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Department of Natural Resources & Environmental Sciences, 310 ERML (S.S.K., S.F.K.), University of Illinois, Urbana, Illinois
Department of Molecular and Integrative Physiology (D.E.B.), University of Illinois, Urbana, Illinois
Address reprint requests to: Schuyler S. Korban, PhD, Department of Natural Resources & Environmental Sciences, 310 ERML University of Illinois, Urbana, IL 61801. E-mail: s-korban{at}uiuc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONDESCRIPTION OF SUBJECTCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Key words: food crops, transgenic plants, plant-based vaccines, oral edible vaccines
Key teaching points:
• Via genetic engineering and molecular biology, genes encoding immunogenic proteins of an infectious agent can be transferred into the nuclear genome of a plant system such that the plant is capable of producing the desired immunogenic protein subunit vaccines.
• The production of antigens in genetically engineered plants provides an inexpensive source of edible vaccines and, in turn, increases the value of plants as novel sources of medicinal drugs.
• Edible vaccines against cholera toxin B subunit, hepatitis B surface antigen, E. coli heat labile enterotoxin, and Norwalk virus capsid protein have been developed and tested for efficacy in animal and human trials. Immunologic responses were provoked in both mice and humans.
• A plant-based oral subunit vaccine for the respiratory syncytial virus has been developed. This virus is a serious pathogen that causes bronchiolitis and pneumonia-type diseases in all human age groups and is a leading cause of viral lower respiratory tract illness in infants and children worldwide.
• This new area of agriculture, referred to as "biopharming," will provide new value to crops grown for the sole purpose of producing and/or delivering biopharmaceutical or medicinal products.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONDESCRIPTION OF SUBJECTCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Plants provide a very attractive alternative that can be scaled up to a high production system for recombinant proteins [2,3,4]. Developing transgenic plants that can express these antigenic proteins will eliminate the need for fermentation facilities and overcome the possible risks of contamination of subunit vaccines by unknown mammalian pathogens that may remain undetected in these cultures. Pathogens that infect plants do not infect humans, whereas mammalian pathogens can infect human and other animal populations. Moreover, to increase production of a subunit vaccine in plants will only require planting more acreage of these value-added plants. If an "edible" plant is used to produce a vaccine, for example, purification to remove host toxins should not be necessary. Also, any required processing of an "edible vaccine," in the form of juice, powder or sauce, would be less complicated and easier than purification.
In recent years, development of plant-based vaccines directed at human and animal diseases [2–5] has opened up an innovative and exciting opportunity for adding new high value to food crops, thus increasing the uses and profitability of these crops. With the tools of genetic engineering and molecular biology, genes encoding immunogenic proteins of an infectious agent are transferred into the nuclear genome of a plant system via genetic transformation protocols, and these transgenic plants are then capable of producing the desired immunogenic protein subunit vaccines. The production of antigens in genetically engineered plants provides an inexpensive source of edible vaccines and, in turn, increases the value of plants as novel sources of medicinal drugs. This new field of biological science, referred to as molecular "biopharming" has received much attention in the past decade and promises to become more important in the next decade.
Background
Thus far, there are several reports on development of transgenic plants that express antigenic proteins of pathogenic human and animal organisms [2–4]. In early work, tobacco was used as a model plant system for expression of antigenic proteins, such as that for hepatitis B surface antigen (HBsAg) [6]. This early study showed that the plant-derived protein had similar buoyant density and antigenicity as do human- and yeast-derived HBsAg, suggesting that the protein assumed typical folding characteristics in the plant. This was followed by a mice immunization study with tobacco-derived recombinant HBsAg (rHBsAg) that demonstrated stimulation of T-cell proliferation. In a later study, it was conclusively demonstrated that B- and T-cell epitopes of HBsAg were preserved when the antigen was expressed in transgenic tobacco [7]. In another effort, constructs carrying the gene encoding the binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) were introduced into tobacco and potato plants [8]. Heat labile enterotoxin is produced by enterotoxigenic E. coli (ETEC), the causal agent of an enteric disease, and also immunogenically interacts with the cholera toxin of Vibrio cholerae. Thus, LT-B is a candidate vaccine against both ETEC and cholera. Transgenic plants expressing the LT-B protein or a modified LT-B protein with a microsomal retention sequence have been developed. The toxin protein was expressed in potato microtubers and at much higher levels in plants containing the fusion protein. Mice immunized by orally feeding with transgenic potato tubers (expressing the recombinant antigen) developed serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) that were specific for LT-B [8].
