Edible plants for oral delivery of biopharmaceuticals

Abstract

Molecular farming is the use of plants for the production of high value recombinant proteins. Over the last 25 years, molecular farming has achieved the inexpensive, scalable and safe production of pharmaceutical proteins using a range of strategies. One of the most promising approaches is the use of edible plant organs expressing biopharmaceuticals for direct oral delivery. This approach has proven to be efficacious in several clinical vaccination and tolerance induction trials as well as multiple preclinical studies for disease prevention. The production of oral biopharmaceuticals in edible plant tissues could revolutionize the pharmaceutical industry by reducing the cost of production systems based on fermentation, and also eliminating expensive downstream purification, cold storage and transportation costs. This review considers the unique features that make plants ideal as platforms for the oral delivery of protein‐based therapeutics and describes recent developments in the production of plant derived biopharmaceuticals for oral administration.

Introduction

The use of plants in medicine can be traced back to antiquity, but the plant molecular biology revolution in the 1980s offered many new opportunities, such as the use of plants as a production platform for recombinant biopharmaceutical proteins. Plants may therefore be considered as an alternative to traditional cell‐based systems using microbial and animal cells. The unique features of plants include cost effective large scale production without the risk of product contamination with endotoxins or human pathogens 1.

Plants are currently used to express antibodies 2, vaccine antigens 3, bioactive peptides 4, diagnostic proteins 5, industrial enzymes 6 and biodegradable plastics 7. Plant based systems for the production of biopharmaceuticals may be developed as bioreactors, i.e. the product is purified from plant tissues and used for oral or parental administration 8 or the plant tissue expressing the biopharmaceutical protein may be used for direct oral delivery 9. In the latter case, the product is expressed in edible plant organs, allowing them to be administered as unprocessed or partially processed material. This eliminates complex downstream processing, which normally accounts for 80% of the total manufacturing cost of a recombinant protein 10. In the developing world, plant derived biopharmaceuticals may also be produced locally near the target population, even if minimal processing of the plant is required 11. These overall cost savings in production, supplies and labour reduce the cost of each dose, making the drug affordable to most of the global population. As well as eliminating most downstream processing costs, the oral delivery of biopharmaceuticals in plant tissues encapsulates the product in a matrix of complex carbohydrates that make up the plant cell wall, protecting sensitive proteins from acid and digestive enzymes in the stomach and thus prolonging their activity 12. The oral delivery of biopharmaceuticals also has several medical advantages over parental administration. First, oral administration is safer because it prevents infections caused by injection. Second, oral vaccines are potentially more efficacious, because the oral route simultaneously promotes mucosal and systemic immunity. Third, oral administration is patient friendly, eliminating the need for needles and syringes. Fourth, oral administration is cost effective because trained medical personnel are not required.

Plants have been developed for the oral delivery of vaccine antigens, to protect against pathogen or toxin challenge 13, autoantigens, to confer tolerance against allergic immune responses or autoimmune diseases 14, and biopharmaceutical proteins, to confer specific functions such as the regulation of blood glucose concentrations 15. This review focuses mainly on the first two applications. For oral vaccination, several phase I clinical trials have been carried out using edible plants, demonstrating the safety and immunogenicity of antigens without the need for a buffer or a vehicle other than the plant cell 16, 17, 18, 19. Several pre‐clinical studies in animal models have also demonstrated the safety and efficacy of tolerance induction for the prevention of allergic reactions 20, rejection immune responses 21 and autoimmune diseases 22. However, the clinical application of oral tolerance strategies is challenging because of the long term need for repeated regular mg doses. Plants could meet this huge demand, given the higher productivity of plants compared with other platforms when both intrinsic yield (per unit biomass) and biomass yield (per hectare per year) are taken into account 1.

Three different approaches are currently used for the expression of pharmaceutical proteins in edible plant organs: transient expression using plants infected with bacterial/viral vectors, stable expression by nuclear transformation and stable expression by chloroplast transformation. Each technique has advantages and disadvantages in terms of yields, scalability, batch processing time and biological containment 8, 23, 24.

