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Over the past decade, scientific advances in molecular biology and immunology have improved understanding of many diseases and led to the development of novel strategies for vaccination. The development of plants expressing vaccine antigens is a particularly promising approach. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and functional in human clinical trials. Future areas of research should further characterize the induction of the mucosal immune system and appropriate crop species for delivery of animal and human vaccines.
Addresses
*†Boyce Thompson Institute for Plant Research, Cornell University,
Ithaca, NY 14853, USA
*Co-operative Research Centre for Conservation and Management of Marsupials, Macquarie University, Sydney, NSW 2109, Australia *e-mail: amw21@cornell.edu
†e-mail: cja7@cornell.edu
Current Opinion in Biotechnology2000, 11:126–129 0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
CPMV cowpea mosaic virus
Ig immunoglobulin
Introduction
Subunit vaccines consist of specific macromolecules that induce a protective immune response against a pathogen. These vaccines are the consequence of advances in molecu-lar biology enabling the location of antigens capable of invoking a protective immune response (vaccinogen), the isolation of the corresponding gene(s), and the production of the vaccinogen in an expression system. Subunit technology has improved existing vaccines and has circumvented some limitations of traditional vaccine production. The specific composition of subunit vaccines increases vaccine safety by circumventing the need to use live viruses or microbes and has thus made them the preferred approach for vaccine man-ufacturers [1]. Unfortunately, traditional subunit vaccines are expensive to produce and not heat stable (making them reliant on the expensive, often capricious and destination-limited series of refrigeration steps, sometimes referred to as the ‘cold chain’, en route from manufacture to vaccination). This limits their availability and use in the low-funded health care systems of developing countries.
Production of vaccines in plants eliminates some current impediments. The simplistic requirement of plants for sun-light, water and minerals makes them an inexpensive means of correctly processing and expressing proteins that can be quite complex. Expression of vaccines in plant tissues elimi-nates the risk of contamination with animal pathogens, provides a heat-stable environment, and enables oral deliv-ery, thus eliminating injection-related hazards.
The first clinical trial with a plant-derived vaccine in 1997 [2••] demonstrated the potential of plant-derived vaccines for expanding the armament against infectious diseases. This review highlights the advances made in the past year towards the development of edible vaccines.
Plant molecular biology and transformation
The potential of molecular biology to increase the value of plants to agriculture, industry and health has led to the application of extensive resources to further develop this technology. The resulting refinement of gene cloning, tis-sue culture and plant transformation produced a springboard from which unprecedented applications could evolve, such as edible vaccines. The process of creating an edible vaccine begins by selecting a suitable vaccinogen. The gene of interest is cloned into an expression cassette that contains plant regulatory sequences capable of driving gene expression and indicating the gene’s terminus. This cassette is then used in plant transformation.
Agrobacterium-mediated transformation
Many techniques have been used to genetically enhance plants, however, literature only reports use of
Agrobacterium-mediated transformation for production of edible vaccines. Agrobacterium is a plant pathogen that in the process of infection transfers a segment of its DNA (T-DNA) into the genome of its host. Molecular biologists have taken advantage of this process to transfer a gene of interest, in a plant expression cassette, into plant genomes. Upon incubation of the transgenic Agrobacteriumwith plant materials, transfer of the T-DNA from the bacterium into the host’s genome occurs through a process similar to con-jugation. During tissue culture, transformed cells are positively selected and regenerated into transgenic plants. The time taken to regenerate a transgenic plant is species dependent and ranges from 6 weeks to 18 months.
Chimeric viruses for vaccine delivery
Autonomously replicating plant viruses can also express foreign genes in plants. Epitopes from vaccinogens have been presented on the surface of plant viruses by mak-ing translational fusions either within or at the 3′
terminus of a coat protein. Following plant generations do not inherit the foreign gene as it is not incorporated into the plant genome. Thus the production of a plant virus-derived vaccinogen requires an extra step of inocu-lation of the host plants with the chimeric virus. Before administration, chimeric virus particles are often purified from host-plant tissues that are unpalatable, containing toxins or are not practical for direct consumption. Nevertheless, the high level of foreign protein expres-sion (up to 2 g/kg of plant tissue) within a short period (1–2 weeks after inoculation) makes this an attractive alternative for vaccine production.
