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REVIEWARTICLE Recent Development and Future Prospects of -Based

Sayed Sartaj Sohrab1,*, Mohd. Suhail2, Mohammad A. Kamal2,3,4, Azamal Husen6 and Esam I. Azhar1,5

1Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; 2King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; 3Enzymoics, Hebersham NSW, Sydney, Australia; 4Novel Global Community Educational Foundation, 7 Peterlee Place, Hebersham, NSW 2770, Sydney, Australia; 5Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; 6Department of Biology, College of Natural and Computational Sciences, University of Gondar, Gondar, Ethiopia

Abstract: Background: Growing world population and continuous disease emergence have invited the development of more efficient new vaccines against a range of diseases. Conventional vaccines are being wildly used in the world but their production requires higher cost, more time and better infrastructure. Thus, the idea of plant-based edible technology has emerged and showed promising results with strong and effective protection against many diseases. have been utilized since more than two decades as pharmaceuticals against many diseases. Methods: Plant-based technology has great potential to express genes and produce clinically important compounds A R T I C L E H I S T O R Y in the desired tissue. Plant biotechnology has played important role in the production of pharmaceutical compounds Received: September 06, 2016 like vaccines, antibodies, , sub-units, growth hormones and enzymes by utilizing genetic modification. It has Revised: January 20, 2017 also been opened a new approach for developing an edible vaccine as an oral delivery. Accepted: January 20, 2017 Results: Edible vaccines have been shown to induce both mucosal as well as systemic immunity. Currently, many DOI: 10.2174/1389200218666170711121810 pharmaceuticals proteins as an edible vaccine have been developed in different plant expression systems and evalu- ated against various life-threatening diseases and some of them have reached advanced phase of the clinical trial and exhibited promising results. Conclusion: In this review, we have discussed about the molecular , edible vaccines, plant base technology and current status of developed edible vaccines in the different plant tissue expression system, mechanism of action and clinical applications with clinical trials stage, significance, requirements, advantage and disadvantage of edible vaccines. Keywords: Plant-based edible vaccine, transgenic plant, immune response, molecular farming, infectious agents, human health.

1. INTRODUCTION instance, easy to transform, expression and purification of complex According to World Health Organization (WHO), globally recombinant proteins with proper folding and stability of their func- more than 20 million deaths annually caused by various diseases; tion; and their bulk production with less cost and time, eco-friendly and among them, Ischaemic heart disease is one of the highest and safe, high scalability, no refrigeration required for storage and causes of death [1]. Vaccines play an important role in controlling eliminates the requirement of trained medical person for delivery of various diseases. Currently, very promising results have been ob- edible vaccine [3]. served against cancer and autoimmune disorder prevention. Con- The development of an edible vaccine in plants is the most re- ventional vaccines are generally made by using live attenuated or cent technology being utilized to produce many desired and clini- killed pathogen and delivered either by injection or oral route. Ad- cally important proteins in different plant tissue expression system ditionally, passive immunization utilizing serum antibody also pro- [4, 5]. The most important objective of this technology is to pro- vides protection against many diseases. Conventional vaccines are duce an edible vaccine and provide protection against many dis- known to be highly effective, although their large scale production eases by inducing specific immune response after oral delivery and is cost effective, time-consuming which ultimately favours the uptake of edible vaccine [6]. Currently, plants are being utilized as quick emergence and re-emergence of outbreaks [2]. Thus, recom- green factory as bio-pharmaceuticals and production of many im- binant and purified antigens as vaccines opened a new approach for portant proteins and enzymes. In 1986, the first transgenic tobacco successful vaccinations to fight against various types of diseases. was developed to express human growth hormone [7] followed by For the bulk production of recombinant vaccines and other clini- another development of transgenic tobacco in 1989 expressing an cally important proteins, currently, the standardized system like antibody with proper assembly of functional complex glycoproteins bacteria, yeast, and mammalian system are being successfully util- by another research group [8]. The structural confirmation of re- ized. The current system has limited benefit as it requires more cost, combinant protein was proved in 1992 by expressing the time and has the limitation of scalability and target integrity. Plant- virus (HBV) surface (HBsAg) in tobacco plant by Mason based edible vaccine development technology provides an alterna- et al. [9]. tive and new approach with many impressive advantages, for Currently, edible vaccine candidates have been developed in multiple plant species including Apple, Arabidopsis, Banana, Beans *Address correspondence to this author at the King Abdulaziz University, (Vicia faba), Canola, Carrot, Clover, Lettuce, Maize, Papaya, , Post Box No-80216, Jeddah-21589, Saudi Arabia; Tel: +966-554627872; Fax: +966-6400-1000; E-mail: [email protected] Peanut, Rice, Spinach, Tobacco, and . The current status of

