Production of pharmaceutical proteins in solanaceae food crops.

The benefits of increased safety and cost-effectiveness make vegetable crops appropriate systems for the production and delivery of pharmaceutical proteins. In particular, Solanaceae edible crops could be inexpensive biofactories for oral vaccines and other pharmaceutical proteins that can be ingested as minimally processed extracts or as partially purified products. The field of crop plant biotechnology is advancing rapidly due to novel developments in genetic and genomic tools being made available today for the scientific community. In this review, we briefly summarize data now available regarding genomic resources for the Solanaceae family. In addition, we describe novel strategies developed for the expression of foreign proteins in vegetable crops and the utilization of these techniques to manufacture pharmaceutical proteins.

An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines.

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs.

Use of the wound-inducible NtQPT2 promoter from Nicotiana tabacum for production of a plant-made vaccine.

The wound-inducible quinolinate phosphoribosyl transferase promoter from Nicotiana tabacum (NtQPT2) was assessed for its capacity to produce B-subunit of the heat-labile toxin (LTB) from enterotoxigenic Escherichia coli in transgenic plant tissues. Comparisons were made with the widely used and constitutive Cauliflower Mosaic Virus 35S (CaMV35S) promoter. The NtQPT2 promoter produced somewhat lower average concentrations of LTB protein per unit weight of hairy root tissue but allowed better growth thereby producing similar or higher overall average yields of LTB per culture batch. Transgenic tobacco plants containing the NtQPT2-LTB construct contained LTB protein in roots but not leaves. Moreover, wounding NtQPT2-LTB transgenic plants, by removal of apices, resulted in an approximate 500% increase in LTB levels in roots when analysed several days later. CaMV35S-LTB transgenic plants contained LTB protein in leaves and roots but wounding made no difference to their LTB content.

Delivery of plant-made vaccines and therapeutics.

As commercial approval of the first, purified, plant-based biopharmaceuticals for parenteral delivery to humans approaches, improved strategies for delivery of plant-made vaccines and therapeutics are required to ensure their further development and to fulfil the prospect of supplying a global solution for affordable medicines. To ensure that this occurs, research should investigate and characterise the host immune system in addition to the effects of adjuvants and carrier vehicles on consistency and efficacy of vaccination. In this review we explore the basic understandings of pharmaceutical delivery and its effect on immunogenicity in an effort to advance the plant-made pharmaceutical platform.

The effect of plant tissue and vaccine formulation on the oral immunogenicity of a model plant-made antigen in sheep.

Antigen-specific antibody responses against a model antigen (the B subunit of the heat labile toxin of enterotoxigenic Escherichia coli, LTB) were studied in sheep following oral immunisation with plant-made and delivered vaccines. Delivery from a root-based vehicle resulted in antigen-specific immune responses in mucosal secretions of the abomasum and small intestine and mesenteric lymph nodes. Immune responses from the corresponding leaf-based vaccine were more robust and included stimulation of antigen-specific antibodies in mucosal secretions of the abomasum. These findings suggest that oral delivery of a plant bioencapsulated antigen can survive passage through the rumen to elicit mucosal and systemic immune responses in sheep. Moreover, the plant tissue used as the vaccine delivery vehicle affects the magnitude of these responses.

The release and induced immune responses of a plant-made and delivered antigen in the mouse gut.

This study investigated the site of release of a model vaccine antigen from plant cells and the corresponding induced immune response. Three plant tissues (leaf, fruit and hairy root) and two formulations (aqueous and lipid) were compared in two mouse trials. A developed technique that enabled detection of antigen release by plant cells determined that antigen release occurred at early sites of the gastrointestinal tract when delivered in leaf material and at later sites when delivered in hairy roots. Lipid formulations delayed antigen release from all plant materials tested. While encapsulation in the plant cell provided some protection of the antigen in the gastrointestinal tract and influenced antigen release, formulation medium was also an important consideration with regard to vaccine delivery and immunogenicity. Systemic immune responses induced from the orally delivered vaccine benefited from late release of antigen in the mouse gastrointestinal tract. The influences to the mucosal immune response induced by these vaccines were too complex to be determined by studies performed here with no clear trend regarding plant tissue site of release or formulation medium. Expression and delivery of the model antigen in plant material prepared in an aqueous formulation provided the optimal systemic and mucosal, antigen-specific immune responses.

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.

Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen.

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65-70 µg LTB g(-1) tissue whilst hairy roots of tomato contained ~10 µg LTB g(-1). Antigen at ~600 ng ml(-1) was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.

Evolution of plant-made pharmaceuticals.