Molecular farming in edible plants

Recombinant proteins can be expressed in edible leaves, seeds, fruits and tubers, depending on the plant species. Many different edible species have been investigated, including alfalfa, carrot, lettuce, tomato, potato, maize, wheat, barley, strawberry, soybean, celery cabbage, spinach, cauliflower, rice and banana 9. The first plant derived edible vaccine was expressed in transgenic potato tubers 25. Potato became an early model to demonstrate the feasibility of orally delivered plant derived vaccines and several clinical phase I studies were completed 18. However, raw potato is unpalatable and cooking has the potential to destroy most protein based vaccine candidates. Therefore, tomato fruits were considered as an alternative delivery vehicle because the fruits are safe and palatable when consumed raw and this platform offers the potential to present vaccines as fruit segments, tomato paste or tomato juice 26. Carrots have also been used to produce vaccines because the proplastids of cultured carrot cells express recombinant proteins 27 and the edible carrot taproot preserves the structural integrity of vaccine candidate proteins 28.

Cereal crops are also widely used for the expression of pharmaceutical proteins because the seeds accumulate recombinant proteins at high levels in a stable, desiccated environment, thus allowing the long term preservation of antigens without the need for a cold chain 29. Maize and, more recently, rice have been used for several phase I clinical studies with the recombinant antigen administered as flour paste, e.g. rice expressing an oral cholera vaccine 30.

Banana has been considered as an ideal host for the production of biopharmaceuticals because it is grown throughout the year in the tropics and subtropics and can be consumed raw. Banana plants are clonally propagated via suckers and therefore provide inbuilt biological containment that reduces the risk of environmental gene flow 31. However, the long development time for transgenic banana plants reduces the feasibility of this species for the oral delivery of biopharmaceuticals 32, 33.

One of the major issues concerning the use of edible plant systems producing biopharmaceuticals is that they must be produced, processed and regulated as pharmaceutical products and should therefore be kept separate from food and animal feed supplies 34. This includes accounting for the risk of admixture by using clear traceability and labelling conventions, and preventing the pollination of food/feed crops by pharmaceutical crops of the same species, which can be addressed by physical separation (growing the pharmaceutical crops in containment or in isolated fields) or biological containment (using species that do not outcross or relying on chloroplast transformation which prevents gene flow because chloroplasts are not found in the pollen of species used for molecular farming). Another approach is the labelling of edible pharmaceutical plants with distinctive features that preserve their identity, facilitate traceability and avoid the contamination of the food supply. As a proof of this concept, transgenic tomato plants expressing neutralizing IgA antibodies against rotavirus were crossed with another transgenic line expressing the Antirrhinum majus Rosea1 and Delila transcription factors in the fruit, thus activating anthocyanin biosynthesis and generating purple fruits 35. The resulting hybrids expressed the recombinant antibodies solely in the purple transgenic fruits making them easy to identify 36.

Mechanism of the mucosal immune response to oral biopharmaceuticals

The gut‐associated lymphoid tissue (GALT) has an area of 300 m² in humans and is therefore the largest immune system tissue in the body 37. Its physiological roles include the ingestion of dietary antigens to avoid immune responses to food and protection against pathogens in the gut 38. GALT also helps to prevents autoimmune diseases by the suppression of pathological reactions against self antigens 39. GALT has thus evolved with strictly regulated immunological mechanisms to suppress unwanted inflammatory responses, promoting a tolerogenic environment, while also protecting the body against pathogens 40. Therefore, GALT plays an important role in the response to oral vaccination and oral tolerance induction.

The immunological mechanisms involved in oral vaccination and oral tolerance induction have been extensively reviewed and are not discussed in detail here 39, 40, 41. Instead, we focus on the factors that direct the immune system towards an active immune response or a tolerogenic one, including the unique characteristics of plant derived biopharmaceuticals that determine their immunogenicity and make them suitable for oral immunization.

