Transgenic plants as edible vaccines — reality and future prospects

This review describes the recent progress in the construction of transgenic plants for vaccine producti,v Transgenic plants are an attractive and cost-effective alternative to microbial systems for the productior о proteins with pharmaceutical value. Advances in biotechnology are enabling plants to be exploitea /· expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. It has recently been shown that genes encoding antigens of bacterial and vira< pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing bacterial antigens stimulated humoral and mucosal immune response, when they were provided as a food. Although the utility of «edible vaccines» to prevent disease remains t< be established, the successful implementation of this strategy can be the first step on the way to modern vaccines of new generation.

Introduction.Research on new vaccines has used molecular biology to identify the antigenic determinants of infectious disease agents and to develop genetic engineering approaches to produce and deliver these antigens as subunit vaccines.In recent studies, tools of plant biotechnology have been added to these efforts.It has been found that transgenic plants provide a novel system for production of recombinant proteins that act as oral immunogens when the plant products are consumed as food.
Many infectious agents colonize or invade epithelial membranes; these include bacteria and viruses that are transmitted via contaminated food or water or by sexual contact.
Vaccines that are effective against these infections must stimulate the mucosal immune system to produce secretory IgA (S-IgA) at mucosal surface such as the gut and respiratory epithelia.In general, a mucosal immune response is more effectively achieved by oral, rather then parenteral, antigen delivery.Several particulate antigens have proven to be effective oral immunogens, including live and killed microorganisms.By comparison with parenteral immunization, oral immunization using subunit or soluble antigens is often inefficient at stimulating an immune response, and requires largeramounts (mg versus μ%> of antigen.
Subunit vaccines based upon recombinant cellculture expression systems are feasible but, for conmercial-scale production, these systems require fei mentation technology and stringent purification pro tocols so that sufficient amounts of recombinan protein can be obtained for oral delivery.Even witK technological improvements, fermentation-based su bunit vaccine production may be prohibitively ex pensive technology for developing countries where novel oral vaccines are urgently needed Transgenic plants that express antigens in their edible tissue might be possible simply through consumption of a «edible vaccine».
In general, research in this field falls into t>)u: general categories.First, experiments have been ccducted to determine the capacity of plants to produce foreign proteins that retain antigenic determinants necessary for effective immunization.Second, the oral immunogenicity of plant-derived proteins has beer• evaluated with special emphasis on th< <c immunogenicity of food samples.Third, research has been conducted Io find an appropriate food crop that could be used for both production and distribution of vaccines, with special emphasis on the developing world.
Hepatitis B surface proteins.The first studies of candidate vaccine expression in transgenic plants have been carried out using the gene encoding hepatitis B surface antigen (HBsAg) [1,3].This protein was chosen because the commercially available vaccine and the associated human immune response have been very well characterized, because the structure of the immunogenic form of that protein was known, and because the availability of a cost-effective recombinant HBV vaccine is a high priority especially for the developing countries.Moreover, the existence of commercially available test systems for HBV detection substantially simplified the procedure of HBsAg detection in plant tissues.
The envelope of hepatitis B virus (HBV) consists of three polypeptides which comprise the large (L), middle (M) and major or small (S or HBsAg) protein components.These three proteins are encoded in a large open reading frame, which is divided into preSl, preS2 and the S gene.The S protein or HBsAg is a major component of the: hepatitis B virions and contains 226 amino acids.The HBV M protein contains additional 55 amino acid residues at the N-terminal of the S protein, usually called the preS2 antigen.Similarly, the L protein contains additional 108-119 amino acids, depending on the serotype of the virion, at the N-terminal of the M protein [9].