 1389-2002/17 $58.00+.00 © 2017 Bentham Science Publishers 832 Current Drug Metabolism, 2017, Vol. 18, No. 9 Sohrab et al. transgenic crops producing vaccine/antigens/antibodies has been Spinach [111-112], Papaya [113, 114], Apple [115], Bean (Vicia listed in Table 1. Many vaccines have been developed in multiple faba) [116], Arabidopsis [108, 117], Soybeans [118, 119] and Clo- expression systems for various human and animal diseases and ver [16]. showed promising results with more efficacy and safety in pre- Different types of genes have been expressed with desirable clinical studies and moved towards the clinical studies and their traits in many plant expression system [4-6, 10, 15-21, 26, 113, results are summarized in Table 2 [10]. The edible vaccines can 120-134] and evaluated as edible vaccine against various viruses be produced locally at low cost and bulk production with less like; Hepatitis B virus, Hepatitis C virus, Influenza viruses, Papil- processing time. They are orally delivered and can be protected loma viruses, Human Immuno deficiency Virus (HIV), Dengue with natural bio-encapsulation which provides protection from virus, Rabies virus, Rotaviruses, Norwalk virus and heat-labile gastric enzymes and longer stability in the gastrointestinal (GI) entero toxin B subunit [5,16,131]. Virus-like particles (VLPs) were tract and taken up by M cells to induce natural immunity [11]. developed in plant systems against many viral diseases known as This technology has significantly attracted both academia and hemagglutinin-based virus-like particles (VLPs), Influenza virus- industry. like particles (VLPs) against pandemic influenza, [47,128], avian H5N1 influenza, [56,131], H7-VLP against H7N9 influenza virus, 2. IMPORTANT DEVELOPMENT IN PLANT-BASED EDI- vaccine against bovine viral diarrhoea and Virus-like particles BLE VACCINE (VLP) against H1N1 influenza virus has also been recently devel- The term edible vaccine was introduced by Charles Arntzen oped [54, 67, 68, 135, 136]. with Hugh Mason and colleagues [12]. First of all nopaline syn- thase and human growth hormone were produced in sunflower and 4. CONCEPT OF EDIBLE VACCINE DEVELOPMENT tobacco plants [7]. The idea of plant molecular farming was bur- The edible vaccine construction requires a transformation and geoning when the proof of concept for recombinant plant-derived regeneration protocols for specific plants. The selected gene is pharmaceutical proteins was reported and published and the idea transformed into plant cells with desired delivery system. The edi- has since expanded to the production of many industrial and agri- ble vaccines should be designed very carefully and eliminate all the cultural recombinant enzymes [13]. Among these, biologically ac- possibility of any pathogenic characters. The productions of con- tive human Interferon-alpha D (IFN) was produced in turnip fol- ventional vaccines are very expensive and require proper purifica- lowing inoculation with mutant cauliflower mosaic virus carrying tion and refrigeration for their storage. The edible vaccine can be human IFN D at its ORFII [13]. In the same year, tobacco plants very useful for children as it can be taken up orally without any expressing either gamma or kappa immunoglobulin chains of the trained medical persons. The edible vaccine can be easily produced mouse were crossed to generate progeny expressing both chains of at a higher scale with low cost [74]. The possibility of carrying out an immunoglobulin, showing the capability of plants to gather het- challenge experiments in specific animal species of interest has erologous biomolecules [8]. Then, human serum albumin (an iden- encouraged and resulted in the enthusiastic development of plant- tical to the authentic human protein) in tobacco and potato plants based edible vaccines [137]. was developed [14]. Since 1989, many