Oral tolerance induction

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the same antigen through the oral route 40. It is based on active immunological mechanisms intended to prevent, suppress or shift adverse immune responses to harmless ones 39. The resulting processes are strictly dose‐dependent: lower doses of the antigen favour the induction of regulatory T cells (T_reg), whereas higher doses favour clonal anergy/deletion, although these mechanisms are not mutually exclusive 38. On the basis of these mechanisms, some plant derived biopharmaceuticals have been used successfully to induce tolerance, thus suppressing immune responses responsible for autoimmune diseases or allergies 42.

Oral vaccination

Plant derived vaccines can also elicit both humoral and cellular immunity at the mucosal and systemic levels 43. Epitope immunogenicity is preserved in plant derived antigens and they can therefore induce high levels of IgA and IgG as well as cell‐mediated responses in certain cases 9. Adjuvants that induce local inflammatory responses may be used to help avoid the induction of tolerance when an active immune response for therapeutic or vaccination purposes is required 44. Examples include the coupling of antigens to subunit B of cholera toxin (CTB), lipopolysaccharides, virus‐like particles and bacterial toxins 45.

Unique features of plant derived biopharmaceuticals that determine their immunogenicity

Plant derived biopharmaceuticals for oral immunization are usually administered as minimally processed biomass that is freeze‐dried and could be ideally formulated as pills 45. Some unique characteristics of biopharmaceuticals presented in this context influence their oral immunogenicity and the amount of bioavailable protein depends on the balance between antigen degradation and antigen release from the plant biomass in the gut. The encapsulation of antigen proteins in plant cells may protect them from degradation in the stomach by allowing them to resist the extreme low pH values and digestive enzymes, resulting in their presentation to GALT and the circulatory system when the plant cell walls are digested 46. The protective effects of the plant cell walls are complemented by other plant carbohydrates, lipids and proteins that occupy the digestive enzymes and delay the degradation of the target antigen, and further protection is offered by the targeting of recombinant antigens to intracellular compartments that provide an extra level of encapsulation 47. Furthermore, the sequestration of recombinant antigens into storage compartments, such as the protein bodies in seeds, could result in the formation of particular antigen aggregates that boost immunogenicity by promoting the uptake of antigens by antigen presenting cells (APCs) 29, 42.

Some secondary metabolites produced by plant cells as a defence response may also increase the immunogenicity of plant derived biopharmaceuticals, by acting as adjuvant compounds. Many studies have shown that saponins, lectins and polysaccharides have immunomodulatory properties that can boost mucosal immunological responses as well as specific cellular and humoral mechanisms 48, 49, 50. Finally, it has been suggested that the unique glycan structures on plant derived recombinant proteins can stimulate their uptake by dendritic cells and thus increase their immunogenicity 51.

Expert overview

Selected studies published in the last 5 years concerning oral tolerance induction and oral vaccination using plant derived biopharmaceuticals are summarized in Table 1.

Table 1.

Representative examples of plant derived pharmaceuticals for oral immunization published in the last 5 years. A single paper has been selected for each target antigen

