Over two billion people worldwide are infected with hepatitis B, a serious liver infection that can result in jaundice, cirrhosis, and liver cancer. Although an injectable vaccine now exists, its expense and requirement for refrigeration makes it unavailable to more than one-third of the world's population, especially in poor countries where the vaccine is in urgent need. Dr. Charles Arntzen, of Arizona State University, has put the idea of a stable, plant-based vaccine forward as an attractive alternative. Now Dr. Arntzen and his colleagues report encouraging results in their effort to create a stable, edible form of the vaccine in their November 2000, Nature Biotechnology progress report.

Traditional vaccines rely on challenging the body's immune system with a weakened or killed form of a pathogen. The immune system "remembers" the chemical characteristics of the pathogen and responds quickly and effectively when exposed to attack by the fully potent form of the organism. More recently, vaccines consisting of a single component of the pathogen, such as the coat protein of a virus, have been equally effective. The current, conventional hepatitis B vaccine utilizes a single protein, HBsAg, produced in yeast, that, when polymerized correctly, forms a complex mimicking the structure of the actual virus. When injected, this complex triggers the body's immune system and provides protection from the disease.

The current hepatitis vaccine extracted from yeast requires chemical modification to become active, increasing the cost of the vaccine, which also must be stored under refrigeration. This has severely limited its utility in third world regions where the disease in rampant. The situation is further complicated by the need for three separate injections of the vaccine at 0, 1, and 6 months of age.

Edible vaccines have the potential to address many of the problems facing synthesis and distribution of vaccines. Plant-based vaccines can be grown locally, reducing the cost and complications of transportation, while the stability of proteins in intact plants removes the need for refrigeration. Furthermore, the edible nature of the vaccines eliminates the need for syringe-based delivery, saving money and reducing the risk of infections.

Although proof of the principle behind edible vaccines had been demonstrated by several groups in animals, it wasn't until 1997 that Arntzen and his colleagues, in collaboration with the University of Maryland's Department of Medicine, were able to test their ideas in humans(1). A test group of 11 human subjects were fed raw potatoes engineered to express the B subunit of the E. coli toxin. Careful monitoring of the test subjects revealed that 10 of the 11 volunteers displayed a four-fold rise in serum antibodies to the toxin. Furthermore, six of the test subjects also displayed a corresponding rise in intestinal antibodies to the toxin. Further studies by Arntzen and other research groups have shown that systemic immune response is possible in humans using other plant-based vaccines.

Edible vaccines work in a very different manner than traditional vaccines. Since standard vaccines are injected parenterally, rather than administered orally, the detector elements of the immune system, the B and T cells, have direct access to large amounts of antigen. In contrast, orally administered vaccines are detected by components of the immune system known as M cells, which are present in the gut. These cells recognize the ingested antigen and transport it to the B and T cells, which in turn mount the immune response. Researchers, including Arntzen, have found that it takes a much higher dose of the antigen to provoke a full immune response when administered in this manner, possibly due to degradation of the antigen in the gut. Unfortunately, production of vaccine antigens in plants often fails to meet the minimum level required to produce an immune response when administered orally.

To overcome this limitation, Arntzen and his colleagues studied various ways to increase plant production of the hepatitis B antigen, HbsAg, in potato. The results of their efforts, along with further proof of the effectiveness of this form of the vaccine, were reported in the November 2000 issue of Nature Biotechnology(2). One of the main concerns about using a plant-based method of administering a hepatitis vaccine has been that transmission of hepatitis is believed to be non-enteric (not through the digestive tract). Although Arntzen and his group had previously shown that the plant-produced form of HBsAg was capable of initiating an immune response when injected into mice, they still needed to establish that the protein contained in potato tissue could stimulate the same response when fed to mice. The ability of plant-produced HBsAg to trigger an immune response when administered orally had been previously established by Hilary Koprowski and his group at Thomas Jefferson University(3). His studies found that HBsAg produced in lupine and lettuce was able to cause the production of HBsAg-specific antibodies when fed to mice and humans, respectively.