The science and policy of pharmaceuticals produced and/or delivered by plants has evolved over the past twenty-one years from a backyard remedy to regulated, purified products. After seemingly frozen at Phase I human clinical trials with six orally delivered plant-made vaccines not progressing past this stage over seven years, plant-made pharmaceuticals have made a breakthrough with several purified plant-based products advancing to Phase II trials and beyond. Though fraught with the usual difficulties of pharmaceutical development, pharmaceuticals made by plants have achieved pertinent milestones albeit slowly compared to other pharmaceutical production systems and are now at the cusp of reaching the consumer. Though the current economic climate begs for cautious investment as opposed to trail blazing, it is perhaps a good time to look to the future of plant-made pharmaceutical technology to assist in planning for future developments in order not to slow this technology's momentum. To encourage continued progress, we highlight the advances made so far by this technology, particularly the change in paradigms, comparing developmental timelines, and summarizing the current status and future possibilities of plant-made pharmaceuticals.

Plant-made vaccines in support of the Millennium Development Goals.

Vaccines are one of the most successful public health achievements of the last century. Systematic immunisation programs have reduced the burden of infectious diseases on a global scale. However, there are limitations to the current technology, which often requires costly infrastructure and long lead times for production. Furthermore, the requirement to keep vaccines within the cold-chain throughout manufacture, transport and storage is often impractical and prohibitively expensive in developing countries-the very regions where vaccines are most needed. In contrast, plant-made vaccines (PMVs) can be produced at a lower cost using basic greenhouse agricultural methods, and do not need to be kept within such narrow temperature ranges. This increases the feasibility of developing countries producing vaccines locally at a small-scale to target the specific needs of the region. Additionally, the ability of plant-production technologies to rapidly produce large quantities of strain-specific vaccine demonstrates their potential use in combating pandemics. PMVs are a proven technology that has the potential to play an important role in increasing global health, both in the context of the 2015 Millennium Development Goals and beyond.

Analysis of a cholera toxin B subunit (CTB) and human mucin 1 (MUC1) conjugate protein in a MUC1-tolerant mouse model.

Since epithelial mucin 1 (MUC1) is associated with several adenocarcinomas at the mucosal sites, it is pertinent to test the efficacy of a mucosally targeted vaccine formulation. The B subunit of the Vibrio cholerae cholera toxin (CTB) has great potential to act as a mucosal carrier for subunit vaccines. In the present study we evaluated whether a MUC1 tandem repeat (TR) peptide chemically linked to CTB would break self-antigen tolerance in the transgenic MUC1-tolerant mouse model (MUC1.Tg) through oral or parenteral immunizations. We report that oral immunization with the CTB-MUC1 conjugate along with mucosal adjuvant, unmethylated CpG oligodeoxynucleotide (ODN) and interleukin-12 (IL-12) did not break self-antigen tolerance in MUC1.Tg mice, but induced a strong humoral response in wild-type C57BL/6 mice. However, self-antigen tolerance in the MUC1.Tg mouse model was broken after parenteral immunizations with different doses of the CTB-MUC1 conjugate protein and with the adjuvant CpG ODN co-delivered with CTB-MUC1. Importantly, mice immunized systemically with CpG ODN alone and with CTB-MUC1 exhibited decreased tumor burden when challenged with a mammary gland tumor cell line that expresses human MUC1.

Current status of plant-made vaccines for veterinary purposes.

Interest is growing for the use of plant-made vaccines for veterinary purposes since the regulatory landscape still enables delivery of either crude extracts or minimally processed plant materials to animals for medicinal purposes. In this article, we highlight the current research directions taken with four diseases considered as important constraints to international trade in animals: avian influenza, Newcastle disease, foot-and-mouth disease and diarrheal disease caused by enterotoxigenic Escherichia coli. We also discuss appropriate plant production platforms with regards to plant species and transformation methodologies, possible areas of development, and the remaining challenges for plant-made vaccines for veterinary purposes.

Tomato (Lycopersicum esculentum).

Tomato (Lycopersicum esculentum) is an important fruit crop in the Americas, southern Europe, the Middle East, and India, with increasing production in China, Japan, and Southeast Asia. It is amenable to producing pharmaceuticals, particularly for oral delivery; for many of the same reasons, it is a popular vegetable. Its fruit does not contain toxic substances and is palatable uncooked; it is easily processed; the plants are able to be propagated by seed or clonally by tip or shoot cuttings; the plants have a high yield of fruit; there is reasonable biomass and protein content; and they are easily grown under containment. This chapter describes Agrobacterium-mediated transformation of the tomato nucleus using cotyledons as explants. We have used this protocol to generate transgenic lines from several tomato cultivars expressing various genes of interest and selectable markers. We also provide protocols for molecular characterization of transgenic lines and batch processing tomato fruit.

Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice.

Yersinia pestis, the causative agent of plague, is an extremely virulent bacterium but there are no approved vaccines for protection against it. Our goal was to produce a vaccine that would address: ease of delivery, mucosal efficacy, safety, rapid scalability, and cost. We developed a novel production and delivery system for a plague vaccine of a Y. pestis F1-V antigen fusion protein expressed in tomato. Immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially-produced F1-V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology.

Oral immunogenicity of a plant-made, subunit, tuberculosis vaccine.

Transgenic plants are a novel way to produce and deliver oral vaccines. Arabidopsis thaliana material shown previously to express the tuberculosis (TB) antigen ESAT-6 fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) was fed to mice and the resulting immune response investigated. The plant-made LTB-ESAT-6 fusion protein induced antigen-specific responses from CD4+ cells and increased IFN-gamma production, indicating a Th1 response. In addition, a Th2 response was induced in the Peyer's patch. This is the first report of an orally delivered, subunit, tuberculosis vaccine priming an antigen-specific, Th1 response.

Risk analysis for plant-made vaccines.

The production of vaccines in transgenic plants was first proposed in 1990 however no product has yet reached commercialization. There are several risks during the production and delivery stages of this technology, with potential impact on the environment and on human health. Risks to the environment include gene transfer and exposure to antigens or selectable marker proteins. Risks to human health include oral tolerance, allergenicity, inconsistent dosage, worker exposure and unintended exposure to antigens or selectable marker proteins in the food chain. These risks are controllable through appropriate regulatory measures at all stages of production and distribution of a potential plant-made vaccine. Successful use of this technology is highly dependant on stewardship and active risk management by the developers of this technology, and through quality standards for production, which will be set by regulatory agencies. Regulatory agencies can also negatively affect the future viability of this technology by requiring that all risks must be controlled, or by applying conventional regulations which are overly cumbersome for a plant production and oral delivery system. The value of new or replacement vaccines produced in plant cells and delivered orally must be considered alongside the probability and severity of potential risks in their production and use, and the cost of not deploying this technology--the risk of continuing with the status quo alternative.

Expression systems and developments in plant-made vaccines.

Delivery of vaccines to mucosal surfaces can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle-associated risks. Transgenic plants are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. In the past few years, new techniques (such as chloroplast transformation and food processing) have improved antigen concentration in transgenic plants. In addition, adjuvants and targeting proteins have increased the immunogenicity of mucosally administered plant-made vaccines. These studies have moved plant-made vaccines closer to the development phase.

Application of Quillaja saponaria extracts as oral adjuvants for plant-made vaccines.

Extracts from the Quillaja saponaria tree are known to provide immune potentiating responses and, hence, can be useful as adjuvants. Partial purification from the crude (food-grade) extract results in Quil A, which is contained in several veterinary vaccines. Further purification can provide concentrated saponin fractions such as QS-21, which is currently under investigation as a potential adjuvant for use in humans. Purified saponins have proven safe and effective when injected and have significantly enhanced the efficacy of some oral vaccines under clinical investigation. Toxicity of the food-grade extract from Quillaja saponaria has limited its use as a parenteral adjuvant; however, this toxicity seems to be abated when delivered orally. It is commonly used within the food and beverage industries and has no documented toxicity in humans at the present levels of consumption. Use of transgenic plants has been proposed as an alternative system for oral vaccine production and administration, and it is likely that an oral adjuvant will be required in most cases. Food-grade saponins have significant advantages for use with plant-made vaccines and are likely to provide a broad adjuvant effect due to the multiple saponin components. A review of the origin, production, biological activity, toxicity and use in the food industry is provided for Quillaja saponaria extract. Previous evaluation of this adjuvant in preclinical studies with plant made vaccines is discussed and a proposed level of experimental use in humans is provided.

Plant cell factories and mucosal vaccines.

Many advances continue to be made in the field of plant-derived vaccines. Plants have been shown capable of expressing a multicomponent vaccine that when orally delivered induces a T-helper cell subset 1 response and enables passive immunization. Furthermore, a plant-derived vaccine has been shown to protect against challenge in the target host. Increased antigen expression levels (up to 4.1% total soluble protein) have been obtained through transformation of the chloroplast genome. In view of these findings, plant-derived vaccines have been proved as valuable commodities to the world's health system; however, before their application, studies need to focus on optimization of immunization strategies and to investigate antigen stability.