genes have been expressed as therapeutic products in various plants based expression system 5. MOLECULAR FARMING IN PLANT SYSTEM by using growing trends and transforming the plants by recombi- Like human, plants can also assemble, modify and secrete de- nant Agrobacterium tumefaciens and “deconstructed” plant viral sired proteins with proper folding. The last 25 years had a signifi- vectors as plants can produce and express multifaceted recombinant cant development in plant science and research has shown promis- proteins in the acceptable shape without loss of their biological ing results in molecular farming with economic productions and the function with better protection (as plants do not carry any patho- potential for large-scale synthesis of valuable candidate recombi- gens of mammals) and current status has been published in many nant proteins and enzymes. Many therapeutic proteins like antibod- reports [3, 5, 15-25]. ies, antigens sub-units, enzymes, hormones, growth regulators have been expressed in various plant species and reviewed in many pub- 3. CURRENT STATUS OF EDIBLE VACCINE lished reports and summarized in the Table 1 [5, 10,17, 22-25]. Currently, only two recombinant proteins as vaccine developed Traditional vaccines are being produced in mammalian or bacterial like monoclonal antibody against HBsAg and New castle disease cell cultures with fermenters-based technology which requires more virus (NDV) vaccine and received regulatory approval from United cost, time and expensive equipment. States Department of Agriculture [16]. Table 1 summarizes the The transgenic plant can be grown in a separate and controlled status of an edible vaccine. The development and commercializa- tion of edible vaccines require an effort and time, there are many condition and the high level of production can be achieved to re- duce the cost of edible vaccine development. Edible vaccines can edible vaccines developed for human and animal diseases; and has be made as capsules to eliminate the need for syringes, trained reached at the various phases of clinical trials [19, 26]. Tobacco medical personnel, and refrigeration which will make the vaccine plant was used for the first time to develop monoclonal antibody by Hiatt and co-workers in 1989 and further this important achieve- even more cost-effective. The production of an edible vaccine will reduce the development cost more as they eliminate or further re- ment drew the attention of researchers to express the medically duce any downstream processing and they can be cultivated on-site important proteins in transgenic crops. In 1990, the first report was for easier distribution and less transportation. Molecular farming in published describing the proof of concept of an edible vaccine [9]. The development of efficacious and affordable pharmaceutical plants can be achieved with different expression systems. For stable expression, the transgenes can be inserted in either the nuclear or compounds in transgenic plants opened new innovations in medical the chloroplast genome [10]. science and plant biology [27-31]. Since 1986, many pharmaceuti- cal therapeutic proteins, antigens, antibodies, sub-units, enzymes, 6. REQUIREMENT OF EDIBLE VACCINE DEVELOP- hormones, growth regulators as edible vaccines against different MENT diseases with various purposes have been produced in different plants like Tobacco [9, 28, 31-72], Tomato [42, 73-76], Banana [77, To develop vaccines, many strategies have been suggested by 78], Potatoes [29, 31, 34, 79-88], Maize [89-96], Rice [97-103], WHO. Vaccines are very valuable for disease and death control lettuce [104], Alfalfa [105, 106], Carrot [107-109], Peanut [110], caused by various infectious agents in approximately 10 million Recent Development and Future Prospects of Plant-Based Vaccines Current Drug Metabolism, 2017, Vol. 18, No. 9 833