Plants for delivery of edible vaccines
Plants for delivery of edible vaccinesWalmsley and Arntzen 127
The mucosal immune system
The success of an edible vaccine requires induction of the mucosal immune system (MIS). The MIS is the primary defense of the surfaces where most human and animal pathogens initiate infection, that is, the mucosal surfaces found lining the digestive tract, respiratory tract and urino-reproductive tract. Induction of a mucosal immune response starts with the recognition of an antigen by spe-cialized cells called M-cells. These cells are localized in the mucosal membranes of lymphoid tissues such as Peyer’s patches within the small intestines. The M-cells channel the antigen to underlying tissues where antigen-presenting cells internalize and process the antigen. The resulting antigenic epitopes are presented on the APC surface, and with the assistance of helper T cells activate B cells. The activated B cells migrate to the mesenteric lymph nodes where they mature into plasma cells and migrate to mucos-al membranes to secrete immunoglobulin (Ig) A. Upon passing through the mucosal epithelial layer towards the lumen, the IgA molecules complex with membrane-bound secretary components to form secretary IgA (sIgA). Transported into the lumen, the sIgA interacts with specif-ic antigenspecif-ic epitopes and neutralize the invading pathogen.
Early developments in plant-derived vaccines
The first demonstration of expression of a vaccinogen in plants occurred in 1990 when Curtiss and Cardineau [P1] expressed the Streptococcus mutans surface protein antigen A (SpaA) in tobacco. After incorporation of the transgenic tobacco tissue into the diet of mice, a mucosal immune response was induced to the SpaA protein. Although the mice were not challenged with the pathogen, the induced antibodies were demonstrated biologically active when they reacted with intact S. mutans. Reports have since fol-lowed of expression of a hepatitis antigen in tobacco and lettuce [3,4], a rabies antigen in tomato [5], a cholera anti-gen in tobacco and potatoes [6,7] and a human cytomegalovirus antigen in tobacco [8•]. Animal trials
demonstrating antigenicity of plant-derived vaccinogens include tobacco- and lettuce-derived hepatitis B surface antigen [9,10], a tobacco- and potato-derived bacterial diar-rhea antigen [11], a potato-derived Norwalk virus antigen [12], and an Arabidopsis-derived foot-and-mouth disease antigen [13]. To date, the data obtained do not allow deter-mination of protective immunity either because the animal model is not susceptible to the disease-causing agent, or because of strict containment issues with the pathogen (as in foot-and-mouth disease).
The use of viruses as carrier molecules for vaccinogens has received much interest [14–19]. Plants and plant viruses were not used until 1993, however, when an epitope from the foot-and-mouth disease virus (FMDV) was expressed on the surface of cowpea mosaic virus (CPMV) [20]. Chimeric plant viruses were proven effective as carrier pro-teins for vaccinogens in 1994 after rabbits raised an immune response against purified chimeric CPMV parti-cles expressing epitopes derived from human rhinovirus 14
(HRV-14) and HIV-1 [21]. Numerous reports have since verified plant viruses as effective alternative vaccinogen expression vectors. Antibodies have been stimulated in mice after injection with plant-virus-derived HIV-1 epi-topes [22–25], mouse zona pellucida epitope [26] and rabies virus epitope [25], whereas complete protection was conferred by a plant-virus-derived canine parvovirus epi-tope in a mink challenge trial [27].
Recent developments in plant-derived vaccines
Recent research has concentrated on meeting the prereq-uisites for application of plant-derived vaccines to the human and animal health industries. Information on dosage, best delivery method and response type, strength and length has been acquired for pathogens including
Vibrio cholerae[28], HIV [29], Pseudomonas aeruginosa[30], murine hepatitis virus [31], and foot-and-mouth disease virus [32]. Further investigation has examined the use of synthetic genes, targeting of vaccinogen expression to spe-cific plant tissues, investigation of the induced immune response and progression to human clinical trials.