The S gene was introduced into cells of tobacco [1] and potato [3] plants and individual transformants were regenerated.When extracts from transgenic plant tissues were examined the presence of HBsAg were revealed by ELlSA using monoclonal antibodies directed against human-serum-derived HBsAg.Further examination of plant-derived HBsAg purified by immunoaffinity chromatography revealed the presence of spherical virus-like particles with an average size of 22 nm.These particles exhibited properties that were very similar to the subvi^al particles obtained from human serum and to the recombinant HBsAg which is formulated in the commercial vaccine produced in yeast cells [1 ].Importantly, HBsAg in the particle form was found to be much more immunogenic than that in the form of the peptide alone [10].
To evaluate the immunogenicity of plant-derived HBsAg it was used for parenteral immunization of mice.Anti-HBsAg antibodies were recovered which reacted with authentic HBsAg from human serum.This was the first indication, that antigenic properties of the protein were maintained in transgenic plants Subsequently, T cells were isolated from mice immunized with plant-derived HBsAg.When grown in culture, these T cells could be activated using со α mercial vaccine as well as a synthetic peptide which mimics the «а» epitope determinant of HBsAg. in total, the immunology studies conducted to date show that the recombinant HBsAg recovered from plant cells retain both B and T cell epitopes [5].
Although recombinant HBV vaccines have shown that HBsAg alone is sufficient to induce a highly protective immunity, experiments in animals have highlighted the potential benefits which might resui* from the inclusion of the preS2 domain in anti-HBV vaccines [11 J.The preS2 domain is also immunogenic in humans and elicit anti-preS2 responses during natural HBV infection, which often occur prior to a ; other anti-HBV response [12].For this reason, me HBV M protein gene (preS2 containing H BsAgjbeen recently expressed in plants 18 ] and physical and immunological properties of this protein wenevaluated [13].These studies have demonstrated th^i plant cells have the capacity to not only synthesize M protein but also to allow it to be assembled in ar immunologically active form. To evaluate the immune response to plant-de rived M protein and to compare it to the response ic HBsAg from serum (preS2 containing HBsAg), HBV vaccine and plant-derived HBsAg, Balb/c mice were immunized intraperitoneally with corresponding antigens.Kinetics of antibody responses were studies j to 14 weeks after primary immunization.The results presented in Fig. 1 indicate that both plant-derived HBV proteins can elicit the anti-HBsAg antibodies in mice and that the plant-derived M protein is nearly as immunogenic as the control preS2 containing HBsAg isolated from serum.Moreover, the presence of anti-preS2 antibodies in the sera of immunized Balb/c mice was detected in mice immunized with the preS2 containing proteins (Fig. 2).These result suggest that plant system can provide an alternative method of producing the HBV M protein suitable f.v vaccination.
Recently, it was also shown that the plantderived HBV M protein given to mice by oral intubation (gavage) stimulated serum antibody response and corresponding specific antibodies were dt tecu .[14].
Escherichia coli heat-labile enterotoxin B su bunit.The choice of which antigens to use in ih initial studies has been strongly influenced by desire to determine if transgenic plant materia ?containing foreign antigens will result in oral immunization and stimulate a mucosal immune response Thus, antigens with the high mucosal immune response have been the early targets for plant based expression.
The binding subunit of the heat-labile enterotoxin of E. coli (LT-B) was an obvious candidate for evaluation in plant expression system since it has been extensively characterized in structural and immunological studies.
The heat-labile enterotoxin (LT) from E. coli is a multimeric protein that is structurally, functionally and antigenically very similar to cholera toxin (CT).It was found that LT has one A subunii (LT-A) and a pentamer of B subunits (LT-B).Specific binding oi the nontoxic LT-B pentamer to the G mi gangliosioes present on epithelial cell surfaces allows entry of th toxic LT-A subunit into cells [15].Antibody Iil terference with binding of the B subunit ίο cells, lhir blocking toxin activity, is the basis of attempts to u < the B subunit as a vaccine component.Because L t is very similar in structure and immunological properties to the CT-B, immunization with CT-B lcaas to cross-protection against enterotoxigenic E. coli LT-B and CT-B are both potent strong oral immunogens.
