel Chargelegue, Patricia Obregon and Pascal M.W. Drake

 Feature Twelve
This article will appear in a forthcoming issue of Trends in Plant Science.

Posted October 12, 2001 · Issue 112

Abstract

The expression of antigens in vegetables and fruits has opened up a new avenue for the development of oral vaccines. In a recent report, an oral multi-component vaccine comprising a viral and a bacterial antigen has been successfully designed and has shown protection in mice. Even though there are still improvements to be made in the areas of expression levels and glycosylation, this is a promising technology for the future and for vaccine production.

In the 21st century, there is still an urgent need for affordable and reliable vaccines; the cost of traditional vaccines (production, maintenance and delivery) are often too high for them to be distributed widely in developing countries. The expression of recombinant proteins in transgenic plants, suggests that inexpensive vaccines could be produced directly "on site." Data have been compiled recently on the development of oral vaccines using transgenic edible plants. Indeed, in the past two years, heat-labile toxin B subunit of E. coli (LTB) [1], Hepatitis B surface antigen [2], respiratory syncytial virus F protein [3], measles virus haemagglutinin [4] and norwalk virus capsid protein [5] have been successfully expressed in plants and delivered orally in animals or humans to determine their immunogenicity. A further appeal of this technology is the possibility of developing an oral vaccine against several pathogens in "one plant," as described recently by Jie Yu and William Langridge [6].

Chimeric Construct

Yu and Langridge expressed a multi-component oral vaccine against acute gastro-enteric diseases, the second major cause of death worldwide after acute respiratory diseases. Cholera toxin subunits A2 (CTA2) and B (CTB) were used as oral adjuvants in the design of a chimeric construct containing two antigens. (Adjuvants increase the immunogenicity of the antigens.) Cholera toxin consists of one A subunit, which contains the active toxin domain (A1) as well as a short sequence (A2) that links the A subunit to five B subunits. Yu and Langridge successfully designed a chimeric construct consisting of a rotavirus immunodominant epitope (NSP4) fused to CTB and an enterotoxigenic E. coli antigen (CFA/I) fused to CTA2. The fusion antigens were synthesized in transformed potato tuber tissues, and assembled into a cholera holotoxin-like structure. Mice were fed five times with 3 g of potato tubers (containing on average 10 µg of the chimeric construct) over 56 days. Oral immunization with this construct induced systemic and mucosal antibody responses against CTB, NSP4 and CFA/I. The authors suggested that the presence of NSP4 antigen induced a specific T helper 1 cell-"type" response that is highly desirable for this type of vaccine. Following challenge with rotavirus, passively immunized mouse pups (by mothers orally immunized with the chimeric construct) were significantly protected against diarrhoea, demonstrating an antibody-mediated protection. It was disappointing that the protective activity of the vaccine against enterotoxigenic E. coli and cholera was not reported as well. Nevertheless this report emphasizes the prospect of developing an oral vaccine against several pathogens.

Limitations

One of the major limitations of the expression of recombinant antigens in transgenic plants remains the achievement of a high yield that is sufficient to confer total protection in humans. Although antibodies can be expressed with yields reaching 8% of total soluble protein [7], the level of expression achieved for other proteins can be as low as 0.01% and rarely exceeds 0.40% of total soluble proteins [8]. Variation in yield between plants might be another concern: as seen in earlier work [5], 150 g of potato can contain between 215 and 751 µg of recombinant proteins. In the present study by Yu and Langbridge [6], although the oral vaccine induced protective activity, 27% of passively immunized animals were not protected three days after challenge with rotavirus (at the peak of diarrhoea events). This result might be explained by poor expression yield and/or yield variation between tuber tissues. Therefore, before human trials can be performed, tight control of expression yield needs to be achieved to reduce variability between plants.

A dramatic increase in the recombinant protein yield in plants can be achieved using chloroplast transformation. Using this approach, two recent reports have described yield of 7% and 46% for human somatostatin [9] and Bt toxin [10], respectively. This method could easily be applied to bacterial antigen expression, but is not suitable for the production of glycoproteins (e.g. viral surface antigens). Indeed, proteins synthesized in chloroplasts do not go through the endoplasmic reticulum and Golgi (where the N-glycosylation of proteins occurs). Therefore, an effort should be made to find other means of improving the expression yield of glycoproteins in nuclear transformed plants.

Plant-specific Glycans

Another major concern about glycoprotein expression in plants is the presence of plant-specific glycans (e.g. α1-3 fucose and β1-2 xylose residues) that might alter the properties of the recombinant protein. Strategies to humanise plant N-glycans have been developed recently, including inhibition of endogenous Golgi glycosyl-transferases or addition of mammalian glycosyl-transferases11. Interestingly, expression of an antibody displaying mammalian-type glycans has been achieved in transgenic tobacco plants by stable co-expression of human β 1-4 galactosyl-transferase [12]. Although it has been shown previously that a recombinant murine monoclonal antibody displaying plant complex glycans is not immunogenic in mice [13]; this technology would be useful for producing glycoproteins that require mammalian-type glycans for their activity or antigenicity (e.g. viral glycoproteins).

Conclusions

The number of medically relevant molecules produced in transgenic plants is increasing exponentially, from recombinant antibodies to oral vaccines. However, the type of oral vaccine described by Yu and Langridge [6] will depend on the licensing of oral adjuvants such as CTB and CTA2 for human use.

There are still improvements to be made in areas such as level of expression and glycosylation. The potentially low cost of production and scale-up to agricultural levels that plants promise, should provide a source for antibodies, vaccines and therapeutic molecules for the population of the whole world.
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Endlinks

Medical Molecular Farming: Production of Antibodies, Biopharmaceuticals and Edible Vaccines in Plants - discusses recent developments and possible environmental concerns. From Trends in Plant Science, 2001, 6:5:219-226. Full text available from BioMedNet.

Plants for Delivery of Edible Vaccines - highlights recent advances. From Current Opinion in Biotechnology, 2000, 11:126-129. Full text available from BioMedNet.

Producing Proteins in Transgenic Plants and Animals - reviews the most recent advances in technologies. From Current Opinion in Biotechnology, 2001, 12:4:411-418. Full text available from BioMedNet.

The Regulation of Biologic Products Derived From Bioengineered Plants - highlight the points of greatest concern for the use of biologics made in bioengineered plants. From Current Opinion in Biotechnology, 2001, 12:3:308-311. Full text available from BioMedNet.

Edible Vaccines - an in-depth discussion from the September, 2000 issue of Scientific American.

Edible Vaccines - an outline of principles, plus a bibliography of research papers.

Harvest of Fear - a Frontline/Nova interview with Charles Arntzen concerning his work on edible vaccines and genetically modified foods.

Gene Transfer in Space and Needle-Free Vaccines: Success of Edible Vaccine May Depend on Picking Right Fruit - two recent articles from The Scientist.

Eating Away Disease - a news article from ABCNews.com.

World Health Organization: Vaccines - information on all major vaccine-preventable diseases, dealing with the disease itself, the vaccine, and research and policy for each. The 1999 Report of the Overview of Vaccine Research at WHO and UNAIDS includes a section on edible vaccines under the section on exploratory vaccine research.

Transgenic Plants - contains an introduction and resource guide. From Colorado State University.

Transgenic Plants and World Agriculture - an online report from the National Academy Press.

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