Fig. (1). Development and mode of action of an edible vaccine. children in developing countries [74]. The transgenic plants can be To develop edible vaccine through transgenic plants, currently two utilized to produce desired proteins at very affordable cost and eas- strategies are being successfully applied known as nuclear and plas- ily available to the masses of poor people in developing countries. tid transformation and transient expression by Agrobacterium me- The injectable conventional vaccines are very expensive and it can- diated technology. Agrobacterium tumefaciens is capable of trans- not be taken without any medically trained personnel and they are forming its own T-DNA into plant cells. To make use of this not easily accessible in developing countries. Due to the re-use of bacterium, the recombinant plasmid can be made and introduce the needles many diseases like hepatitis B virus (HBV), hepatitis C gene into a plant cell by co-cultivation of desired plant tissue with virus (HCV) and HIV can be easily transmitted and to maintain the Agrobacterium. This is known as plant transformation system and efficacy and transportation to remote localities of the traditional developed plant is known as transgenic plant [138-140]. vaccine, refrigeration is required. Therefore, there is an urgent need for an edible vaccine which will be less costly easily applicable and 7.1. Selection of Plant Candidates for an Edible Vaccines Pro- deliver to needy people without refrigeration. By utilizing the plant duction expression system, the monoclonal antibodies can be with low cost To develop an edible vaccine, appropriate plant selection is at higher scale and it is estimated that only 100 dollars are required very important. There are many plant species have been used for to develop one-kilogram antibodies as compared to 3 million dol- transformation and expression of desired genes. There is a need to lars by using another system. Additionally, plant expression system check the availability of easy plant regeneration and transformation provides other benefits like reducing unwanted contaminations as it protocols for specific plants species and the desired plant products frequently occurs in the animal cell culture system. can be easily used as an edible vaccine without going for cooking. 7. STRATEGY FOR DEVELOPMENT OF EDIBLE VAC- The most acceptable part of plants is as fruit and salad which can be CINE eaten as raw without the loss of vaccine property. The edible vaccine can be developed by transforming the de- 7.2. Plastid Transformation sired plants with the desired gene. The selection of the desired plant A new and alternative technology is available targeting chloro- can be raw as after cooking the protein gets denatured and tomato, plast genome apart from nuclear genome transformation. Chloro- cucumber, and banana are most preferable plants for edible vaccine plast has the smaller genome with more than 100-250 genes and can development. Initially, the desired gene should be selected for spe- be incorporated into algae by easy transformation. The most impor- cific protein expression and then cloned into plant transformation tant character of the chloroplast is that does not go out the crossing vector. The developed vector can be utilized to introduce the de- sired gene with the help of Agrobacterium mediated delivery sys- of transgenes via pollen transfer and it is maternally inherited and tem. The transformed plants producing desired proteins are known produces stable protein. By transforming multiple gene copy num- as transgenic plants. The developed edible vaccine can be taken up ber by biolistic process or polyethylene glycol treatment of proto- orally as an antigen to induce immunity against targeted pathogen. plasts in the chloroplast, higher amount (up to 70% of total leaf Edible vaccines are considered as the cheapest vaccines as they protein) of recombinant protein can be expressed and purified [141, require less cost for their bulk production and easy for oral delivery. 142]. To transform plastid, a target gene can be placed with marker 834 Current Drug Metabolism, 2017, Vol. 18, No. 9 Sohrab et al. gene in-between the two flanking sequences originated from the mature fruit and rapidly spoils after ripening. There will be a vari- chloroplast genome for homologous recombination induction be- ability of dosage and this is not suitable for infants. tween plastid genome and vector [130]. For chloroplast transforma- tion, some selected plants have been used like tobacco, carrot, petu- 10. SIGNIFICANCE OF EDIBLE VACCINE TECHNOLOGY nia and lettuce [143]. This is one of the most recently developed technologies [4- 5,129]. Edible vaccines are highly useful as compared to the con- 8. EDIBLE VACCINE-MODE OF ACTION ventional vaccine as it can be delivered orally and it is more con- An outline of edible vaccine development and the mode of venient for children. The edible vaccine does not require storage action have been provided in Fig. (1). The edible vaccine induces facility for their preservation and this will be more acceptable and both mucosal and systemic immune response. The desired pro- successful in those countries without the strong infrastructure. Ad- teins in the form of vaccine will be released from plant tissue and ditionally by removing this storage barrier will add more benefit to absorbed in the intestinal wall after delivery. The cellular and save approximately $300 million per year and this will help in pro- gastric enzymatic degradation of vaccine can be protected by bio- viding vaccine for extra 10 million children [148] and in an experi- encapsulation. The vaccine is absorbed in the form of antigen on ment it was found that rice edible vaccine developed in rice was the intestinal cell wall and taken by M cells and pass to macro- secure for approximately 18 months at room