The potato has since been used for production of antigenic proteins against various pathogens and/or diseases. The cholera toxin B subunit (CTB) against Vibrio cholerae was successfully expressed in potato tubers and found to accumulate at high enough levels that induced both mucosal and serum immune responses in mice [9]. The potato was also used for the production and delivery of the human insulin antigen [9]. Mason et al. [10] optimized the LT-B enterotoxin antigen and expressed it in potato tubers as well. Later, Tacket et al. [11] conducted the first human clinical study where they demonstrated that humans given a plant-derived oral vaccine (fed raw transgenic potato tubers carrying the recombinant LT-B antigen) produced both serum IgG- and mucosal IgA-specific antibodies in humans. The capsid protein of the Norwalk virus was also expressed in potato tubers and found to be immunogenic in test mice as well [12]. Again, a human clinical trial was conducted by feeding 24 healthy adult volunteers two or three doses of these potato tubers and found that 19 of 20 volunteers fed the transgenic potato (carrying the capsid protein of the Norwalk virus) developed an immune response, although the level of serum antibody increases was reported to be modest [13].
In a recent study, Yu and Langridge [14] produced the first multicomponent vaccine in potato against three enteric diseases including cholera, rotavirus and ETEC. Although the amount of the recombinant fusion proteins used in their immunological studies was about 3.3 µg/g potato tuber, mice fed these tubers produced both serum and mucosal antibodies against the three human diseases.
Among other food crops used for the development of plant-based vaccines, both lupine and lettuce were used to express a hepatitis B surface antigen (HbsAg) either in the pods or leaves, respectively, and these tissues were found to be useful systems for production and delivery of this antigen vaccine [15]. Soybean was used for production of the glycoprotein B antibody of the herpes simplex virus 2 (HSV-2) [16], while corn was used for production of an LT-B subunit vaccine [17]. Recently, Stöger et al. [18] expressed the single-chain Fv (ScFv) antibody of the human carcinoembryonic antigen (CEA), a marker antigen to diagnose tumor onset, in both rice and wheat grains. While, Mor et al. [19] used tomato to express the human acetylcholinesterase (AChE) that provides protection against organophosphate poisoning.
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DESCRIPTION OF SUBJECT |
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RSV disease occurs throughout the world and is more severe in underdeveloped countries where it results in increased mortality. In adults, it usually takes the form of a "common cold," but can be more severe. In recent years, RSV infections have been increasingly noted in nursing homes and in other group settings serving the institutionalized elderly [22]. The virus enters the human body mainly through the nose and eyes, but also through the mouth [20]. In the northern hemisphere, the yearly peak season for RSV infections occurs from December through March and, in urban centers, the virus is detected from the Fall through the Spring [20].
There is no consistently effective treatment available for RSV infections, and these infections can occur repeatedly in the same individual. A major difficulty in developing an RSV vaccine that works via the serum immune system has been the fact that natural infection confers, at most, only temporary protection against reinfections [23]. Another problem is that the mechanism by which even partial immunity to RSV develops is not well understood [23].
Although our ultimate goal is to develop this RSV edible oral vaccine in the apple, we elected to use the tomato first for the production and delivery of such a vaccine. The tomato is one of the most important vegetable crops of commercial importance in the U.S. USDA figures report that tomato continues to show an increase in planting acreage (fresh market acreage for 1999 was up 15% over 1998 and 13% greater than 1997) and a rise in production (1999 production was 23% higher than 1998 and 16% above 1997) (USDA Agricultural Statistic Board, NASS, USDA, Sept. 1999). Developing new uses for tomato (by creating a specialty crop) will expand the market for this important food crop and provide a competitive advantage to U.S.-produced tomato specialty crops.