LT-B also has recently been expressed in plan s although the levels of expression were low [2 j Th* oral immunogenicity of recombinant LT-B was tesuc in mice and compared with bacterial LT-B.Vvh'к given orally to mice by gastric intubation, the planiderived antigen stimulated humoral and mucosa; immune responses with titers comparable to the bacteria-derived LT-B.In addition, the antibodies produced against the tobacco-derived LT-B were abl to neutralize LT activity, indicating the potentia' protective value of the immune response.
The oral immunogenicity of unpurified recombinant LT-B was also assessed by feeding raw transgenic potato tubers to mice.After only four feedings of 5 g tuber samples to mice, mucosal and serum antibodies were recovered.No immune response was observed in animals that were fed nontransformed tubers.
It should be also noted that CT and LT are excellent oral adjuvants, which stimulate immune responses against co-fed antigens at concentration., well below those that cause diarrhea.
Norwalk virus capsid protein.Further evidence to support the concept of edible vaccines have recently been obtained in experiments with plant-derived Not walk virus capsid protein (NVCP).Norwalk virus is a member of the Caliciviridae family and causes epidemic acute gastroenteritis in humans.As in the case of HBV, expression of NVCP in plant cells yields .. protein that self-assembles in plant cells into virut like particles.The plant-derived NVCP was orally immunogenic in mice.Extiacts of tobacco leaf expressing NVCP were given to CDl mice by gavage and the treated mice developed both serum IgG and secretory IgA specific for Norwalk virus-like particles.Furthermore, when potato tubers expressing NVCP were fed directly to mice, they developed serum IgA specific for Norwalk virus [7 ].
Vaccines for animal diseases.Edible vaccines can also provide efficient and humane strategies for disease prevention in production of companion animals, as well as feral populations.It is practically possible to generate vaccines against viral and bacterial infections by expressing corresponding antigens in plant tissues edible for animals.The already mentioned LT-B subunit is the most likely candidate for the first commercial vaccine, as enterotoxigenic E. coli strains readily infect animals as well.Admittedly, vaccines for animals are a more likely target for edible-vaccine technology in the near future than vaccines for human as the latter need more detailed inspections for safety.
Recently, transgenic plants have been generated that expressed the gene encoding the glycoprotein (G-protein) that coats the outer surface of the rabies virus [6, 14 |.Although the immunogenicity of these material has yet to be evaluated, it is encouraging, to note that ba t containing some G~protein produced in a more traditional in vitro system was effective in immunizing raccoons orally, providing protection against «street virus» challenge.
Future prospects.The research conducted to date has demonstrated that transgenic plants have the capacity to synthesize and accumulate subunit antigenic proteins that retain immunological properties of their native counterparts.In the case of HBV proteins and NVCP, virus-like particles accumulated in plant cells.It is very significant as the particulate form is very important in determining immunogenic properties and has greater oral immunogenicity than soluble proteins.
Studies remaining to be conducted will involve the evaluation of dosage requirements for plantdelivered vaccines.Successful experiments conducted thus far have used proteins (LT-B and NVCP) with very high oral immunogeniciity.It will be necessary to determine if other proteins, which may not be normally transmitted orally, will be as effective in inducing an oral response.From this point of view the results of the oral immunization with the plantderived HBV M protein are rather encouraging.Multi-subunit vaccines, including oral adjuvants such as LT or CT (or derivatives thereof), and various fused proteins could be also used for enhancing the oral response.
It is well recognized that most food proteins do not trigger an immune response.In general it is due to the induction of a state of immune tolerance.It will be necessary to determine if food-based vaccines also would induce oral tolerance to the desired antigen If so, controlled use and dosage will be a requirement for edible vaccines.
The type of plant material that would best serve as an edible vaccine also has yet to be determined First studies has focused primarily on tobacco and potato, but other plants such as corn, soybeanbananas and others are currently under research.
Lastly, a thorough study of the safety oi the future edible vaccines needs to be undei'cken.Rc searches in this area are likely to nci ease oui understanding of the basic mechanisms, which can be applied to the development of the new generation o. vaccines.