temperature [99] and phage and B cells and moves towards the antigen presenting cells they are needle-free vaccine [149] and by using this edible vaccine and after that they will be moved and present to T and B cells in technology many blood borne infections can be controlled. Trans- the payer’s patches and gut-associated Lymphoid tissues passed genic rice has the less environmental impact as their pollen does not on to macrophages, and local lymphocyte population generating scatter long distance [146]. Moreover, by using edible vaccine serum IgG, IgE responses. The plasma cells generated by the technology the possibility of unwanted contaminants can be elimi- activation of immune cells and they move towards lymph nodes nated as it happens during animal cell culture. for clonal amplification followed by distribution to other mucosal surfaces. When any pathogenic infection takes place then the 11. FUTURE PERSPECTIVES OF EDIBLE VACCINE memory helper T cells swiftly induces antibody production and The future of plant-based edible vaccine technology is very secretion and the released antibody immediately neutralizes the promising as this provides cost effective, safer vaccine and elimi- invading antigen [86, 133]. nates storage requirements as well as trained a medical person for their delivery. The edible vaccine can be developed in bulk with 9. ADVANTAGES AND DISADVANTAGES OF EDIBLE low cost with lees processing time. This vaccine can be developed VACCINE even at the site of utilization in a safe manner. It is well known that An edible vaccine has many advantages like delivery of the the traditional vaccine played an important role in making better vaccine, particularly by the oral route, is in high demand [144]. It health for world population but their bulk production is expensive can be used as raw in the form of fruits and vegetables. Edible and time-consuming, therefore, the edible vaccine technology is an vaccines can be dried and used as powered form which does not alternative approach to resolving these issues. By utilizing this require cold storage. The edible vaccine does not require any ad- technology, more effective, better and safer immunization, as well juvant to enhance immune responses and can be delivered effec- as disease prevention can be provided to the community especially tively. The edible vaccine also induces mucosal immunity as for the life-threatening disease affecting bigger masses of the popu- compared to traditional vaccines. The edible vaccines can be pro- lation of globally like AIDS, dengue, malaria, heart disease, intesti- duced, transported and stored at very less cost as compared to nal disease as well as respiratory diseases and disorders. Moreover, traditional vaccines [99]. The edible vaccines can be developed in in the future, multi-component vaccine as an adjuvant can be devel- seeds and oils and seeds can be dried and aqueous extracts of oils oped by crossing two generation of plants harboring multiple types can be stored without any refrigeration [145]. Transgenic plants of genes expressing clinically important antigens [150, 151]. The producing edible vaccines can be grown in soil and it does not future of edible vaccine also depends on the standard set by WHO require costly equipment and machines. The plants can be grown especially on the cost effectiveness, purity, and safety with more easily and the production cost is less and does not require any efficiency [122]. Various types of system has been used to express special premises to manufacture them and does not require sterili- the desired genes in different expression system against multiple zation. The edible vaccines can be administrated orally and re- diseases [152-160]. The acceptance and growing of transgenic move the use of injections and requirements of trained medical crops at a larger scale in developing countries is also a concern, and person. The edible vaccine does not have any risk of contamina- if this is accepted by the society then the edible vaccine production tion and does not require sterile conditions and reduces the spread and vaccination against many diseases will be possible globally [74, of another disease [11, 146]. Multiple antigens can be expressed 78]. in one vaccine by combining the various genes known as a Multi- Currently, microalgae are being used as very important and component vaccine. The edible vaccine does not have any possi- valuable source of active molecules like carotenoids, chlorophyll, bility of the reformation of an infectious organism as they do not enzymes, fatty acids, phycobiliproteins and carotenoids. Microalgae involve attenuated pathogens. Mass level production can be done can be utilized in the future for the recombinant protein expression, for edible vaccine as compared an animal system [147]. There is purification, pharmaceuticals, immune regulators, growth factors, no fear of pathogen infection in plants while in the animal, if any hormones, and many other products, like anticancer agent Taxol infection happens then antibodies will be developed against those and they can be used as an edible vaccine. They have many alterna- pathogens too and co-migratory proteins will affect the purifica- tive advantages as for large scale production and fast transformation tion of the protein. and rapid growth and stable expression levels with proper folding The edible vaccine produced in Beans, Potato, Rice, and Spin- and accumulation of multiple antigens as vaccines and effective ach cannot be used as raw; and during cooking, the antigenic prop- delivery through the oral route. They can be utilized as a tablet for erty lost. The banana based edible vaccine requires 2-3 years to get easy administration and immunogenic response [161, 162]. Recent Development and Future Prospects of Plant-Based Vaccines Current Drug Metabolism, 2017, Vol. 18, No. 9 835