In our first set of experiments, we developed various plant expression constructs carrying the RSV-F antigenic protein gene. These constructs consisted of the universal constitutive cauliflower mosaic virus (CaMV) 35S promoter along with enhancer elements or leader sequences added to the RSV-F protein gene. The CaMV 35S-driven gene constructs carrying the antigenic F-protein were transformed into apple leaf protoplasts to determine whether the F-protein could be correctly processed in a plant cell. Using SDS-PAGE and Western analyses, the correct size protein (68 kd) was found in transformed apple protoplasts [24]. Upon immunoblotting with a monoclonal antibody against the F-protein, it was confirmed that the F protein was expressed in apple. Furthermore, the level of expression of the F-protein increased in constructs carrying the leader sequence AMV RNA4 and the P268 enhancer element [24]. This suggested that we could successfully enhance the level of expression of the F-protein in transgenic plants by using these newly designed gene constructs developed in our laboratory.
Then, gene constructs (one containing the constitutive CaMV 35S promoter and the other containing the fruit-specific E-8 promoter) carrying the antigenic F protein also have been transferred into tomato using Agrobacterium-mediated transformation, and transgenic tomato plants have been recovered. The presence of the transgene was confirmed in 71 out of 74 tomato plants growing in the greenhouse using Southern blotting (Fig. 1). The plants appeared normal and were not stunted in growth. All plants were fertile (Fig. 2), and harvested fruit was analyzed using ELISA. With the E-promoter, the F-protein was expressed only in the fruit of all transgenic plants carrying the F-antigenic protein gene. Variability in the expression level of the F-protein among plants (even between and within each of the two constructs) was observed, ranging from 9.0 to 32.5 µg/g of fruit fresh weight (Fig. 3). Fruits from the highest F-protein expressing plants (transgenic line #120 with 32.5 µg/g of fruit fresh weight in plants transformed with the E-8 promoter construct) were fed to mice, and among 25 orally immunized mice, 22 showed significant immune responses and produced anti-RSV-F antibodies [25]. Pre-immunized and control mice did not produce detectable anti-RSV-F antibodies. This demonstrated successful oral immunization of mice and showed the fruit-derived RSV-F was active as an oral immunogen. T1 plants from the two highest F-protein expressing T0 lines were later grown in the greenhouse, and the expected 3:1 segregation ratio was observed for presence/absence of the transgene (following Southern blot hybridization), suggesting that the F-protein transgene is transmitted to its progeny and that it is stably inherited in a Mendelian fashion in the progeny.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONDESCRIPTION OF SUBJECTCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Plant-based vaccines will provide a new use for food crops as these crops can then be grown for the sole purpose of producing and/or delivering biopharmaceutical or medicinal products. This leads to a new area of agriculture, now referred to as "biopharming," whereby agricultural crops with added-value are grown in specialized areas and specifically used as "factories" for production and delivery of edible vaccines and/or other antimicrobial agents. Nevertheless, there are some limitations to these recombinant antigens produced in plants that should be overcome before this technology can make it to the commercial sector. Among these limitations is the need to achieve high enough yield of the recombinant antigen in the plant tissue, and another limitation is dealing with the presence of plant-specific glycans that might alter the properties of the recombinant protein [26]. Research efforts to alleviate both of these limitations are currently underway in various laboratories.
The use of foods as vehicles for production and delivery of human vaccines is an exciting and novel field of biotechnology and should pay dividends for both human health and the agricultural sector in the near future.
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ACKNOWLEDGMENTS |
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REFERENCES |
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This article has been cited by other articles:
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  J. Benyacoub, F. Rochat, K.-Y. Saudan, I. Rochat, N. Antille, C. Cherbut, T. von der Weid, E. J. Schiffrin, and S. Blum Feeding a Diet Containing a Fructooligosaccharide Mix Can Enhance Salmonella Vaccine Efficacy in Mice J. Nutr., January 1, 2008; 138(1): 123 - 129. [Abstract] [Full Text] [PDF]
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