Disease	Pathogen	Antigen	Construct	Expression platform	Expression level	Animal model	Main immunological responses	Reference.
AIDS	HIV	p24, Nef	Fusion protein of the two antigens	Tobacco/transplastomic	Up to 40% TSP	Mice	Induction of specific IgG antibodies Induction of specific IgG antibodies	57
		p24	‐	A. thaliana/nuclear; carrot/nuclear A. thaliana/nuclear; carrot/nuclear	17–366 ng g–1 FW in A. thaliana; 90 ng g–1 FW in carrot 17–366 ng g–1 FW in A. thaliana; 90 ng g–1 FW in carrot	Mice	Induction of specific IgG antibodies	70
		gp120 and gp41	Multiepitopic protein derived (multi‐HIV) from both antigens Multiepitopic protein derived (multi‐HIV) from both antigens	Tobacco/transplastomic	ND	Mice	Induction of specific antibodies and T helper specific responses Induction of specific antibodies and T helper specific responses	42, 71
Anthrax	Bacillus anthracis	PA	‐	Indian mustard/nuclear; tobacco/transplastomic	0.3–0.8% TSP in mustard; 2.5–4% TSP in tobacco0.3–0.8% TSP in mustard; 2.5–4% TSP in tobacco	Mice	Induction of specific IgG and IgA antibodies and protection against anthrax	53
Botulism	Clostridium botulinum	BoHc	45 kDa C‐terminal part of BoHc	Rice/nuclear	100 μg/seed100 μg/seed	Mice	Induction of specific IgA and IgG antibodies and protection against botulinum neurotoxin. Induction of specific IgA and IgG antibodies and protection against botulinum neurotoxin	59
Cervical cancer	HPV	HPV16 L1	‐	Tobacco/nuclear	0.22–0.31% TSP	Mice	Induction of specific IgA and IgG antibodies and of cell‐mediated immune response. Induction of specific IgA and IgG antibodies and of cell‐mediated immune response	72
		HPV16 L1, E6, E7	L1 based cVLPs containing a string of T‐cell epitopes E6 and E7 fused to their C‐terminus. L1 based cVLPs containing a string of T‐cell epitopes E6 and E7 fused to their C‐terminus	Tomato/nuclear	0.05–0.1% TSP	Mice	Induction of specific antibodies, long term protection, inhibition of tumour growth and tumour reduction. Induction of specific antibodies, long term protection, inhibition of tumour growth and tumour reduction	73
Cholera	Vibrio cholerae	CTB	CTB lacking N‐glycosilation (CTB/Q)	Rice/nuclear	2.35 mg g–1 of seed	Mice/MacaquesMice/Macaques	Induction of CTB‐specific IgG and IgA antibodies and protection against CT‐induced diarrhoea. Induction of CTB‐specific IgG and IgA antibodies and protection against CT‐induced diarrhoea	59
Gastroenteritis	Enterotoxigenic Escherichia coli. Enterotoxigenic Escherichia coli	LTB; ST	Fusion protein of the two toxins	Tobacco/nuclear	Up to 0.05% TSP	Mice	Induction of specific mucosal and systemic antibodies. Induction of specific mucosal and systemic antibodies	54
	Human rotavirus	VP2, VP6, VP7	Virus‐like particles (VLPs) 2/6/7	Tobacco/nuclear	Up to 0.15% TSP	Mice	Induction of specific IgA and IgG antibodies. Induction of specific IgA and IgG antibodies	74
		ARP1	‐	Rice/nuclear	170 μg/seed (11.9% of seed protein). 170 μg/seed (11.9% of seed protein)	Mice	Neutralizing activity and protection against rotavirus. Neutralizing activity and protection against rotavirus	75
Hepatitis B	Hepatitis B virus	HBsAg	‐	Potato/nuclear	Up to 0.05% TSP	Mice	Induction of specific antibodies and of stable immunological memory. Induction of specific antibodies and of stable immunological memory	76
Influenza	Influenza virus	M2e peptide	Flagellin of Salmonella typhimurium fused to four tandem copies of M2e peptide. Flagellin of Salmonella typhimurium fused to four tandem copies of M2e peptide	N. benthamiana/transient. N. benthamiana/transient	Up to 30% TSP (˜1 mg g–1 LFW). Up to 30% TSP (˜1 mg g–1 LFW)	Mice	Induction of specific serum antibodies and protection against influenza virus challenge	60
		H3N2 nucleoprotein	‐	Maize/nuclear	Up to 70 μg g–1 seed. Up to 70 μg g–1 seed	Mice	Induction of specific antibodies. Induction of specific antibodies	77
Malaria	Plasmodium falciparum	Pfs25	N‐terminal fusion of the protein with CTB	Alga/transplastomic	0.09 % TSP	Mice	Induction of specific IgG and IgA antibodies	52
	Plasmodium vivax	MSP‐1, CSP	Fusion protein of the two antigens	Brassica napus/nuclear	ND	Mice	Induction of specific IgG1 antibodies and Th1‐related cytokines	78
Toxoplasmosis	Toxoplasma gondii	GRA4	‐	Tobacco/transplastomic	0.2% TSP (6 μg g–1 LFW)	Mice	Induction of specific mucosal and systemic immune response	79
		SAG1	N‐terminal fusion of SAG1 to 90‐kDa heat shock protein of Leishmania infantum	Tobacco/transplastomic	100 μg g–1 LFW	Mice	Induction of specific antibodies, reduction of the cyst burden	80
Tuberculosis	Mycobacterium tuberculosis	CFP10, ESAT6	‐	Carrot/nuclear	0.024% TSP for CFP10; 0.056% TSP for ESAT6	Mice	Induction of cell‐mediated and humoral immune responses	81
		Ag85B, MPT64, MPT83, ESAT6	‐	Potato/nuclear	ND	Mice	Induction of specific IgA and IgG antibodies and Th1‐associated immune responses	82
Allergic asthma	‐	Der p 1	Bioencapsulation of allergen (45–145 aa) into protein bodies	Rice/nuclear	Up to 90 μg/seed	Mice	Inhibition of allergen‐specific Th2, cytokine synthesis, IgE and IgG and bronchial hyper‐responsiveness	66
		Der p 2	‐	Tobacco/nuclear	0.5% TSP	Mice	Decrease of allergen‐specific IgE and IgG1, IL‐5 and eotaxin, eosinophil infiltration, hyper‐responsiveness; Induction of regulatory T cells	65
Allergic conjunctivitis	‐	Cry j 1, Cry j 2	Modified antigens obtained by deconstruction, fragmentation and shuffling	Rice/nuclear	0.5–1.3 mg g–1 seed	Mice	Suppression of allergic conjunctivitis. Reduction of allergen‐specific IgE, inflammatory cells and cytokines	20
Haemophilia A	‐	HC and C2 domain of FVIII	Antigen fusion to CTB	Tobacco/transplastomic	80 μg g–1 LFW for CTB‐HC; 370 μg g–1 LFW for CTB‐C2	Mice	Suppression of allo‐antibody formation; reversal of inhibitor formation. Induction of regulatory T cells	21
Haemophilia B	‐	FIX	Antigen fusion to CTB	Lettuce/transplastomic	0.48–0.63% TSP (up to 58.38 μg g–1 LFW)	Mice	Suppression of allo‐antibody formation. Induction of regulatory T cells	62
Pompe disease	‐	GAA	N‐terminal (410 aa) GAA fused to CTB	Tobacco/transplastomic	0.13–0.21% TSP (up to 6.38 μg g–1 LFW)	Mice	Suppression of anti‐GAA antibody formation	64