Table 1. Status of Transgenic crops producing vaccine/antigens/antibodies.

Recombinant Protein/Antigens/Vaccines Production System References

Rabies virus glycoprotein Tomato [12]

Human IFN D Turnip [13]

Norwalk virus vaccine Potato, Tobacco [28, 34, 42]

Rabies virus glycoprotein Tomato, Tobacco [28,30,57,76]

HIV antigen Tobacco [31]

Human Lactoferrin Potato [31]

Colon Cancer antigen Tobacco [32]

Human Somatotropin Tobacco [33]

Bacillus anthracis protective antigen Tobacco [35, 38,40]

Measles virus Hemagglutinin Tobacco [36]

Hepatitis B virus-IgG Tobacco [37]

Tet C (Tetanus vaccine antigen) Tobacco [39]

Norwalk virus capsid protein Tomato [42]

Human papillomavirus -16-L1 capsid protein (CP) gene Tobacco [45]

Diarrhea Tobacco [46]

Influenza Antigen Tobacco [47,54, 56, 68, 128,135]

Virus like particles (VLP)-H1N1 Tobacco [47,68,128]

Recombinant Norwalk Virus Tobacco [49]

P.yoelii merozoite surface protein Tobacco [50]

Japanese encephalitis virus envelop protein Tobacco [51]

Hepatitis B surface antigen-(HBsAg) Potato, Tobacco, Lettuce [ 53,81, 84-85,88,104]

Cholera Ctox A and Ctox B subunits Potato, Tomato, Tobacco, Rice [55,70, 78,79, 83, 99]

Serum albumin Tobacco [58]

Atherosclerosis Tobacco [59,61]

HCV-capsid protein Tobacco [60]

Bacterial polysaccharides Tobacco [62]

Rabies virus- monoclonal antibody E559 Tobacco [63]

Malaria vaccine Tobacco [64]

Monoclonal antibody Tobacco [65]

Monoclonal antibodies for cancer Tobacco [66]

H7N9 influenza virus Tobacco [67]

Monoclonal antibody - Ebola GP1 protein Tobacco [69]

Rotaviruses Tobacco [71]

(IgA) and M (IgM) Tobacco [72]

Yersinia pestis (Plague) Tomato [73]

Hepatitis B Surface antigen-(HBsAg) Cherry tomato [75]

Table (1) contd…. 836 Current Drug Metabolism, 2017, Vol. 18, No. 9 Sohrab et al.

