Vaccines are for dinner

Transgenic plants have been sought not only as bioreactors but also as potential scaffolds for oral vaccines. Tobacco was initially exploited for the successful expression of Streptococcus mutans surface protein A as a potential dental caries vaccine (1) and for hepatitis B surface antigen (2) as a vaccine bioreactor alternative for viral hepatitis B. Today, a number of edible plants (Table 1), including potatoes, tomatoes, maize, and soybeans, have been genetically modified to express a variety of vaccine targets, including hepatitis B surface antigen (2), Norwalk virus particles (3, 4), heat-labile enterotoxin B subunit (5, 6), and others (7–11) that can benefit both humans (6–8) and livestock (9–11). The advantage of edible vaccines is that often the plant products, whether leaves, fruit, or seed, can be readily consumed with limited or no processing. Viable oral platforms such as edible products suitable for human use are in demand to deliver vaccines (12). Obviously, the fact that food products are consumed obviates many of the health concerns that arise in the oral vaccination of humans (as reviewed in ref. 13). Edible vaccines can also have the advantage of circumventing cold-storage issues (the “cold chain”), because plant tissues can be dried or, as when the seeds are targeted, have low moisture content. Likewise, water- or oil-based plant extracts can provide additional storage convenience. In this issue of PNAS, Nochi et al. (14) describe a rice-based oral vaccine that potentially addresses many of these topics. At hand is the need for vaccine development and strategies to aid underserved nations with the ability to produce vaccines locally in a cost-effective manner. Because rice is produced in many such areas, this current work shows the feasibility of propagating rice-based vaccines that are truly edible vaccines, unlike in the earlier work with tobacco that ultimately provided the mechanisms for edible vaccine development (1, 2). In addition, this approach breaks the cold-chain barrier that for many conventional vaccines drives up cost and creates storage problems. In fact, it is estimated that removal of this barrier could give an added benefit of as much as $300 million per year, which could provide vaccines for an additional 10 million children (15). In this regard, the work by Nochi et al. shows that their transgenic rice was stable for >18 months at room temperature or at 4°C. Because these vaccines are needle-free (16), they have the added advantage of eliminating the associated waste and potential for dissemination of bloodborne infections. The propagation of transgenic rice also has a lesser impact on the environment because of rice's limited pollen scattering, unlike with maize or wheat (13). Thus, Nochi et al. have addressed many of the issues that prevent vaccines from coming to market, including cost, cold-chain, needle-associated, and regulatory concerns.

Table 1.

Edible transgenic plant vaccines

Vaccine	Edible plant	Ref.
Norwalk virus particle	Potato	3
	Tomato	4
Heat-labile enterotoxin B subunit	Potato	5
	Maize	6
	Soybean	20
Cholera toxin B subunit	Rice	14
	Potato	21
Enterotoxigenic Escherichia coli fimbrial subunit	Soybean	11
Japanese cedar pollen peptide	Rice	19

The authors' examination of the type of vaccine produced is also a significant achievement. A major obstacle to furthering the field of edible vaccines is the need to produce effective immunity. Typically, vaccines applied to mucosal surfaces in the absence of adjuvant fail to stimulate an immunogenic response and resort to the default pathway, or tolerance (as reviewed in ref. 12). Obviously, we need to be tolerant of food; the genetic modification of foods may be potentially problematic and could result in food allergies, thus jeopardizing future consumption of the unmodified food in the diet. For rice, a major worldwide staple, this would be especially problematic. Nochi et al. (14) nicely show that oral immunization with the transgenic rice encoding the cholera toxin B subunit (CTB) does not stimulate a serum IgG response against rice storage protein or, presumably, a secretory IgA response. This will be a key element in the success of transgenic rice. Moreover, they show that the encoded CTB is resistant to the gut environment because of its expression in endosperm that normally is resistant to gastrointestinal digestion. In overcoming these obstacles, Nochi et al. show modest fecal IgA and serum anti-CTB antibody responses after the mice were orally immunized with the equivalent of 75–150 μg of rice-generated CTB per dose, given six times over a 10-week vaccination schedule. When compared with CTB rice-immunized mice, the mice orally dosed with recombinant CTB showed equivalent serum IgG antibody responses but weak fecal IgA responses. This latter finding is surprising, but the authors did show protection against native cholera toxin challenge, as evidenced by reduced intestinal water content (diarrhea). This level of protection was equivalent to that in mice given the rice CTB, whereas mice given native rice or PBS were not protected.