Ag85B antigen 85B; AIDS acquired immune deficiency syndrome; ARP1 anti‐rotavirus llama heavy chain antibody; BoHc heavy chain of botulinum type A neurotoxin; CFP10 10‐kDa culture filtrate protein; Cry j 1, Cry j 2 Cryptomeria japonica allergen 1 and 2; CSP circumsporozoite protein; CTB cholera toxin B subunit; cVLPs chimeric virus‐like particles; Der p 1, Der p 2 Dermatophagoides pteronyssinus allergen 1 and 2; ESAT6 early secreted antigenic target‐6; FIX coagulation factor IX; FVIII coagulation factor VIII; FW fresh weight; GAA acid alpha glucodisase; gp41, gp120 envelope glycoprotein 41 and 120; GRA4 dense granule protein 4; HBsAg hepatitis B surface antigen; HC heavy chain; HIV human immunodeficiency virus; HPV human papillomavirus; HPV16‐E6, −E7, −L1 human papilloma virus 16 E6, E7 and L1 proteins; LFW leaf fresh weight; LTB B subunit of heat‐lable toxin; MSP‐1 merozoite surface protein‐1; Nef negative regulatory factor; ND not determined; p24 24 kDa capsid protein; PA protective antigen; Pfs25 25 kDa Plasmodium falciparum surface protein; SAG1 surface antigen 1; ST heat‐stable toxin; TSP total soluble proteins; VLPs virus‐like particles; VP2, VP6, VP7 rotavirus capsid proteins 2, 6, 7.