Recombinant Protein/Antigens/Vaccines Production System References

Hepatitis B surface antigen-(HBsAg) Banana [77-78]

Human rotaviruses-VP7 Potato [82,83]

Newcastle disease virus (NDV) protein Potato [86]

Salmo salar (SasalFN 1) protein Potato [87]

Gastroenteritis virus vaccine Maize [89,91-92]

Avidin Maize [90]

LT-B (Heat labile toxin B) Maize kernels, Potato, Soybean [93, 96, 119]

Trypsin Maize [95]

Japanese cedar pollen peptide Rice [97]

Diabetes mellitus Rice seeds [100]

JEV envelop protein Rice [101]

LTB Rice [102]

Antiviral lectin griffithsin (GRFT)-for HIV Rice endosperm [103]

-amylase Alfalfa [105]

Mannheimia haemolytic GS60 antigen Alfalfa [106]

DTP subunit vaccine Tobacco and carrot cells [107]

HIV-1 subtype Cp24 protein Arabidopsis, Carrot [108]

UreB (urease) protein against Helicobacter pylori Carrot [109]

Urease subunit B gene of Helicobacter pylori Peanut [110]

Rabies antigen Spinach [111]

HIV-1 Tat protein Spinach [112]

Synthetic anticysticerosois vaccine Papaya [113-114]

Respiratory syncytial virus (RSV)-F Apple [115]

Human cytomegalovirus (HCMV) Beans (Vicia faba) [116]

Gastroenteritis virus Arabidopsis [117]

Bovine viral diarrhea Panax ginseng [136]

HIV-1 Tobacco [152]

Hirudin from Hirudo medicinalis Canola [153]

LTB Peperomia pellucida Loc [154]

Dengue virus Not Available [155]

Human 1-antitrypsin Rice [156]

Tricosanthin- Tobacco [157]

leukotoxin GFP-fusion protein (Lkt50-GFP) Clover [158]

Hepatitis B surface antigen-(HBsAg) Tobacco [159]

Rotavirus VPI Potato [160]

Abbreviations: HBV: hepatitis B virus, HCV: hepatitis C virus, HIV: human immunodeficiency virus, HBsAg: Hepatitis B Surface antigen, VLPs: Virus like particles.

Recent Development and Future Prospects of Plant-Based Vaccines Current Drug Metabolism, 2017, Vol. 18, No. 9 837

Table 2. Plant-based vaccines, antibodies and therapeutic proteins in clinical development.

Plant Product Plant Host Expression System Indication Administration Route Product Stage

Vaccines Esherichia coli LT-B Potato Transgenic Diarrhoea Oral Phase 1 Maize Transgenic Phase 1 Norwalk virus CP Potato Transgenic Diarrhoea Oral Phase 1 HBsAg Lettuce, Transgenic Hepatitis B Oral Phase 1 Potato Transgenic Phase 1 Rabies virus GP/NP Spinach Transient (viral vector) Rabies Oral Phase 1 Newcastle disease virus Tobacco cell Transgenic Newcastle Subcutaneous USDA approved Suspension disease Personalized anti idiotype ScFVs Tobacco Transient viral vectors) Non-Hodgkin’s Subcutaneous Phase 1 lymphoma Personalized antiidiotype Tobacco Transient Non-Hodgkin’s Subcutaneous Phase 1 (ongoing) DcFVs lymphoma H5N1 influenza HA Tobacco Transient (Agrobacterial H5N1 “avian” Intramuscular Phase 1 (ongoing) VLP binary vector) influenza Phase 2 (Health Canada approved H5N1 Influenza HA/1 Tobacco Transient H5N1 Intramuscular Phase 1 ongoing H1N1 Influenza HA C1 Tobacco Transient H1N1 “swine” Intramuscular Phase 1 ongoing influenza Antibodies Anti-CD20 Duckweed Transgenic Non-Hodgkin’s Intravenous Pre-Clinical Lymphoma rheumatoid arthritis Anti-Streptococcus Tobacco Transgenic Dental caries Topical Phase 2; EU Surface antigen I/III approved Anti-CCR 5 Tobacco Transient HIV Topical Pre-clinical Anti-HIV gp120 Tobacco Transgenic, HIV Topical Pre-clinical Transient binary vector Anti-HBsAg scFV Tobacco Transgenic Hepatitis B Not applicable On market Therapeutic and dietary proteins Glucocerebrosidase Carrot cell Transgenic Gaucher disease Intravenous Phase 3 suspension Insulin Safflower Transgenic Diabetes Subcutaneous Phase 1/2