The selection of CTB as a candidate vaccine for testing in rice is appropriate because of its adjuvant properties (17). Nochi et al. (14) were able to show that the rice CTB could enter via the gut sampling cells or M cells (18), which are responsible for continually sampling the gut contents for aberrant antigens. A number of pathogens can also enter via these cells to cause infection, thus subverting this defensive mechanism. A major obstacle for successful oral transgenic plant vaccines is the potential outcome of tolerization rather than immunity, but this is true with any oral vaccine given in the absence of mucosal adjuvant. Tolerance, or lack of responsiveness, occurs when a particular antigen or vaccine is fed without costimulation of the innate immune system. Consequently, instead of being immunized, the host becomes actively unresponsive, and subsequent challenge with the antigen leaves the host unresponsive. Taking advantage of this known behavior, Takagi et al. (19) produced transgenic rice that encoded a fusion protein between soybean seed-storage protein glycinin AlaB1b and known allergen peptides from Japanese cedar pollen for targeted expression in the rice seed endosperm. When mice were fed with this transgenic rice expressing the pollen peptides known to be reactive for T cells, they were resistant to Japanese cedar pollen challenge, showing reduced serum histamine release, reduced allergic IgE antibodies, and reduced Th2 cells that support the allergic response. The authors also showed that cooking the transgenic rice did not affect its ability to tolerize the host (19). Although this outcome would be expected because T cell peptides are required for tolerization, the investigators show that oral feeding with transgenic rice can potentially treat autoimmune diseases (19), which is an advantage of unadjuvanted oral vaccines (12). CTB also has been used to induce oral tolerance (reviewed in ref. 17). Although immunity or tolerance can be driven by CTB, presumably by interaction with host M cells on Peyer's patches, a major limitation of any edible vaccine will be the required coadministration of a mucosal adjuvant, unless mucosal adjuvants can be successfully coexpressed with the desired edible vaccine. Being able to demonstrate immunity using their transgenic rice represents a significant accomplishment for Nochi et al.

The greater protein content of rice is an advantage over some of the starch-based edible vaccines described previously (Table 1) and for heat-labile enterotoxin B subunit (LTB; ref. 5). LTB is very similar to CTB in exhibiting adjuvant activity. When maize-derived LTB was fed in three 1.0-mg doses to human volunteers, seven of nine volunteers produced a serum IgG response, whereas only four individuals showed a fecal IgA response (6). Protein-based seeds such as soybeans have the unique advantage of a high protein content (35–40%), as opposed to rice and maize that have 8–10% protein. When LTB was expressed in soybeans, as much as 2.4% of the total seed protein was LTB (20). Mice fed or parenterally immunized with soybean-derived LTB showed IgG and IgA anti-LTB antibody responses that could protect against diarrhea. Thus, these collective studies demonstrate, regardless of the plant-derived vaccine used, the importance of developing coexpressed mucosal adjuvants with the edible vaccine. Although Nochi et al. (14) show no anti-rice storage protein response, neither the expressed CTB nor the recombinant CTB have the adjuvant potency of native cholera toxin. However, when mice were fed wild-type rice in conjunction with native cholera toxin, an anti-rice storage protein antibody response was elicited (14), suggesting that the expression of highly potent mucosal adjuvants may be problematic if these were to stimulate immune responses to the food product, be it rice, maize, wheat, or soybeans. Perhaps plant-derived alternative adjuvants need to be sought, or alternative adjuvants need to be added exogenously to the prepared edible vaccine.

The future of edible vaccines will depend on producing sufficient quantities.

The future of edible vaccines will depend on the feasibility of producing sufficient quantities of immunogenic vaccines. The edible vaccines that innately possess immunogenicity and do not require additional adjuvant will probably be the first successful vaccines for human or livestock use. The selection of the transgenic plant platform will largely depend on the vaccine and the region where it will be propagated. The grain-based vaccines, as described by Nochi et al. (14), will be the most likely candidates because pollen dispersion or potential contamination of normal food supplies with transgenic pollen can be controlled. The future use of these vaccines also will depend on the development of stable transgenic lines that effectively maintain the vaccine expression for subsequent plant generations. In addition, edible vaccines may have to withstand food processing and possibly cooking. Nevertheless, a demand for the development of edible vaccines persists because they can eliminate many of the problems associated with conventional vaccines, including storage issues, injection risks and associated waste, high production costs, and ease of distribution in underserved areas. Who knows? It may nice to have a little vaccine with supper tonight!