To test the immunogenicity of the potato-generated HBsAg, Arntzen and his fellow researchers used a line of potatoes that had been engineered to express the gene encoding HBsAg from the tuber-specific patatin promoter. This particular line of transgenic potatoes accumulated 1.1 µg of HBsAg per gram of fresh tuber. Mice were fed a total of 16.5 µg HBsAg over a course of three weeks, along with 30 µg (total) of cholera toxin (CT) to act as an adjuvant (general immune stimulant). Unlike the control mice fed only regular tubers and CT, the experimental mice displayed a primary serum antibody response that peaked at 73 mIU/ml three weeks following the last dose. Furthermore, a high-level recall response was observed only in the experimental mice upon injection of a subimmunogenic dose of commercial hepatitis vaccine, indicating that the feeding of transgenic tubers had established immune memory in the mice.

Although these results were encouraging, Arntzen and his fellow workers believed that a stronger immune response could be triggered if they were able to increase the amount of protein produced by the potato plants per gram of tuber. A number of factors influence the level of protein produced in transgenic plants. Modifying the promoter, 5'-untranslated, and 3'-polyadenylation signal regions has, in all cases, modified the expression of a transgene in plants. However, it has also been observed that the level of expression of an introduced gene varies between transgenic individuals carrying the same construct. This is thought to be due, in part, to the location within the genome of transgene insertion (positional effects) as well as the number of copies of the gene that insert (copy number). To account for any variation caused by these last two factors when conducting comparisons between different constructs, Arntzen devised a method of calculating the amount of protein produced per number of transcripts present for each of the expression constructs under study. To further control for any individual effects, data was collected from several individual transgenic plants representing each construct.

In an attempt to increase the expression of HBsAg in potato, Arntzen and his colleagues tested the introduction of a number of signaling peptides and 5'- and 3'-untranslated regions (UTRs) in constructs driven by the nominally constitutive cauliflower mosaic virus (CaMV) 35S promoter. Sequences tested included 5'-UTRs from tobacco etch virus and tobacco mosaic virus, and 3'-UTRs from the soybean vspB and potato pinII genes. After normalizing to transcript levels as described above, Arntzen found that the use of different 5'-UTRs had little effect on expression levels, but the introduction of the vspB and pinII 3'-UTRs increased the amount of HBsAg protein significantly. Unfortunately, the highest expressing lines also exhibited stunted growth and reduced tuber formation, indicating that high levels of the introduced protein may be phytotoxic. Interestingly, the introduction of endoplasmic reticulum (ER) retention signals also increased the amount of HBsAg protein per unit mRNA. The authors theorized that retention of protein in ER might lead to increased subunit interaction, stabilizing the protein and protecting it from degradation.

The results reported by Arntzen and his group represent encouraging support of the concept of plant-delivered vaccines. However, there remain significant obstacles before an edible hepatitis B vaccine becomes a reality. The possible phytotoxicity of hepatitis antigen to the plant production source will have to be overcome. It may be possible to work around this obstacle through the use of adjuvant, possibly also expressed within the plant. This may make it possible for the body to detect and respond to a lower dose of antigen. As the research advances, the issue of dosage control will become significant—just as with standard vaccines, it will be critical to ensure that patients do not receive too much or too little of the antigen. On the more aesthetic side, it may be necessary to find a more palatable form of the vaccine, as the taste of raw potato is not particularly pleasing to many. Efforts are already underway to make banana-based edible vaccines. However, any form of stable, edible vaccine would be welcomed in many parts of the world. With the results of his recent paper, Dr. Arntzen has taken this vision one step closer to reality.

Sources:

1. Langridge WHR. 2000. Edible vaccines. Scientific American 283: 66-71.

2. Richter LJ, Thanavala Y, Arntzen CJ, and Mason HS. 2000. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nature Biotechnology 18: 1167-1171.

3. Kapusta J, et al. 1999. A plant-derived edible vaccine against hepatitis B virus. FASEB Journal 13:1796-1799.

Claire Granger
Biologist
alesia_sun@yahoo.com

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