Gastric lipase Maize Transgenic Cystic fibrosis, Oral Phase 2 pancreatitis Lactoferrin Maize Transgenic Gastrointestinal Oral Phase 1 infections Intrinsic factor A. thaliana Transgenic Vitamin B12 Oral Phase 2 deficiency

CONCLUSION plant-based vaccine development. However, lots of more research The idea of an edible vaccine has attracted many researchers needs to be performed in this twenty-first century to remove any and received more publicity. Plant-based vaccine development possible environmental risk factors related to living organism, dem- technologies have made significant progress during last five years, onstration of a safety issue, regulatory issues, commercial scale and have proven that the potential vaccine can be developed in a production, manufacturing, processing, commercialization and plant cell. Therefore, various efforts have made towards the en- more public awareness to get acceptance for uses of an edible vac- hanced expression, accumulation, stability and biological function cine. The demonstration of safety and public acceptance will lead of expressed proteins in plant tissue as well as a purification proc- towards the successful application and uses/implications of plant- ess. This novel development has facilitated the promising future of based edible vaccine technology, and by using this technology vac- 838 Current Drug Metabolism, 2017, Vol. 18, No. 9 Sohrab et al. cination can be done not only for human but also in animals against expression technologies in plants. Biotechnol. Lett., 2015, 37(2), various types of diseases at the global level. 265-279. [18] ucka, M.; Kowalczyk, T.; Szemraj, J.; Sakowicz, T. Plants as an LIST OF ABBREVIATIONS alternative source of therapeutic proteins. Postpy. Higieny. I. Medycyny. Dowiadczalnej (Online), 2015, 69, 362-373. HBV = Hepatitis B virus [19] Aryamvally, A.; Gunasekaran, V.; Narenthiran, K. R.; Pasupathi, HCV = Hepatitis C virus R. New strategies toward edible vaccines: An overview. J. Diet. IFN  = Human Interferon alpha Suppl., 2016, 1-16. VLPs = Virus like particles [20] Led, K.; Luthar, Z. Production of vaccines for treatment of infectious diseases by transgenic plants. Acta. agriculturae. CONSENT FOR PUBLICATION Slovenica., 2016, 107(1), 191-217. [21] Phan, H. T.; Conrad, U. Plant-based vaccine antigen production. Not applicable. Methods. Mol. Biol., 2016, 1349, 35-47. [22] Abdoli Nasab, M.; Jalali Javaran, M.; Cusido, R. M.; Palazon, J. CONFLICT OF INTEREST Purification of recombinant tissue plasminogen activator (rtPA) protein from transplastomic tobacco plants. Plant. Physiol. The authors declare no conflict of interest, financial or other- Biochem., 2016, 108, 139-144. wise. [23] Merlin, M.; Pezzotti, M.; Avesani, L. Edible plants for oral delivery of biopharmaceuticals. Br. J. Clin. Pharmacol., 2016. ACKNOWLEDGEMENTS [24] Tschofen, M.; Knopp, D.; Hood, E.; Stöger, E. Plant molecular First author would like to gratefully acknowledge the research farming: Much more than medicines. Annu. Rev. Anal. Chem. (Palo facility provided by Special Infectious Agents Unit, King Fahd Alto Calif), 2016, 9(1), 271-294. 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