Oral vaccination

Recent studies concerning plant derived oral vaccines have covered a broad range of diseases, including respiratory infections, gastroenteritis, hepatitis, HIV/AIDS, cervical cancer, malaria, botulism and toxoplasmosis. In order to develop more efficacious vaccines, antigens have been combined with mucosal adjuvants either by protein fusion (e.g. 52) or co‐administration (e.g. 53). Vaccine efficacy can also be enhanced by developing fusion proteins, combining different antigens representing the same pathogen (e.g. 54). Broader spectrum vaccines representing more than one pathogen have also been produced in plants, e.g. the combined expression of CTB and LTB in maize to induce simultaneous immune responses against enterotoxigenic Escherichia coli (ETEC) and Vibrio cholera in mice by oral administration 55.

In some cases, plant derived oral vaccines have been used to deliver booster doses after the primary dose has been administered by injection, e.g. the recently reported oral delivery of wafers made from maize seeds expressing Hepatitis B virus surface antigen (HBsAg) to mice previously immunized by intramuscular injection with the same antigen 56. This vaccination scheme induced long term systemic and mucosal immune responses, providing additional protection over the conventional parenterally administered vaccine. Another example concerns the oral administration of a partially purified HIV p24–Nef fusion protein from transplastomic tobacco leaves as a booster to mice previously immunized by a subcutaneous injection with either p24 or Nef 57. This treatment also induced strong antigen‐specific serum IgG responses (IgG1 and IgG2 subtypes) associated with cell‐mediated Th1 and humoral Th2 responses, respectively.

Table 1 clearly highlights that the preferred animal model for the testing of plant derived vaccines is the mouse, although there is a single exception 58. Oral delivery is the preferred mucosal route, although there are also two examples of intra‐nasal delivery 59, 60. Most of the reported pre‐clinical studies in animal models confirmed that an immune response was induced by plant derived vaccines but only a limited number of studies have demonstrated the efficacy of the vaccine by including a pathogen challenge 53, 59.

Oral tolerance induction

Plant derived pharmaceuticals used for oral tolerance induction have predominantly targeted autoimmune diseases, especially autoimmune diabetes 40, 61. However, another interesting application is the use of oral tolerance as a form of prophylactic tolerance induction, to prevent rejection responses that hamper replacement therapies in genetic disorders, such as haemophilia A and B 21, 62, 63 and Pompe disease 64. These diseases are currently treated by regular replacement therapy, but complications occur in some patients due to the development of neutralizing antibodies against the therapeutic proteins, which are recognized as non‐self‐antigens 40. Preparations of transplastomic leaves expressing coagulation factor VIII, coagulation factor IX and acid alpha glucodisase (GAA) have been used to treat mouse models of the three diseases listed above, resulting in the suppression of unwanted adaptive immune responses and the induction of oral tolerance in each case.

The last 5 years has also seen the use of transgenic plants producing heterologous allergens as a form of allergen‐specific immunotherapy, e.g. for the treatment of allergic asthma 65, 66 and allergic conjunctivitis caused by Japanese cedar pollen 20, 67.

Perspectives

The oral delivery of biopharmaceuticals is a topic of intense current research. More than 60% of current small‐molecule drugs are taken orally 68 but only 40 protein and peptide drugs are administered in this manner 69. Recent progress in the development of plant derived pharmaceuticals is encouraging and the oral administration of edible plants expressing biopharmaceutical proteins could therefore achieve a significant step towards the goal of developing safe, cost‐effective and efficacious biopharmaceuticals for oral delivery. We speculate that the greatest potential of plant derived biopharmaceuticals reflects their cost–effective production. For this reason, the first therapeutic targets are likely to include autoimmune diseases, given the large amount of autoantigen that must be consumed to achieve oral tolerance induction, and low cost vaccines for oral delivery in the developing world.

We envisage that one of the major limits in the use of plant platforms for direct oral delivery of biopharmaceuticals may be the regulatory barriers to commercialization, enhanced by the intrinsic fragmented nature of this approach which relies upon different production platforms.

Competing Interests

All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work.

Merlin, M. , Pezzotti, M. , and Avesani, L. (2017) Edible plants for oral delivery of biopharmaceuticals. Br J Clin Pharmacol, 83: 71–81. doi: 10.1111/bcp.12949.