One ever-present mission is to increase the level of trans-gene expression within transgenic plants. Mason et al. [33••] increased expression of the B subunit from the
Escherichia coli heat-labile enterotoxin by constructing a plant-optimized synthetic gene. Analysis of the native cod-ing region revealed codes or sequences that may have caused inefficient processing or premature degradation of the gene products in plants. These were removed during construction of the synthetic gene. Using the native gene for comparison, the synthetic gene increased antigen accu-mulation in leaves and tubers by 3–14-fold. Antigenicity of the resulting vaccinogen was demonstrated through mice feeding trials [33••].
IgG and IgA proved capable of improving the clinical symptoms caused by intranasal infection with an attenuat-ed rabies virus strain.
Brennan et al. [34•] further characterized the immune response induced by mucosal delivery of a plant-derived vaccine. A chimeric CPMV expressing a vaccinogen from
Staphylococcus aureus was delivered mucosally (either intranasally or orally) to mice with or without an adjuvant. Unlike past studies, sampling was not restricted to serum and/or feces. Lavage fluids were also collected from the bronchia, intestine and vagina. Vaccinogen-specific IgA and IgG could be detected at each of these sites regard-less of presence or absence of adjuvant. This is the first report of a plant-derived vaccinogen inducing mucosal immune response at distant mucosal sites without the need of an adjuvant.
The first human clinical trials for a transgenic, plant-derived antigen were planned, approved (US Food and Drug Administration) and performed in 1997 [2••]. Transgenic potatoes constitutively expressing a synthetic bacterial diar-rhea vaccinogen (the B subunit of E. coli heat labile toxin LT-B) were orally delivered to human volunteers in Phase I/II clinical trials. Variability of vaccinogen expression in transgenic tissues was accounted for by batch processing of the potato sample. Thus, each participant received potato cubes from a random sample of tubers either non-transgenic control tubers or transgenic tubers. The 11 ‘test’ participants received 50–100 g of raw transgenic potato and the three ‘control’ participants received 50 g of raw untransformed potato. Prior to and at multiple time points after ingestion of the potato, serum and fecal samples were taken and ana-lyzed for LT-B specific antibodies. A significant rise in LT-B antibodies was displayed by 10 of the 11 test participants, whereas no LT-B specific antibodies were detected in the control participants. The serum antibody levels induced by ingestion of the transgenic potatoes were comparable to those measured when volunteers were challenged with 106
virulent enterotoxigenic E. coli (ETEC) organisms [36]. Thus, plant-derived recombinant LT-B delivered in edible plant tissues was protected against digestion and proved capable of inducing an immune response in humans. Phase one and two trials are currently in progress using orally deliv-ered potato-derived hepatitis B surface antigen (HBsAg) as a booster for the commercial hepatits B vaccine, and potato-delivered Norwalk virus virus-like particles (VLPs) for a viral diarrhea vaccine. In the three human trials performed with plant-derived vaccines, plant cells have proven suffi-cient for vaccinogen protection against digestion, the vaccinogen has induced systemic and mucosal immune responses without aid of adjuvants, and no adverse effects of genetically modified materials have been demonstrated ([1]; T Thanavala et al., unpublished data).
Conclusions
The technology of using plants and plant virus for expres-sion and delivery of vaccines has reached an exciting stage
in its development. The list of plant-derived vaccinogens continues to grow and includes viral, bacterial, enteric and non-enteric pathogen antigens. In an effort to increase expression levels, stability and ease of harvest, synthetic genes have been constructed [33••] and expression has been targeted to specific tissues [34•]. Variability of vac-cinogen expression in plant tissues has been circumvented by batch processing [2••] and recent investigations have determined the efficacy of plant-derived vaccinogens to induce immune responses, as well as the immune response type, location and duration. Both oral and nasal vaccina-tions have demonstrated the ability to induce a mucosal immune response; however, the oral route requires either protection of the vaccinogen against digestion or excessive vaccinogen amounts. Above all, initial clinical studies have demonstrated plant-derived vaccinogens to be safe and functional. Plant-derived vaccines show great promise for increasing the ease and decrease the cost of vaccine deliv-ery to humans and animals. Remaining issues include if the route of vaccination determines the location of the immune response and the elucidation of appropriate crop species for the delivery of animal and human vaccines.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest ••of outstanding interest
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