Amanda M. Walmsley

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.

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.

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.

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.

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.

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.

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 containedxa0~65–70xa0μg LTBxa0g−1 tissue whilst hairy roots of tomato containedxa0~10xa0μg LTBxa0g−1. Antigen atxa0~600xa0ngxa0ml−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.

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 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.

Improved expression of recombinant plant-made hEGF

Key messageThe yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF.AbstractHuman epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34xa0%, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24xa0% of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.